Global climate change in recent decades has triggered prolonged and severe droughts in many parts of the world. Consequently, irrigation with low-quality water sources was increased, and fertilization practices accelerated soil salinization [1]. Saline stress is more likely to affect areas with increased temperature, lower rainfall, and high evaporation and transpiration rates. Salinity and other abiotic stresses are considered a significant threat to modern agriculture, compromising the growth and development of essential crops [2]. In addition, salinity stress triggers changes in physiological responses and biochemical pathways in plants, decreasing leaf area, photosynthesis, and transpiration [3, 4]. Another significant effect is the increased production of reactive oxygen species (ROS) due to the destabilization of cellular homeostasis, causing damage to proteins, DNA, and lipids [5]. When the Na⁺ and Cl^– contents in the soil are high, the ability of the plant to absorb and transport other mineral nutrients, especially NO₃^–, K⁺, and Ca₂⁺, declines because these nutrients directly interfere with the pH of the soil [6, 7].

Throughout evolution, plants have developed adaptation strategies and mechanisms to overcome high salinity in the soil through specialized channels denominated Na⁺/H⁺ antiporters (NHXs) [5]. Briefly, NHXs feature multifunctions related to salt tolerance, development, cell expansion, growth performance, and disease resistance of plants [8]. NHXs are classified as ubiquitous transmembrane proteins belonging to the monovalent cation/proton antiporter 1 (CPA1) superfamily, whose members are found in prokaryotes and eukaryotes [9]. NHXs have some notable features: homodimers in the cell membrane and conserved regions that can transport Na⁺ and H⁺ to 10–12 transmembrane regions in the N terminus [10]. Another important feature of NHX proteins is that they can be classified into three groups according to their cellular location: NHX proteins can be localized in the plasma membrane, vacuole, and endosome [11, 12, 13]. Eight NHX members have been identified in Arabidopsis thaliana; four genes (AtNHX1, AtNHX2, AtNHX3, and AtNHX4) are predicted in the vacuole, two genes (AtNHX5 and AtNHX6) localize in the endosome, and two genes (AtNHX7 and AtNHX8) on the plasma membrane [9, 11, 14]. During the evolutionary process, the NHX family maintained highly conserved regions, indicating that this family is essential in the mechanisms involved in the development and survival of organisms [13, 15, 16].

Subsequent studies have reported the involvement of NHX proteins in salt stress response processes [17, 18, 19]. Gaxiola et al. [20] first reported the identification of AtNHX1 in A. thaliana. NHXs are vital for plant salt tolerance and preventing Na⁺ buildup in the cytosol. Vacuolar NHX proteins, such as AtNHX1, sequester Na⁺ into vacuoles, maintaining osmotic balance and water uptake, while under salt stress, plasma membrane NHX proteins, such as Salt Overly Sensitive 1 (SOS1) (AtNHX7), expel Na⁺ into the apoplast via the Salt Overly Sensitive (SOS) pathway [20]. Recent progress in next-generation sequencing (NGS) has facilitated the identification of NHX genes in many plant species, including Oryza sativa [21], Beta vulgaris L. [22], Vitis vinifera [23], Triticum aestivum L. [16], Canavalia rosea [24], and Lonicera japonica Thunb. [25]. Among the NHX genes identified, some successful examples have been identified that mitigated the effect of saline stress [26, 27, 28, 29, 30, 31, 32, 33]. In transgenic plants of Arachis hypogaea L. transformed with the AtNHX1 gene, a significant tolerance was observed under saline stress conditions [26]. In another study, it was reported that under saline stress conditions (150 mM NaCl), tomato plants transformed (T1: cv. PED) with the TaNHX2 gene via Agrobacterium mediation showed highly efficient development under such circumstances [27]. Additionally, it is also worth mentioning that physiologically, these plants also showed an increase in the chlorophyll content and the relative water content, further enhancing tolerance in these plants. For example, Yarra and Kirti [28] suggested in another study using Solanum melongena L. that TaNHX2 gene overexpression triggered plant improvements under saline stress (200 mM NaCl). Moreover, an accumulation of proline, improvement in photosynthetic efficiency, and stable water and chlorophyll contents in the leaf were observed in these transformed plants. Studies aimed at transgenic tobacco plants transformed with the TaNHX1 and TaNHX3 genes (Triticum aestivum) exhibited increased salinity tolerance [29, 30, 31]. Indeed, the overexpression of the NHXs gene in these studies contributed to plant resistance against saline stress, demonstrating greater efficiency in scavenging ROS and consequently improving the antioxidant activity enzyme [32, 33].

The common bean (Phaseolus vulgaris L.) stands out among the important agronomic crops. Phaseolus vulgaris L. is considered a fundamental food in human nutrition because it is highly nutritious [34]. Meanwhile, despite being cultivated in different environments, the production and development of common beans can be affected by abiotic stresses. Therefore, an urgent need exists to develop new and efficient strategies for breeding abiotic stress tolerance improvement in common beans. The generation and availability of access to the high-quality genome of the common bean [35] provide an unprecedented opportunity to perform a genome-wide identification of essential genes such as NHX. Hence, this study identified and comprehensively characterized the number, exon/intron distribution and conserved motifs, chromosomal locations, cis-regulatory elements analysis, phylogenetic relationships, and NHX gene duplication throughout the P. vulgaris L. genome. In addition, tissue-specific and saline-responsive NHX gene expression patterns were also observed. This study can enhance the understanding of the NHX genes and provide essential information for further functional studies of NHX and future molecular breeding of common beans.

Different strategies for acquiring salinity tolerance have been developed during the evolution of plants; however, these all involve a complex mechanism linked to the physiology and expression of certain genes [13, 52]. The bioinformatics tools allied with genomics can identify new genes and metabolic pathways that are key resources for improving adaptability, for example, of common bean crops to the present and future climate change scenarios. Recently, the NHX genes were noted in plants to encode important membrane transporters involved in the development and subsequent tolerance to salt stress [8]. In this study, bioinformatics automatically identified nine PvNHX genes (PvNHX1–9) in the P. vulgaris L. genome. With the advent of the NGS, NHX gene members have been identified in many species, including eight in A. thaliana [20], seven in Morus atropurpurea [53] and O. sativa subsp. Indica [54], nine in Camellia sinensis [55], and ten in Punica granatum L. [18], among others. Thus, a difference can be observed in the number of these genes compared to other plant species, suggesting that duplication and loss of specific NHX genes have played an essential role in the evolutionary process.

Previous investigations demonstrated that Na⁺/H⁺ exchangers may be categorized into two primary types—SOS (salt overlay sensitive) and NHX—according to the protein’s subcellular location (reviewed by Ayadi et al. [56]). The intracellular NHX class can be found in the tonoplast or endosomal compartments and plays crucial functions in response to saline stress. Consequently, we hypothesize that comprehending the subcellular localization of the protein may assist us in deducing the biological function of the PvNHX proteins (Table 1). Moreover, these proteins are primarily situated in the vacuole, chloroplast, nucleus, and cell membrane of common beans, suggesting a role in eliminating harmful chemicals from cells, such as removing Na+/K+ under unfavorable saline circumstances. It has been previously revealed that all known members of A. thaliana are also situated in the vacuole [57, 58].

Examining the exon–intron structure revealed that the structures of nine PvNHX genes are diverse, with variable numbers of exons and introns, indicating pivotal evolutionary changes within the p. vulgaris genome (Fig. 1). PvNHX1 lacks introns, whereas the remaining eight PvNHXs contain a greater number of introns. It is speculated that PvNHX1 facilitates rapid transcriptional activity in plant development or response to unfavorable environmental conditions [59]. Meanwhile, the number of introns/exons can affect gene transcription levels, whereby genes with fewer introns/exons are expressed faster [60, 61]. Additionally, we discovered that PvNHX1, 6, and 9 (paralog pairs; 1–6, 1–9) have fewer introns, indicating that these PVNHX proteins in common beans have recently evolved with fewer introns (Fig. 1; Table 2). This corroborates that the number of introns has decreased with time, implying that more recently developed, newer proteins have fewer introns than more ancient ones [62].

Furthermore, similar results were obtained in other plant species, such as M. atropurpurea [53] and P. trichocarpa [63], revealing that the NHX genes are highly conserved in plants, possibly related to their similar functions. Additionally, these nine PvNHX proteins presented the highly conserved amiloride binding domain (FFI/LY/FLLPPI motif; Fig. 1b). Thus, following this rationale, similar results were observed in P. trichocarpa [63], V. vinifera [23], and P. granatum [18]. This implied that the motif (FFI/LY/FLLPPI) in the transmembrane region of PvNHX in the common bean is sensitive to the Na⁺ of the substrate.

The findings on the chromosomal structure and duplication analysis of the common bean suggest that the majority of pvNHX genes are located on distinct chromosomes and do not adhere to a singular pattern (Fig. 2). Gene duplication is arguably crucial for the expansion of gene families and the diversification of their functions [64]. Throughout plant evolution, mechanisms such as segmental duplication, tandem duplication, and transposition events contribute to the growth of gene families [65]. Duplication analysis indicated that the PvNHX genes were associated with syntenic blocks (Fig. 2), suggesting that segmental duplication events significantly contribute to the expansion of PvNHX in common beans. The Ka/Ks ratio values for the PvNHX gene pairs varied from 0.25 to 1.21 (Table 2). Various selection categories for duplicated genes are indicated by the Ka/Ks ratio, wherein Ka/Ks $\mathrm{>}$1 signifies positive selection, Ka/Ks = 1 indicates neutral evolution, and Ka/Ks 1 represents negative selection [66]. The four pairs of paralogous genes had Ka/Ks ratios below 1. This discovery indicates that negative selection pressure influenced most PvNHX genes.

In the present study, the phylogenetic tree was constructed to determine the evolutionary relationship between the PvNHX genes in P. vulgaris L., O. sativa, Z. mays, A. thaliana, V. vinifera, P. trichocarpa, M. truncatula, and G. max (Fig. 3). As per previous reports, these results corroborate the grouping of NHX sequences into three distinct clades [22, 67, 68]. The PvNHX protein sequences were categorized as follows: the PvNHX2 sequences were in the plasma membrane clade. The PvNHX1, PvNHX3, PvNHX4, PvNHX5, PvNHX6, PvNHX7, PvNHX8, and PvNHX9 sequences were in the plasma membrane clade (Fig. 3). Meanwhile, as shown in phylogenetic tree analysis, no NHX proteins were observed in the endosome clade. These results were similarly observed in G. max [69]. Furthermore, this result suggests that the nine PvNHX proteins have an evolutionary relationship with other dicot and monocot plants and may have similar functions as their orthologs. Additionally, based on the phylogenetic analysis, the group Vac contains more PvNHX genes. In this sense, we suggested that these genes were the most conserved during the evolutionary process.

Significant orthologous relationships between the common bean and soybean can be attributed to their evolutionary histories, i.e., P. vulgaris L. and G. max diverged from a common ancestor. Moreover, these bean variants underwent a whole-genome duplication (WGD) event around 56.5 million years ago, subsequently diverging from each other around 19.2 million years ago [35]. In evolutionary terms, G. max underwent an independent WGD around 10 million years ago. No association has been shown for the PvNHX2 and pvNHX8 genes in P. vulgaris L. and A. thaliana, O. sativa, or G. max, indicating that these genes may have emerged post-divergence of these species (Fig. 4; Supplementary Table 3). These genes were only identified in the P. vulgaris L. genome, and we suggest that they played an essential role in the evolution of P. vulgaris L. Conversely, the PvNHX4, PvNHX6, and PvNHX9 genes, together with their orthologues, are found in every examined species. This suggests these genes originated from the same ancestor before separating eudicots and monocots. Furthermore, these genes are related to genes found in O. sativa, A. thaliana, and G. max. Overall, these findings imply that these genes may have a crucial adaptive function in salt-stress conditions.

A unique promoter region regulates each gene in plants. Promoters are non-protein coding regions in genes located upstream of the transcription start site (TSS). Promoters exhibit conserved DNA elements that, along with transcription factors, are essential regulators of gene expression suppression or activation in different tissues, organs, or according to various stages of plant development [70]. As already mentioned, putative cis-acting elements related to light response, hormones, stress, and development were identified in the promoter regions of PvNHX genes in the common bean (Fig. 5). These elements are also a strong indication that biotic and abiotic stresses and different hormones could regulate them. Akram et al. [17] reported that Gossypium hirsutum and G. barbadense stress-responsive cis-regulatory elements exceed the other identified elements. In turn, a more significant proportion of light-responsive elements was observed in C. rosea, corroborating these findings [24]. Considering these results, this information demonstrates the plasticity of the different roles that NHX genes can play during the growth and development of plants and between other numerous physiological processes.

It is estimated that the microRNAs (miRNAs) are small (20–24 nucleotides) non-coding RNA molecules that regulate gene expression by binding to target gene transcripts to inhibit their translation or promote mRNA degradation [71]. This study identified 22 miRNAs from five families targeting seven PvNHX genes (File S5). The PvNHX2, PvNHX4, PvNHX5, PvNHX8, and PvNHX9 genes were collectively targeted by PvumiRN2588. In addition, PvNHX2 and pvNHX7 were targeted by PvumiRN395. The miR395 family is an ancient and highly conserved miRNA family [72]. Previous studies showed that miR395 expression is important during stress since it controls sulfation-mediated hormonal activity [73]. Additionally, miR395 showed regulatory effects on plant salt stress tolerance [74]. Moreover, PvNHX genes were targeted by other miRNA families such as miR67, umiR56, and umiRN2597.

PvNHX2 primarily interacts with five proteins: SOS2, SOS3, AVP1, CBL1, and CBL10 (Fig. 6). SOS2 and SOS3 regulate intracellular Na⁺ and Ca²⁺ homeostasis by participating in the regulatory mechanism of salt stress. SOS1 is crucial in extruding harmful Na⁺ from cells and is vital for plant salt tolerance. HKT1 imparts salt tolerance, while RCD1 protects chloroplasts from elevated ROS levels. HAK5, a high-affinity potassium transporter 5, plays a crucial role in the high-affinity potassium absorption noted in plant roots in saline circumstances [75]. Furthermore, calcineurin B-like (CBL) can engage with and regulate the CBL-interacting protein kinases (CIPKs) to facilitate Ca²⁺ signal transduction. The genetic interaction between the CBL4 and CBL10 double mutant in Arabidopsis exhibited increased salt sensitivity compared with the cbl4 and cbl10 single mutants, indicating that CBL4 and CBL10 each regulate distinct salt-tolerance pathways [76]. We propose that these proteins and PvNHX proteins collaborate and participate in sodium ion transport across the membrane and the response to salt stress.

It is important to mention that the specificity of gene expression in different plant tissues and developmental stages provides essential information about the possible functions of genes. The various expression patterns displayed by the nine identified PvNHX genes and other analyses presented in this manuscript indicate that the modulation of these genes is differentiated in terms of the specific tissue and possibly the type of stress (Figs. 7,8). Previous research showed that one of the strategies of plants under salt stress is sequestering Na⁺ to the vacuoles, alleviating Na⁺ toxicity in the cytoplasm [77]. NHXs are crucial in sequestering Na⁺ into vacuoles to maintain Na⁺ homeostasis and improve plant salinity tolerance [78].

In this way, significant changes in the expression levels for the cultivar TR43477 (salt-susceptible) of some genes (PvNHX2, 3, 6, 7, and 9) in leaf tissues under salt-stress were observed (Fig. 8). We can speculate that the salt-tolerant cultivar Ispr showed an increased expression level for PvNHX3 and PvNHX8 genes in roots and leaves, respectively. PvNHX3 and PvNHX8 are part of the Vac clade, suggesting that the NaCl-resistant mechanism of the common bean Ispr cultivar mainly involves the sequestration of Na⁺ into vacuoles. Vac-class NHXs are essential for the salt tolerance of plants [22]. In G. max, GmNHX genes demonstrated elevated expression levels in roots compared to shoots during salt stress [69]. Similar results were observed in Zygophyllum xanthoxylum [79] and Puccinellia tenuiflora [80]. The expression levels of the Vac pvNHX8 gene in leaves in the Ispr cultivar (tolerant-salt) were significantly higher under salt stress, implying that PvNHX8 can sequester Na⁺ in leaves during salinity stress. The present study could be enhanced if further analysis were performed on physiological and molecular aspects in different tissues and responses to various abiotic stresses in the common bean.

This study provides conclusive evidence that the expression of some genes, such as PvNHX2, 4, and 5 in roots, and PvNHX1, 4, and 6, was unaffected by salt stress, suggesting these genes may not contribute to the physiological responses associated with salt tolerance in P. vulgaris L. This indicates that this transcriptional diversity may be attributed to the genetic structures of the cultivars and the physical and chemical properties independent of NHX proteins. Therefore, by aiming to create a strategy that can mitigate the effects of climate change, this study shows that in silico analyses are relevant tools and contribute to identifying traits and genes related to stress salt in common beans.

The recent progress in plant genome-wide analyses has facilitated the identification and isolation of important genes and an evaluation of their roles in regulating yield and salt stress responses. This study identified nine putative pvNHX genes in the P. vulgaris L. genome. Phylogenetic analysis revealed that these PvNHX genes are grouped into two classes: Vac- (PvNHX1, 3, 4, 5, 6, 7, 8, and 9) and PM-class NHXs (PvNHX2); meanwhile, the same clade motif compositions and exon/intron structures are conserved. Synteny event comparisons in the NHX genes in P. vulgaris L., A. thaliana, O. sativa, and G. max identified several orthologous genes. In addition, the synteny between P. vulgaris L. and G. max was conserved. The expression patterns differed across the various P. vulgaris L. tissues; PvNHX7 and PvNHX9 were the highest expressed genes in most analyzed tissues. The expression profiles of PvNHX genes in salt-tolerant (Ispir) and salt-susceptible (TR43477) genotypes of P. vulgaris L. revealed that some pvNHX genes were affected by salt stress. This study provided candidate genes for further functional research, which is essential for understanding the mechanism through which NHX genes regulate salt stress responses in P. vulgaris. Finally, to ensure food security and sustainable standard bean production worldwide, identifying new genes and varieties tolerant to saline stress will be extremely important for maintaining the yield and quality of this bean species in the future.

All data reported in this paper will also be shared by the corresponding author upon reasonable request.

EMB, and JO, performed in silico analysis and wrote the manuscript. TBS, conceived, provided technical support, and critical review. SGHS, conceived, provided technical support, and revised the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

This study was financed by UNIPAR (grant no. 40163/2023) and partly financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. BEM and JO were supported by a CAPES fellowship.

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/FBS26725.