Soil salinization, driven by climate change and human activities, is one of the most severe environmental challenges worldwide. It leads to a reduction in arable land and poses a significant threat to both the yield and quality of crops (Cheng et al., 2023; Schroeder et al., 2013). High concentrations of salts in agricultural soils result in the accumulation of reactive oxygen species (ROS) in plant cells, impairing the plant’s ability to absorb water and essential mineral nutrients, which in turn inhibits growth (Petrov et al., 2015). Consequently, the development of salt-tolerant crop varieties has been a major goal in global crop improvement efforts (Ashraf and Munns, 2022; Katerji et al., 2003; Shams and Khadivi, 2023; Shams et al., 2023). Potassium (K⁺) is a key cation in plant cells, comprising 2-10% of the dry weight of plants. It is essential for plant growth and development (Leigh and Wyn Jones, 1984), and its presence enhances the plant’s ability to adapt to a variety of biotic and abiotic stresses, including drought and salinity (Zörb et al., 2014). Sodium (Na⁺) is one of the most common soluble cations in saline-alkaline soils, and when its concentration exceeds a certain threshold, it induces ionic toxicity. Furthermore, Na⁺ disrupts the balance of K⁺ within the plant, causing cellular damage. Therefore, maintaining a high cytoplasmic K⁺/Na⁺ ratio is crucial for maintaining ion homeostasis within plant cells.

Plants tolerate sodium (Na⁺) toxicity through two main mechanisms: sodium exclusion and tissue tolerance (Zhu, 2003). Ion-selective transport capacity is a key factor determining a plant’s ability to adapt to saline environments, with K⁺/Na⁺ transporters playing a critical role in this process. Key transporters, such as the Na⁺/H⁺ antiporter (SOS1), Na⁺/H⁺ exchangers (NHXs), and high-affinity K⁺ transporters (HKTs), form a complex network that regulates the uptake, transport, and compartmentalization of Na⁺ (Van Zelm et al., 2020; Volkov, 2015). Thus, identifying and characterizing Na⁺/K⁺ transporters is crucial for understanding how plants maintain ion homeostasis and confer salt tolerance. Among the K⁺/Na⁺ transporters, the high-affinity K⁺ transporter (HKT) family plays a critical role in both plant mineral nutrition and salt stress regulation. The first HKT protein was cloned from wheat in 1994 (TaHKT2;1) (Schachtman and Schroeder, 1994a). Initially characterized as a high-affinity K⁺ transporter, HKT proteins were subsequently shown to transport other ions, such as Na⁺ (Rubio et al., 1995). Studies have demonstrated that HKTs are involved in regulating plant salt tolerance, and under conditions of severe K⁺ deficiency, they can facilitate Na+ uptake, providing an adaptive mechanism for coping with short-term K⁺ shortages.

The HKT (High-Affinity K⁺ Transporter) family belongs to the Trk/Ktr/HKT superfamily and is characterized by a distinct structure composed of four transmembrane domains, a pore domain, and additional transmembrane units (MPM1-MPM4) (Riedelsberger et al., 2021b). Phylogenetic analysis has divided the HKT family into two subgroups: Class I and Class II (Gomez-Porras et al., 2012; Haro et al., 2010; Platten et al., 2006). The primary difference between these subgroups lies in the presence of a serine residue (SGGG) or a glycine residue (GGGG) in the first pore loop (Mäser et al., 2002). Generally, Class I HKTs (SGGG) function as Na⁺ uniporters, while Class II HKTs (GGGG) mediate Na+ and K⁺ symport. However, within these two classes, there exists considerable functional diversity, which has yet to be fully explained at the molecular level. To date, multiple HKT genes have been identified and shown to play a role in salt tolerance across various species. In Arabidopsis, a single gene, AtHKT1, has been characterized, and its overexpression enhances salt tolerance by facilitating the transport of Na⁺ from the roots to the shoots (Berthomieu et al., 2003b; Mäser et al., 2002b; Rus et al., 2004). In rice, OsHKT1;4 restricts the movement of Na⁺ from the leaf sheath to the leaves under salt stress, thereby improving salt tolerance. In maize, ZmHKT1 transports Na⁺ in the xylem to promote salt tolerance, while ZmHKT2 contributes to salt tolerance by regulating K⁺ levels in the (Cao et al., 2019; Jiang et al., 2018; Zhang et al., 2023). Mutation of the wheat TaHKT1 gene results in increased Na⁺ content in the leaves of transgenic lines, highlighting its role in regulating Na⁺ transport from the roots to the leaves (Byrt et al., 2014b). Collectively, these findings demonstrate the involvement of HKT genes in plant responses to salt stress.

The expression patterns of HKT genes in response to varying K⁺ and Na⁺ conditions further elucidate their functional roles. For example, in rice, Class I HKT gene, OsHKT1;1 showed higher expression in the shoots compared to the roots, with increased expression in the roots under high Na+ conditions and decreased expression in the shoots. Additionally, OsHKT1;1 expression increases in both the leaves and roots under high Na⁺ conditions. In barley, HvHKT1;5 expression is elevated in the roots under low K⁺, high K⁺, or high Na⁺ conditions (Huang et al., 2020). In contrast, Class II HKT genes, such as OsHKT2;1 and OsHKT2;2 in rice, exhibit increased expression under low K⁺ or low Na⁺ conditions, but decreased expression under high K⁺ or high Na⁺ conditions (Oomen et al., 2012). In barley, HvHKT2 expression increases in the leaf sheath, leaves, and roots under low K⁺ conditions, but decreases in the leaf sheath and roots under high Na⁺ conditions, while it increases in the leaves (Mian et al., 2011). These findings suggest that the HKT gene family plays a crucial role in enhancing plant salt tolerance and ion transport. However, the underlying molecular mechanisms contributing to the observed functional diversity remain unclear.

Halophytes, defined as plants capable of completing their life cycle in saline environments exceeding 200 mM NaCl, represent invaluable genetic reservoirs for deciphering plant salt adaptation mechanisms. Their unique ion homeostasis strategies, particularly through specialized transporters, provide critical insights for crop salt tolerance improvement (Cheeseman, 2015; Munir et al., 2022; Ungar, 2023). A paradigmatic example is Suaeda salsa, which employs a sophisticated Na⁺ compartmentalization strategy. In this species, SsHKT1;1 functions as a dual-affinity potassium transporter (Shao et al., 2014), Coordinating with SsSOS1 (plasma membrane Na⁺/H⁺ antiporter) and SsNHX1 (vacuolar Na⁺/H⁺ exchanger) to establish tissue-specific Na⁺ gradients (Wang et al., 2020). Similar mechanisms have been characterized in Thellungiella salsuginea, where TsHKT1;2 modulates root-shoot Na⁺ partitioning through xylem loading regulation (Ali et al., 2013, Ali et al., 2012). Despite these advances, the functional diversification of HKT transporters in coastal pioneer species remains underexplored. Ipomoea pes-caprae is a perennial vine belonging to the Convolvulaceae family and the Ipomoea genus. It is widely distributed along tropical and subtropical coastal regions and is highly regarded for its medicinal and ecological properties. Its exceptional tolerance to abiotic stresses has also attracted considerable scientific attention (Cheng et al., 2021; Liu et al., 2020; Zhang et al., 2018). Therefore, the identification of functional genes involved in the response of IPC to extreme environmental conditions, and the application of these gene resources to the genetic improvement of salt-tolerant crops, holds significant potential for agricultural advancements. A high-quality reference genome for IPC was provided by our previous genome sequencing efforts (Cheng, 2023), establishing a foundation for the study of its salt tolerance mechanisms. In the present study, bioinformatics approaches were employed to identify two members of the HKT gene family in the IPC genome. The chromosomal locations, gene duplication events, evolutionary relationships, gene structures, and cis-regulatory elements of these HKT genes were also investigated in IPC and several closely related Ipomoea species. Furthermore, real-time quantitative PCR (qPCR) was performed to analyze the expression profiles of IpcHKT1;1 and IpcHKT1;2 in different tissues, providing insights into their roles in salt stress responses. Additionally, a yeast heterologous expression system was utilized to validate the ability of IpcHKT1;1 and IpcHKT1;2 to co-transport Na⁺ and K⁺ ions. Collectively, these findings provide valuable information for further understanding the involvement of IpcHKT1;1 and IpcHKT1;2 in salt stress tolerance and offer insights into the functional characterization of these genes.

The increasing frequency of extreme environmental conditions such as high temperatures, freezing, drought, flooding, and high salinity has had a significant impact on global agricultural production and food security. Among these, soil salinization is a challenging environmental issue affecting 20% of the world’s irrigated land (Ali and Salem, 2024; Mukhopadhyay et al., 2021; Saifullah et al., 2018; Ventura et al., 2015). Excessive soil salinization not only reduces the productivity of salt-sensitive crops, such as sweet potatoes, wheat, and corn, but also decreases the productivity of salt-tolerant crops, such as cotton, barley, and sugar beet. By 2025, drought and salinization are projected to affect 50% of arable land (Wang et al., 2003). Studies have shown that the combination of high temperatures and high salinity has a detrimental effect on root growth in plants. In tomatoes, plants grown under high salinity, high temperature, or a combination of both showed reduced root growth (Rivero et al., 2014). A similar effect was observed in barley, where the combination of high temperature and salt stress inhibited root growth more than salt stress alone (Faralli et al., 2015). In wheat, seedlings subjected to both heat and salt stress showed greater inhibition of root growth compared to plants treated with salt alone (Keleş and Öncel, 2002) (Hamada and Khulaef, 1995). Quinoa also exhibited greater reductions in growth and yield under the combined stresses of drought, salinity, and high temperature compared to individual stressors (Abbas et al., 2023). These reports suggest that adverse environments, such as high salinity and high temperature, can cause severe damage to plant growth, particularly root development. However, IPC, a vine species that grows along beaches, is capable of developing highly developed roots even in high-temperature and high-salinity environments (Grigore and Vicente, 2023). Therefore, studying this plant is of significant importance for improving the ability of sweet potatoes and other crops to withstand high-temperature and high-salinity stresses.

The HKT family of proteins has been reported to regulate various developmental and stress responses in plants (Riedelsberger et al., 2021a). Previous studies have indicated that HKT family genes control the concentration and balance of K⁺ within plant cells. To date, members of the HKT family have been identified in various species, including Arabidopsis thaliana (Berthomieu et al., 2003a; Chandran et al., 2023; Mäser et al., 2002a; Rus et al., 2004), Oryza sativa L (Imran et al., 2021; Jabnoune et al., 2009; Oomen et al., 2012; Wang et al., 2024), Zea mays L (Cao et al., 2021; Jiang et al., 2018; Zhang et al, 2023), Triticum aestivum L (Byrt et al., 2014a), and Hordeum vulgare L (Huang et al., 2020; Mian et al., 2011). In this study, we identified 10 HKT family members across 5 species within the Ipomoea genus, including IPC. We found that the number of HKT genes in species of the Ipomoea genus was consistent, with only two members present, located on the same chromosome and tightly linked. This suggests that the HKT genes in the Ipomoea genus share a common evolutionary origin. It is likely that the ancestral species of Ipomoea underwent a tandem duplication event in its evolutionary history, and this characteristic has been retained across different Ipomoea species, indicating that this tandem duplication event may have conferred an adaptive advantage for regulating and enhancing the function of HKT genes in plants.

Further analyses of the physicochemical properties, gene structure, and cis-regulatory elements suggest that the functions of the HKT genes in the Ipomoea genus may differ. For example, the promoter region of IpcHKT1;1 contains five Box4 elements and two G-box elements, while IpcHKT1;2 has the opposite configuration, with two Box4 elements and five G-box elements. In addition, the promoter region of IpcHKT1;1 uniquely contains eight A-box elements, which are absent in other HKT genes ( Figure 3B ). It has been reported that A-box elements in the Solanum lycopersicum DREB family cis-elements may play a role in responding to salt and heat stress (Maqsood et al., 2022). Subcellular localization assays in tobacco leaves further support the idea that both IpcHKT1;1 and IpcHKT1;2 are membrane-bound transporters localized to the plasma membrane ( Figure 4 ). However, the results also indicate that IpcHKT1;2 contains additional short peptide chains at the N-terminus and longer peptide chains at the C-terminus compared to IpcHKT1;1, suggesting potential functional or regulatory differences between the two proteins ( Figure 2C ). These findings imply that the two HKT genes in IPC may have undergone functional differentiation. The divergence in their functions may be related to environmental selection pressures, as IPC (a species within the Ipomoea genus) grows in coastal environments and is exposed to extreme conditions such as high salinity and heat (Fita et al., 2015; Kotula et al., 2020). This likely led to the evolution of regulatory mechanisms to adapt to these challenging conditions. Additionally, this research provides a basis for improving the salt tolerance of Ipomoea species, such as sweet potato, through the incorporation of genes related to salt tolerance from IPC.

The expression of HKT genes is typically influenced by stress conditions, such as high sodium or low potassium concentrations (Huang et al., 2020; Huang et al, 2020; Jaime-Pérez et al., 2017; Su et al., 2003). However, there does not appear to be a universal pattern across species. For instance, high Na⁺ concentrations may upregulate the expression of certain HKT gene members while downregulating others (Jiang et al., 2018). Similarly, in some species, gene expression is upregulated in the shoots and downregulated in the roots, while the opposite effect is observed in other species (Oomen et al., 2012). This suggests that the expression and regulation of the HKT family is complex, providing plants with various mechanisms to adapt to stress conditions. The investigation of HKT expression responded to salt treatment in this study support also showed this tendency ( Figure 5 ). Under normal conditions, the expression of IpcHKT1;1 remained relatively stable across different tissues. However, under salt stress, its expression was downregulated in roots, stems, and leaves, followed by a gradual recovery. In contrast, under normal conditions, the expression of IpcHKT1;2 was significantly higher in the stems (more than 20 times that in the roots) and in the leaves (more than 6 times that in the roots). After 4 hours of salt stress, the expression of IpcHKT1;2 increased more than 40 times in the roots, with notable upregulation in both stems and leaves. After 72 hours, its expression remained high, suggesting that IpcHKT1;2 may play a role in IPC’s long-term adaptation to high salinity. Additionally, when the expression of IpcHKT1;2 began to decrease after 12 hours of salt stress, IpcHKT1;1 expression began to recover across all tissues. This suggests that IpcHKT1;1 and IpcHKT1;2 may adopt distinct, possibly even antagonistic, strategies in response to salt stress, and there may be a regulatory interplay between these two genes in IPC.

To date, several plant HKT genes have been cloned and expressed in yeast systems to assess their selectivity in K⁺ and Na⁺ transport. Heterologous expression of AtHKT1;1, HvHKT1;5, Ni-OsHKT1;5, and Po-OsHKT1;5 proteins in yeast has demonstrated their role as Na⁺/K⁺ symporters (Haro et al., 2005; Horie et al., 2001; Schachtman and Schroeder, 1994b). However, the heterologous expression of OsHKT2;1 and TaHKT2;1 results in Na⁺/K⁺ symport activity under low Na⁺ concentrations, but they function as Na⁺ transporters under high Na⁺ concentrations (Oomen et al., 2012). Additionally, EcHKT1;1 and EcHKT1;2 act as Na⁺/K⁺ symporters in yeast, even at elevated Na+ concentrations. Furthermore, Ca2⁺ and Mg2⁺ have been shown to also pass through EcHKT1;1 and EcHKT1;2 via membrane transport (Fairbairn et al., 2000; Liu et al., 2001). In Ipomoea batatas (sweet potato), the IbHKT1 gene acts as a Na⁺/K⁺ symporter in yeast, and transgenic sweet potato roots overexpressing IbHKT1 exhibit a significant increase in K⁺ uptake, suggesting that IbHKT1 plays a role in K⁺ absorption in plants (Park et al., 2017). In the current study, heterologous expression of IpcHKT1;1 and IpcHKT1;2 in yeast defective strains (CY162 and AXT3K) revealed that these genes from IPC exhibit weak K⁺ transport activity and, in the presence of high Na⁺ concentrations, possess Na⁺/K⁺ symporter activity ( Figure 6 ). These findings are consistent with those observed in sweet potato, suggesting that the functions of HKT proteins within the Ipomoea genus are relatively conserved.

In summary, we conducted a genome-wide identification of HKT genes in IPC, followed by a comparative analysis of HKT genes in representative species of the Ipomoea genus, including chromosomal localization, phylogenetic analysis, gene structure, protein structure, and cis-element analysis. The results revealed that HKT genes within the Ipomoea genus are highly conserved in terms of both their origin and function. Furthermore, we performed subcellular localization, salt stress response, and yeast functional complementation assays for the IpcHKT genes. These experiments demonstrated that IpcHKT genes are localized to the plasma membrane, function as Na⁺/K⁺ symporters, respond to salt stress, and play a crucial role in the salt tolerance of IPC. This study enhances our understanding of the evolutionary conservation of HKT gene structure and function in the Ipomoea genus, and provides new insights into the role of HKT genes in plant salt tolerance mechanisms.