Salinity, a major environmental factor affecting crop productivity worldwide, especially in drylands, where irrigation practices often contribute to soil salinization [1]. High salinity disrupts plant physiological processes by inducing osmotic stress, ion toxicity, and nutrient imbalance, leading to reduced seed germination, stunted growth, and lower yields [2]. Excessive sodium (Na⁺) and chloride (Cl⁻) affect essential metabolic functions, impairing photosynthesis and enzymatic activities [3]. Barley (Hordeum vulgare L.) is an important cereal crop cultivated widely, serving as a staple food, feed, and key ingredient in brewing. Despite its adaptability to diverse climates, barley growth and productivity are significantly limited by soil salinity, a common abiotic stress that adversely affects plant water uptake, nutrient balance, and overall physiological functions [1]. Sodium toxicity is a primary factor in salinity stress, leading to ionic imbalances that disrupt cellular homeostasis and metabolic processes [3].

High-affinity potassium transporters (HKTs) are a category of important membrane proteins essential for maintaining potassium (K⁺) homeostasis in plants, especially under conditions of low soil K⁺ availability or salinity stress. These transporters mainly help the absorption and distribution of K⁺ and Na⁺, which are crucial for various physiological processes, including osmotic regulation, enzyme activation, and maintaining membrane potential [4]. HKTs are divided into two subclasses on the basis of their ion selectivity: Subclass I members primarily transport Na⁺, while Subclass II members are more selective for K⁺ [5]. The foremost HKT gene, HKT1, was recognized in wheat, where it arbitrates high-affinity K⁺ uptake and low-affinity Na⁺ transport [6]. Further research indicates that HKTs are widely existing across plant species such as Arabidopsis, rice, and barley, with their expression often regulated by environmental factors like salt stress, K⁺ deficiency, and osmotic stress [7,8]. In rice, OsHKT1;1 controls Na⁺ exclusion from shoots, enhancing salt tolerance by preserving a favorable Na⁺/K⁺ ratio [9]. Similarly, in wheat, the homolog TaHKT1;5-D is associated with improved Na⁺ elimination from leaves, aiding salinity tolerance in Triticum durum [10]. In maize, ZmHKT1;1 influences Na⁺ uptake and translocation, with its overexpression resulting in increased salt sensitivity [11]. The barley HKT1 gene was further identified as corresponding to HKT1;1 through phylogenetic analysis and encoded a transporter that is selective for Na⁺ [12]. Knockdown of HvHKT1;1 using barley BSMV-induced gene silencing enhanced sensitivity to salt and Na⁺ buildup in leaves and roots of barley lines, compared to controls, highlighting this gene’s crucial role in barley’s salt tolerance [12].

In nearly all previous cases, researchers have focused only on the full-length protein to measure transport activity. However, Imran et al. [13] reported the presence of several functional mRNA variants in rice OsHKT1;1. What about barley HvHKT1;1? In this study, we aimed to identify HvHKT1;1 mRNA variants in a barley cultivar, Haruna-nijo, and characterized their expression profiles and transport activities using qPCR analyses and two-electrode voltage-clamp (TEVC) experiments with Xenopus laevis (X. laevis) oocytes.

The HKTs play a crucial role in salinity tolerance of plants by regulating the homeostasis of Na⁺ and K⁺, which is important for maintaining cellular functions in salty environments [4,8,17]. HKTs mainly facilitate Na⁺ and K⁺ selective transport across cell membranes, helping to reduce the toxic effects of excess Na⁺ in plant tissues [18]. Specifically, HKT1-type transporters, localized in the plasma membrane of root cells, help remove Na⁺ from the xylem sap, decreasing its movement to the shoots and protecting photosynthetic tissues from salt damage [4]. Additionally, HKTs contribute to osmotic adjustment and ion balance, which are essential for supporting growth and productivity during salt stress [17].

HKT subfamily 1 transporters play a key role in plant salinity tolerance by facilitating Na⁺ transport, preventing toxic ion buildup, and redistributing Na⁺ to vacuoles or less sensitive tissues [10]. A key function of these transporters is Na⁺ retrieval from the xylem, reducing its excessive accumulation in shoots and maintaining ionic balance [8]. AtHKT1;1, a passive Na⁺ transporter, is primarily expressed in xylem parenchyma, where it helps in Na⁺ unloading, thus enhancing salt tolerance [4,19]. Its presence in leaf phloem suggests an additional role in Na⁺ recirculation from shoots to roots [20]. In durum wheat, Na⁺ removal from leaves is a key function for salt stress adaptation, controlled by the loci Nax1 and Nax2, which encode Na⁺-selective transporters TmHKT1;4-A2 and TmHKT1;5 [10,21,22]. Similarly, OsHKT1;5 in rice regulates unloading Na⁺ from the xylem and phloem to protect young leaves under salt stress [9,23,24]. It has also reported that OsHKT1;1 is involved in Na⁺ exclusion from shoots and recirculation to roots [25,26]. In X. laevis oocytes, HvHKT1;1 exhibited Na⁺-selective transport, further confirming the specificity of HKT transporters in regulating Na⁺ homeostasis [12]. Previously, it has been reported that OsHKT1;1 produced several mRNA splicing variants [13]. However, only a few studies have reported on HvHKT1;1, and its detailed physiological functions and variants have not yet been studied. We assume that, like OsHKT1;1, other HKT1;1 may also produce variants. Based on this assumption, in this study, several variants of HvHKT1;1 (-V1, -V2, and -V3) were confirmed in addition to the HvHKT1;1 full-length (HvHKT1;1-FL) in a salt-tolerant barley cultivar, Haruna-nijo (Fig. 1).

In this study, HvHKT1;1-FL expressed in Xenopus oocytes exhibited higher inward rectifying currents in 96 mM Na⁺ (Fig. 4A), as previously mentioned [12], with little or no currents similar to the water-injected negative control in 96 mM K⁺, Li⁺, Cs⁺, or Rb⁺ solutions (Fig. 4A). Additionally, analysis of reversal potential, ion permeability ratio, and high Vmax, low Km values indicated efficient Na⁺ transport by HvHKT1;1-FL with low affinity (Table 1 and Fig. S4A,B). Moreover, reversal potential analysis confirmed the effective transport of Na⁺ by HvHKT1;1-FL, but not Cl⁻ (Figs. 5A,B and S5A,B). In contrast, oocytes expressing HvHKT1;1-V1, -V2, and -V3 also showed notable Na⁺ currents, though with more moderate inward currents compared to HvHKT1;1-FL, and minimal transport of other ions (Fig. 4B–D). Additionally, the low Vmax and high Km values of these splice variants suggest reduced Na⁺ transport efficiency, implying alternative ion selectivity or decreased functionality relative to the full-length HvHKT1;1 (Fig. S4C–H). Furthermore, reversal potential analysis and ion permeability ratios indicated that HvHKT1;1 variants (-V1, -V2, -V3) functioned as Na⁺ channels (Table 1; Figs. 5C–H and S5C–H), but their selectivity was less than that of HvHKT1;1-FL.

It has been reported that AtHKT1;1 shows higher expression in roots than in shoots [27]. However, OsHKT1;1 is highly expressed after 12 h of salt stress treatment in shoots and shows no difference in expression in roots [25]. Additionally, Imran et al. [13] reported a gradual decline in OsHKT1;1 expression in shoots following salt stress exposure, with no clear pattern in roots. Moreover, Ali et al. [28] observed that EpHKT1;1 transcript upregulated more slowly in response to salt treatment. It has been demonstrated that CmHKT1;1 transcript level in roots exceeds that in the control, peaking at 6 h [29]. Moreover, LbHKT1;1 exhibited the greatest expression levels in both shoots and roots under salt stress conditions [30]. Similarly, in S. linearistipularis, exposure to NaCl increased the expression of SlHKT1.1 in both roots and shoots [31]. A previous study reported increased HvHKT1;1 expression in roots under salt stress conditions [12]. In this study, the HvHKT1;1-FL mRNA was the most abundant among the identified variants in roots, sheath, and leaves (Fig. 2). Furthermore, all variants, including the full-length, showed higher expression in the leaves of Haruna-nijo (Fig. 2). Under salt stress, HvHKT1;1-FL and HvHKT1;1-V3 display high transcript levels in leaves at 2 h and in sheaths at 12 h, with minimal root expression (Fig. 3A,D). HvHKT1;1-V1 follows a similar pattern but at lower levels (Fig. 3B). In contrast, HvHKT1;1-V2 shows the lowest expression across tissues (Fig. 3C). Though the variants are missing a partial or a full transmembrane domain, this missing domain does not affect the expression, as also reported in the case of OsHKT1;1 and OsHKT1;3 variants [13,15]. Overall, transcript levels are highest in sheaths over time, while root expression remains low across all variants, indicating tissue-specific and time-dependent regulation of HvHKT1;1 in barley. In conclusion, this study offers a comprehensive analysis of barley HvHKT1;1 and its splice variants, revealing distinct expression patterns and Na⁺ transport properties. HvHKT1;1-FL exhibits the highest expression and transport efficiency, whereas the variants display reduced Na⁺ transport, implying regulatory roles in sodium homeostasis. Expression profiling under salinity stress highlights tissue-specific and time-dependent regulation. Comparative studies among cereals reveal both conserved and divergent features of HKT1 transporters. In rice, OsHKT1;1 and OsHKT1;3 produce multiple splice variants that are stress-responsive and may fine-tune Na⁺ transport under salinity [13,15,25]. Some OsHKT1;1 variants are upregulated during early salt exposure and show reduced ion conductance, suggesting a potential role in Na⁺ uptake temporally rather than being non-functional by-products. Similarly, wheat transporters such as TaHKT1;5-D and TmHKT1;4-A2 are single full-length isoforms, but their alleles lead to differences in Na⁺ exclusion capabilities and salt tolerance levels [10,21,22]. In contrast, the barley HvHKT1;1 variants identified here appear to share Na⁺ selectivity with OsHKT1;1 variants but display distinct expression timing and tissue specificity, implying a species-specific regulatory mechanism. Their induction at early time points under salinity (especially in leaves) suggests possible adaptive modulation rather than transcriptional noise. However, functional attenuation (lower Vmax, higher Km) may also reflect partial loss of transport capacity, consistent with non-essential or regulatory variant roles. Therefore, the barley HvHKT1;1 variants may represent a combination of adaptive and functionally reduced forms, a pattern that aligns with OsHKT1;1’s stress-induced splicing diversity but diverges from the more stable single-gene expression observed in wheat HKT1;5. These findings enhance our understanding of Na⁺ transport in barley and offer insights for enhancing salinity tolerance in crops. Although our electrophysiological and expression data clarify variant-specific transport functions, we did not evaluate their effects on whole-plant Na⁺/K⁺ homeostasis or growth under salinity. However, previous studies have shown that HvHKT1;1 regulates tissue-level Na⁺ distribution and contributes to salt tolerance in barley [12]. But the previous report didn’t consider HvHKT1;1 variants, which were characterized in the present study, may differentially affect plant performance under saline conditions. Future research should investigate the physiological roles of these variants and their potential applications in crop breeding and genetic engineering to enhance stress resilience.