NHX gene families have already been identified and functionally characterized for several plants, including Arabidopsis, Rice, wheat, sweet beet, cotton and other [9, 45, 46, 47, 48]. However, the NHX genes in C. sinensis has not been studied yet. In this study, the gene structure, phylogenetic relationship, genomic distribution and expression of NHX genes in C. sinensis were all analysed at the genomic level. A total of 9 NHX genes have been identified in C. sinensis based on the Na+/H+ exchanger domain (Table 1). However, there are 7 NHX genes in Oryza sativa (Os), Solanum lycopersicum (Sl), Medicago truncatula (Mt), Sorghum bicolor (Sb), Zea mays (Zm). Gossypium hirsutum (Gh), Glycine max (Gm) have 23 and 12 NHX genes and Solanum tuberosum (St) has 5. Gene duplication and loss specific to different subfamilies of NHX over the course of evolution could explain these differences in the number of NHX genes in plants.
In-silico studies based on subcellular localizations showed that NHXs are grouped into three classes (Vac-, Endo-, and PM-class). In Arabidopsis, both NHX7 and NHX8 are localized in the plasma membrane  whereas in tea, TEA006997.1 localized in the plasma membrane, TEA011468.1 is localized in endosome and the others in the vacuole (Table 1). Members in each of the classes from algae to higher plants, showed that the NHX families were fairly similar, indicating that NHXs had conserved functions throughout the evolutionary process . The function of NHX transporters may be influenced by their subcellular localization. Members of the NHX family, which are found on both the plasma membrane and tonoplast, help to maintain ionic homeostasis by excluding and compartmentalizing excess Na+. Furthermore, endomembrane-bound NHX members have been discovered to be important for cellular cargo trafficking, growth development, and protein processing regulation [15, 50]. The exon/intron structural diversity, which plays an important role in the evolution of gene families, brings to the evidence for phylogenetic groupings. In C. sinensis, TEA021179.1 possesses a greater number of introns (18) and exons (19) while TEA012938.1 has lesser number of introns (12) and exons (18) than the rest of the 5 genes present in Vac-class. However, in Populus trichocarpa, Vac-class NHXs (PtNHX1-5) contain 14 exons and the Endo-class NHX (PtNHX6) has 22 exons, while the PM-class NHXs (PtNHX7 and PtNHX8) displays 23 exons . Similarly, for NHX genes in Glycine max (Gm), seven members of GmNHX contain 14–15 exons, whereas the rest three members have 20 exons . These findings suggested that NHX gene families in plants have a fair share of structural diversity.
The putative amiloride-binding site and membrane-spanning pore in the NHX gene families, which contain the amino acid sequence “FFIYLLPPI” , have been found to be highly conserved [6, 16, 18]. In the presence of the drug amiloride and/or its derivatives, this domain inhibits the cation/H+ exchange . In the motif study, the amiloride-binding site is located in the N-terminus of motif 3 and it is found in 6 NHX genes of C. sinensis out of the 9. However, motif 3 is not conserved in TEA023041.1, TEA011468.1 and TE006997.1 (Fig. 2). The C-terminus of NHX proteins was diverse in contrast to the conserved N-terminus. Studies have shown that the deletion of the C-terminal hydrophilic region results in increased Na+/H+ transport activity, implying that the C-terminus is important not only for subcellular localization but also for transport activity regulation [8, 53]. Additionally, conserved motif analysis also showed that motif 8 and motif 14 conserved in all the NHX genes (Fig. 2). Similar results are observed in Arabidopsis  and Oryza sativa . The phylogenetic analysis indicated that, the NHXs in P. trichocarpa , S. bicolor , and B. vulgaris  showed three phylogenetic clusters based on their location in the cell; we found the same results for tea NHX transporters. So according to these findings, the NHX family genes have remained relatively conserved throughout evolution.
Cis-acting regulatory elements function as key molecular switches in transcriptional regulation of gene activities that control a variety of biological processes such as hormonal response, abiotic stress response and development [54, 55]. Hormones including ABA, ethylene, SA and IAA play significant roles in plants development and stress response [56, 57, 58, 59]. In this study, cis-acting regulatory elements related to transcription factors were identified to be randomly distributed across the promotor region of the 9 tea NHXs (Additional File 1: Table S2). One ABA-responsive element (ABRE) has been discovered in 6 NHXs (TEA012938.1, TEA012286.1, TEA012245.1, TEA000661.1, TEA025916.1, TEA011468.1) of C. sinensis (Additional File 1: Table S2). Whereas in poplar, one or two ABREs were observed . This analysis showed that NHX genes may play a role in the ABA signaling pathway. Furthermore, ARE (anaerobic induction), DRE (drought-responsive cis-acting element), LTR (low-temperature responsive element), MBS (drought response,) and STRE (stress-response) were identified as stress responsive regulatory elements in tea. Similarly, in PtNHXs from poplar and SbNHXs from S. bicolor are also found to contain similar elements . The results indicated that the identified regulatory elements in this study aid in understanding their roles in various abiotic and biotic stress-related pathways.
Further to understand the distribution pattern of the tea NHXs, the genomic distribution mapping was performed. Tandem duplication events were absent across the tea NHXs (Fig. 5). The duplication of genes increases the functional divergence, which is an essential factor in adaptability under changing environmental conditions . The dN/dS ratio indicates the selection pressure on amino acid substitutions, with a ratio less than 1 indicating purifying selection and a ratio greater than 1 indicating positive selection. Wang et al  found that positive selection of a gene during evolution increases its potential and transcription levels under stress conditions in T. aestivum and TaBT1. Whereas in tea, the dN/dS ratios provided conclusive evidence that strong purifying selection pressure existed during evolution, allowing a variety of factors to regulate the genes (Additional File 1: Table S3).
In plants, sodium-proton antiporters facilitate both Na+/H+ and K+/H+ exchanges, contributing to stress tolerance as well as K+ nutrition [62, 63, 64]. NHXs have been reported to enhance salinity tolerance in different species, such as Arabidopsis , B. vulgaris , S. lycopersicum [66, 67], H. vulgare , Z. mays , T. aestivum , G. max , O. sativa [72, 73] and S. bicolor . The expression data for various tissues and stress conditions showed that the tea NHXs may be involved in developmental processes and abiotic stress responses. Our study revealed that in C. sinensis, the NHX genes express differentially in 8 different tissues where TEA012938.1, TEA012286.1 and TEA000661.1 genes showed the highest level of expression in all the tissues and belonged to Vac-class (Additional File 1: Table S5). The different expression patterns in various tissues (Fig. 8), indicated that the NHX gene family provide opportunities to break the functional constraint from the original gene during the course of evolution.
Based on data from other species, functional annotation and interaction analysis of NHX proteins can help us predict their potential regulatory roles. An interaction network was built using Arabidopsis as the model plant and 5 tea NHX proteins described as responsive to salt stress in GO annotations as well as in expression analysis. The electrochemical gradient of protons across tonoplasts, generated by two vacuolar H+-pumps, H+-APTase and H+-PPase, has been shown to drive the Vac-class NHXs [6, 74, 75] In this analysis, all the tea genes considered for building the interaction network, belongs to the Vac-class. By increasing cation accumulation, co-expression of ZxNHX and ZxVP1 genes can improve salt tolerance in transgenic plant species such as Lotus corniculatus , Alfalfa , and sugar beet . These finding suggested that when plants were exposed to salt stress, Vac-class NHXs might work together to transport Na+ across tonoplasts. Calcineurin B-like (CBL) is well known for its ability to interact and modulate CBL interacting protein kinases (CIPK), which then mediate Ca+ signal transduction [34, 79]. During the salinity response, CBL regulates NHX7 (SOS1) and CIPK mediates the Ca2+ signalling pathway . A salt-stress elicited Ca2+ signal activates a protein kinase complex consisting of CBL4 (SOS3) and CIPK24 (SOS2), and the complex then phosphorylates and activates the SOS1 protein to extrude Na+ out of the cell in Arabidopsis under salt stress . In transgenic tobacco, overexpression of SOS1 gene increased salt tolerance by maintaining a higher K+/Na+ ratio . In the current study, CLBs are hypothesized to interact with TEA006066.1, TEA012938.1, TEA012286.1, TEA21179.1, and TEA012245.1 but not with CIPK (Fig. 7). Similarly, NHX7 (SOS1) interactions with CBLs were predicted in poplar  and S. bicolor . However, in the future, yeast two hybrid research will need to confirm these proteins interactions.
Ion transporters are important in many biological processes, including ion uptake and sequestration, energy provision and cell expansion . Previous studies in plants found that Na+/H+ antiporters as important members in transporters, mediate the coupled exchange of Na+ or K+ for H+ in all cellular compartments [83, 84]. The NHX genes primarily use two proton pumps, the H+-ATP enzyme and H+-PPase, to produce H+ electrochemical gradients that transport Na+ from the cytoplasm to vacuoles or outside the cell, there by maintain Na+ ion stability and avoiding the toxic effect of Na+ accumulation in cells [85, 86].
Stress response analysis showed that each tea NHX genes were responsive to abiotic stresses of drought, cold and salt. Under PEG treatment, the expression of TEA012938.1, TEA012245.1,TEA00066.1, TEA023041.1, TEA011468.1 reached the highest level at 12h (Fig. 10), and TEA012938.1, TEA023041.1 and TEA011468.1 also responded to salt stress in varying degree, demonstrating these genes may be associated with salt and drought stress. MeJA was found to be linked to salt tolerance in few studies [87, 88]. MeJA expression analysis revealed that TEA012938.1 and TEA000661.1, expressed high levels throughout the MeJA treatment (Fig. 12), demonstrating these genes may respond to MeJA hormone regulation. Further in the study, the expression levels of TEA012938.1, TEA023041.1 and TEA011468.1 were significantly up-regulated by various concentrations of NaCl over a 48-h period and 72-h period (Fig. 11), and their expression levels under high-salt stress were relatively higher than those under either mild or moderate-salt stress. In Reaumuria trigyna, the expression levels of RtNHX1 in leaves showed an increase and reached a high level at 3 hours, and then reduced after 6 hours when exposed to high salt stress (200 mM NaCl) . A similar expression pattern was found in sweet potato, where IbNHX2 was significantly up-regulated at 4 hours after treatment of 200 mM NaCl . Another study  found that the transcription level of TaNHX3 in both leaves and roots sharply increased at 24 hours and then gradually decreased after 48 hours over a 96 hours period in different wheat cultivars subjected to salt stress. Moreover TEA012938.1 belonging to the Vac-class NHX, showed the highest level of expression for all the salt stress condition. The study showed that the expression levels of Vac-class NHXs are significantly higher than other class genes thereby confirming that Vac-class NHXs might play critical roles in salt tolerance. The study also notices that TEA012938.1 and TEA023041.1 showed significant expression levels under all abiotic stress conditions thereby providing a comprehensive understanding of the functions of NHXs in C. sinensis.