Comparative analysis of salt tolerance and associated physiological traits in wild and cultivated barley at the seedling stage

Abstract

Background

Soil salinity poses a significant threat to global agricultural production. Wild barley (H. spontaneum), the ancestor of cultivated barley, offers valuable genetic diversity for stress adaptation. This study compared salt tolerance among six barley varieties (two wild barley, two salt-tolerant, and two salt-sensitive cultivated barley), by employing analysis of root/shoot growth and physiological traits under salt stress conditions.

Results

To explore the physiological basis of salt tolerance differences among six barley varieties, multiple indicators including photosynthetic capacity, ion homeostasis, stomatal regulation, and reactive oxygen species (ROS) scavenging ability were analyzed. Additionally, tissue damage was assessed using histological staining and fluorescence imaging techniques, and the accumulation differences of sodium ions (Na⁺), potassium ions (K⁺), and reactive oxygen species were visualized. Salt-tolerant cultivated barley maintained superior photosynthetic performance and root Na⁺/K⁺ homeostasis compared to salt-sensitive varieties, but no significant differences were observed in stomatal regulation and ROS scavenging ability. Wild barley performed better than cultivated barley in all measured parameters, exhibiting enhanced photosynthetic capacity, stomatal regulation, more stable ion homeostasis, and higher ROS scavenging ability.

Conclusion

By integrating multiple physiological indicators, this study clarified the differences in physiological characteristics among wild barley, salt-tolerant cultivated barley, and salt-sensitive cultivated barley. Additionally, we identified specific physiological limitations of current salt-tolerant cultivated varieties compared to wild barley. These findings provided reference for future breeding strategies to develop salt-tolerant barley.

Backgrounds

Climate change intensifies abiotic stresses that threaten global agriculture, among which soil salinization represents a major constraint, adversely affecting seed germination, plant growth, and crop yield [1]. Currently, more than one-third of the world's irrigated agricultural land is impacted by salinity [2]. A thorough understanding of the physiological mechanisms underlying salt tolerance in crops is therefore essential for improving rapid evaluation systems, identifying reliable selection indicators, and ultimately facilitating the screening of germplasm resources and the development of salt-tolerant cultivars.

Salt stress elicits a range of detrimental effects in plants, including disrupted metabolism and growth inhibition [3]. In response, plants must readjust their physiological and biochemical processes, modifying growth and developmental patterns [4]. Salinity leads to increased ionic concentrations in the soil, thereby decreasing soil water potential and rapidly imposing osmotic stress on plants, which impairs root water uptake, leading to dehydration at the cellular level. The osmotic challenge triggers the rapid synthesis and accumulation of abscisic acid (ABA), which subsequently promotes stomatal closure to minimize transpirational water loss [5]. In addition to its direct impact on plant-water relations, osmotic stress disrupts normal cellular metabolism through various mechanisms. Prolonged cellular dehydration inhibits cell division and elongation, thereby constraining the growth of both roots and shoots. Furthermore, osmotic stress increases cell membrane permeability and compromises membrane integrity, predisposing plants to subsequent ion toxicity and oxidative damage [5]. Ion toxicity, particularly from Na⁺, causes long-term damage. Due to its structural similarity to K⁺, Na⁺ competes for entry into plant tissues [6], disrupting Na⁺/K⁺ homeostasis. Elevated cytosolic Na⁺ inhibits enzymatic activity, compromises protein synthesis, and disrupts osmotic balance, while K⁺ deficiency impairs functions like stomatal regulation [7]. Tolerant plants typically maintain a low cytoplasmic Na⁺/K⁺ ratio through mechanisms like selective ion uptake, restriction of root-to-shoot Na⁺ translocation, and vacuolar Na⁺ sequestration [8]. Consequently, the Na⁺/K⁺ ratio has become a key indicator for evaluating salt tolerance.

Salt stress induces multiple photosynthetic disorders [9]. The accumulation of sodium ions leads to stomatal closure, thereby restricting the diffusion of CO₂ [10]. Internal ionic toxicity and osmotic stress within leaves act synergistically to disrupt chloroplast ultrastructure, inhibit chlorophyll biosynthesis, and reduce the photochemical efficiency of photosystem II (PSII) [11]. Consequently, key physiological parameters such as relative chlorophyll content (SPAD value) and net photosynthetic rate are often significantly suppressed under salt stress conditions.

Furthermore, the accumulation of salt ions leads to the overproduction of reactive oxygen species (ROS) in shoots and roots, including hydroxyl radicals (·OH), superoxide anions (O₂·⁻), hydrogen peroxide (H₂O₂) and single-oxygen (¹O₂). This disrupts ROS homeostasis, leading to oxidative stress [12]. When ROS accumulation exceeds the cellular scavenging capacity, it causes irreversible oxidative damage to lipids, proteins, and DNA, potentially triggering programmed cell death [13]. Therefore, the ROS level is widely used as an indicator of salt stress severity. The increased concentration of ROS is partly balanced by antioxidant enzymatic scavenging compounds, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) [14]. These enzymes form a crucial defense system against oxidative damage [15]. SOD acts as the first line of defense, catalyzing the dismutation of O₂⁻ into O₂ and H₂O₂ [16], while POD and CAT detoxify H₂O₂ by converting it into H₂O and O₂ [17, 18].

Barley is the fourth most important cereal crop globally and the most salt-tolerant cereal crop [19]. However, domestication has reduced genetic variation in cultivated barley. In contrast, wild barley still thrives across an astonishingly wide ecological habitats, including highlands and deserts [20, 21], allowing it to accumulate substantial natural genetic variations that helps combat various abiotic stresses, such as salt stress. Studies indicate that wild barley has greater drought tolerance and accumulates less Na⁺ under salt stress than its cultivated counterpart, making it a vital resource for understanding salt tolerance mechanisms [22, 23]. Therefore, this study aimed to explore the physiological characteristics underlying superior salt tolerance in wild barley by comparing wild and cultivated barley under salt stress.

Materials and methods

Plant materials and growth conditions

Six barley varieties were used in this study: two salt-tolerant wild barley varieties (SRY01 and Tam) [23], two salt-sensitive cultivated varieties (Franklin and Gairdner) and two salt-tolerant cultivated varieties (YNP5 and YNP7) [23], all provided by the Agricultural College of Yangzhou University.

Seeds were surface-sterilized, germinated on moist filter paper in a thermostatic incubator (25 ℃, in dark) for 24 h, and then sown (4 seeds per pot) in black pots (5 cm × 5 cm × 10 cm) filled with potting soil. At the two-leaf stage, the seedlings were transferred into plastic containers filled with 300 mM NaCl. The control group was transferred to plastic containers filled with water. Plants were harvested for measurement two weeks after the initiation of NaCl treatment. Fresh plant samples from each replicate were randomly selected for the analysis of malondialdehyde (MDA), hydrogen peroxide (H₂O₂) content, and Na⁺, K⁺ distribution, as well as antioxidant enzyme activities.

Whole-plant physiological assessment

Six plants per variety from each treatment and control group were harvested to measure aboveground and belowground dry/fresh weights. Roots were carefully extracted from the pots and gently washed to remove residual growth medium, minimizing root loss. Plant material designated for dry weight determination was oven-dried at 70 °C for 48 h [17].

The relative chlorophyll content (SPAD value) was determined using a chlorophyll meter (Minolta Co., Ltd., Japan). Measurements were taken at the upper, middle, and lower sections of the second fully unfolded leaf and averaged. The net photosynthetic rate was measured on the second fully unfolded leaf using an LI-6800 portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA; console version: Bluestem v1.4.02) equipped with a 2 cm × 3 cm leaf chamber. The chamber conditions were set as follows: fan speed 10,000 rpm, flow rate 500 μmol s⁻¹, overpressure 0.1 kPa, relative humidity 50%, leaf temperature 25 °C, chamber CO₂ 400 μmol⁻¹ and light intensity 1000 μmolm⁻² s⁻¹. The experiments were designed as a completely randomized experiment, with three replicates per treatment and two plants per replicate.

H₂O₂ and MDA content analysis

For the assessment of H₂O₂, prewashed 70 mg shoots and roots were separately blended into 5 ml of TCA at a 0.1% (w/v) ratio under ice–cold conditions. The mixture was then centrifuged at 12,000 rpm for 15 min. The supernatant (0.5 mL), 1 mL of 1 M potassium Iodide (KI), and 0.5 mL of 1 mM potassium phosphate buffer (pH 7) were added, and spectrophotometric readings were taken at 390 nm [24].

Malondialdehyde (MDA) levels were measured using thiobarbituric acid (TBA). Frozen foliage (0.1 g) samples were mixed with 0.1% (w/v) trichloroacetic acid (TCA) and centrifugation was done at 15,000 rpm. The supernatant (0.5 mL) and 20% TCA (2 mL) containing 0.5% TBA were then heated for 30 min at 95 ℃ and cooled to 40 ℃. Centrifugation was performed at 15,000 rpm for 10 min. The spectrophotometric values of solution were recorded at 532 and 600 nm [24].

Antioxidant enzymes activity determination analysis

Fresh shoot and root samples (0.1 g) were homogenized in ice-cold phosphate buffer (50 mM, pH 7.0). The homogenate was centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was collected and stored at −80 °C for subsequent enzyme activity assays [25].

To analyze the POD activity, the enzyme extract (0.1 mL) was mixed with a reaction mixture containing pyrogallol, phosphate buffer at pH 6.8, and 1% H₂O₂. Variation in spectrophotometric values was recorded at every 20 s for 120 s at 420 nm, as described by Chance and Maehly (1954) [26]. The control solution was prepared by mixing double-distilled water as an alternative to the enzymatic extract. The procedure described by Chance and Maehly (1954) [26] was adopted for the estimation of catalase (CAT) activity. The solution consisted of 3 mL of phosphate buffer (50 mM) at pH 7.0, H₂O₂ (15 mM). In this solution, 0.1 mL of enzyme extract was added to initiate the reaction. At every 20 s interval, variations in absorbance were observed in the reaction mixture at 240 nm [27]. The ability of superoxide dismutase (SOD) to minimize photochemical reduction of nitroblue tetrazolium (NBT) was measured. In the solution phosphate buffer (50 mM) with a pH of 7.8, methionine (13 mM), NBT (75 mM), EDTA (0.1 mM) and 20 mM riboflavin were included. The solution was exposed to two fluorescent light tubes (40 mol m⁻¹ s⁻¹) for 10 min. The absorbance was recorded using a UV–visible spectrophotometer at a wavelength of 560 nm [24, 28].

Measurement of K⁺ and Na⁺ content

For K⁺ and Na⁺ content analysis, dried plant samples were ground and passed through a 0.5 mm sieve. A 0.1 g aliquot of the powdered sample was digested in concentrated H₂SO₄ (18.4 M) for 1.5 h. After cooling, 30% H₂O₂ was added, and digestion continued until a clear solution was obtained to ensure complete decomposition of H₂O₂. The K⁺ and Na⁺ concentrations in the digest were measured using inductively coupled plasma mass spectrometry (ICP-MS). Ionic content was measured simultaneously in roots and shoots, with three biological replicates established for each treatment and control within each variety.

Histochemical staining of O₂·⁻, H₂O₂ and cell permeability

Histochemical detection of superoxide anion (O₂·⁻) and hydrogen peroxide (H₂O₂) was performed using nitrobule tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB), respectively. Briefly, intact barley leaves were vacuum-infiltrated for 30 min with either 50 mM sodium phosphate buffer (pH 7.5) containing 0.2% (w/v) NBT or 10 mM sodium phosphate buffer (pH 6.5) containing 1 mg mL⁻¹ DAB. The leaves were then incubated in the dark at 37 °C for 1 h (NBT) or 12 h (DAB). Subsequently, chlorophyll was removed by boiling the leaves in ethanol for 10 min, followed by preservation in fresh ethanol. Cell permeability was assessed using Evans Blue staining. Leaves were vacuum-infiltrated with 50 mM sodium phosphate buffer (pH 7.5) containing 0.1% (w/v) Evans Blue for 30 min and incubated in the dark at 37 °C for 24 h. After washing with a 9:1 mixture of ethanol and glycerol solution, chlorophyll was cleared by boiling in ethanol for 30 min, and leaves were stored in fresh ethanol. All stained leaves were photographed under uniform lighting conditions [24].

Confocal laser scanning microscopy measurements

To quantify in vivo reactive oxygen species (ROS), 5-mm-long root sections were incubated with 25 μM 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) in the dark for 30 min. After incubation, the root sections were mounted on microscope slides with mounting medium and sealed with a coverslip. Root sections were imaged by using Leica SP8 spectral confocal laser scanning microscope. Three biological replicates were used, with two root sections per plant. The imaging settings were as follows: 488 nm laser excitation; PMT1, 500–600 nm (for DCF fluorescence). Imaging of Na⁺ and K⁺ distribution was performed using 20 μM Na⁺ dye CoroNa Green AM (Thermo Fisher Scientific) and 20 μM K⁺ dye APG-2 AM (asante potassium green-2 AM, Abcam Biotechnology company). The CoroNa Green AM and APG-2 AM fluorescent dyes were dissolved in pure DMSO, and then respectively diluted with buffer solution (10 mM KCI, 5 mM Ca²⁺-MES, pH 6.1) and TES {2-[(2-Hydroxy-1,1-bis (hydroxymethyl) ethyl) amino] ethanesulfonic acid; 10 mM, pH 7.5}, respectively, to the working concentration. As described above, barley root sections were prepared and incubated with CoroNa Green AM + FM 4–64 [(N-(3-Triethylammoniumpropyl)−4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide)] or APG-2 AM + FM 4–64 in 1.5 mL Eppendorf tubes for 2 h and 2.5 h in darkness, respectively. After incubation, root sections were rinsed in ddH₂O and mounted on microscope slides for confocal imaging. The settings of Na⁺ and K⁺ confocal imaging were as follows: 488 nm laser excitation; PMT1: 500–550 nm (for Na⁺ confocal imaging) or 520–560 nm (for K⁺ confocal imaging). Image J 1.42 software was used to analyze fluorescence intensity of obtained images [27].

Measuring stomatal aperture using scanning electron microscopy

Leaf discs (5 mm in diameter) were excised from the second fully expanded leaves. The discs were fixed in 2.5% glutaraldehyde and imaged using a GeminiSEM 300 scanning electron microscope. Stomatal aperture was measured and calculated using ImageJ 1.42 software.

Statistical analysis

The data was analyzed by one-way ANOVA, and all experiments employed a completely randomized design. Differences between means were determined with Tukey’s Honestly Significant Difference (HSD) Test at 0.05 level. All analyses were conducted using SPSS software. All data were expressed as means ± SE (n = 3). The results were visualized graphically using Origin 2024b.

Results

Salt stress impairs growth and photosynthetic performance in barley varieties

Following a two-week exposure to 300 mM NaCl, both wild and cultivated barley varieties exhibited significant growth inhibition (Fig. 1A). Salt stress significantly (p ≤ 0.05) reduced shoot and root fresh and dry biomass compared to controls (Fig. 1A-E). The salt-tolerant cultivated barley YNP5 and YNP7 exhibited a markedly milder degree of leaf chlorosis compared to the salt-sensitive barley Franklin and Gairdner, while the leaves of wild barley SRY01 and Tam maintained a green coloration (Fig. 1A).

Fig. 1.

Effects of salt stress on the growth of six barley varieties. A Plant morphology of six barley varieties under control and salt stress condition. Fresh and dry weight of barley shoots (B, C) and roots (D, E) under control and salt stress conditions. Data are means ± SE (n = 3)

The second fully unfolded leaf was used to measure the net photosynthetic rate and SPAD value. The results showed that salt stress significantly reduced the net photosynthetic rate across all varieties. Salt-tolerant barley exhibited a higher photosynthetic rate than salt-sensitive barley, with the wild barley demonstrating an even superior rate (Fig. 2A). The SPAD value was significantly higher in wild barleys under salt stress compared to their respective controls, whereas it was lower in the salt-sensitive cultivated varieties Franklin and Gairdner (Fig. 2B). The SPAD values of YNP5 and YNP7 under salt stress were not much different from the controls (Fig. 2B). The changes in SPAD values corresponded with photosynthetic rates.

Fig. 2.

Photosynthetic rate (A) and SPAD values (B) of barley varieties under control and salt stress conditions. Data are means ± SE (n = 3)

Wild barley exhibits improved stomatal regulation under salt stress

Scanning electron microscopy of the second leaf revealed that salt stress reduced stomatal aperture in all six varieties (Fig. 3A, B). Under salt stress, the salt-tolerant varieties YNP5 and YNP7 maintained smaller stomatal apertures than the sensitive varieties Franklin and Gairdner, but the difference is not significant. The stomatal aperture of two wild barley varieties were significantly smaller than those of salt-tolerant cultivated barley, exhibiting a more efficient stomatal closure response under salt stress.

Fig. 3.

Effect of salt stress on stomatal aperture of six barley varieties. One out of six typical images are shown for each variety in (A). Scale bar = 2 μm. (B) Stomatal aperture was measured. Data are means ± SE (n = 3)

Wild barley exhibits enhanced oxidative stress tolerance and reduced cellular damage under salt stress

Exposure of plants to salt stress leads to excessive accumulation of reactive oxygen species (ROS) [29, 30], inducing toxic responses in plants. Malondialdehyde (MDA) is a key product of lipid peroxidation, accumulates directly reflecting the degree of cell membrane damage [31]. Here, we measured H₂O₂ and MDA content to assess the salt tolerance of different varieties. Compared with control conditions, all varieties exhibited higher levels of H₂O₂ and MDA under salt stress (Fig. 4A-D) than under control conditions, but with significant variation in the increase. The tolerant cultivated varieties YNP5 and YNP7 consistently maintained lower root H₂O₂ and shoot MDA contents than the sensitive ones (Fig. 4B, C). The wild barley accumulated less H₂O₂ and MDA in both root and shoot than the salt-tolerant cultivated barley (Fig. 4A-D).

Fig. 4.

Shoot and root H₂O₂ (A, B) and MDA content (C, D) of six barley varieties under control and salt stress condition. Data are means ± SE (n = 3)

In order to visualize the accumulation of ROS and the extent of cellular damage induced by salt stress, histochemical staining was performed on leaves using 3,3’-diaminobenzidine (DAB), nitroblue tetrazolium (NBT), and Evans Blue. Under control conditions, no visible leaf damage or pigment accumulation was observed in any variety. However, the extent of pigment accumulation varied among varieties under salt stress, reflecting differences in leaf damage. The salt-tolerant cultivated barley showed less damage than the salt-sensitive barley, while the wild barley exhibited less pigment accumulation than the salt-tolerant cultivars (Fig. 5A-F).

Fig. 5.

Analysis of antioxidant enzyme activities. POD, CAT, and SOD activity in shoot (A, C, and E, respectively) and root (B, D, and F, respectively) of six barley varieties under control and salt stress conditions. Data are means ± SE (n = 3)

To clarify the active oxygen scavenging capacity of barley varieties with varying salinity tolerance, we measured the activity of key antioxidant enzymes (Fig. 6A-F). The results showed that these activities were markedly higher in wild barley, the salt-tolerant cultivated barley showed higher shoot POD and root SOD activities than the salt-sensitive cultivated barley (Fig. 6A, F). Furthermore, the wild barley exhibited the highest enzyme activity in both roots and leaves compared to cultivated barley. These results indicate that the higher antioxidant enzyme activity in wild barley helps maintain reactive oxygen species homeostasis.

Fig. 6.

Histochemical staining of leave O·²⁻ (A, B), H₂O₂ (C, D) and cell permeability (E, F) on barley varieties under control and salt stress conditions

Maintenance of a low Na⁺/K⁺ ratio is associated with salt tolerance in barley varieties

Salt stress markedly increased Na⁺ accumulation in both roots and shoots across all varieties, although the magnitude of this increase varied substantially among varieties (Fig. 7A, B). The salt-tolerant cultivated varieties accumulated significantly less Na⁺ in both roots and shoots than the salt-sensitive cultivated barley, and the wild barley accumulated the lowest Na⁺ content and Na⁺/K⁺ ratio among all the barley varieties (Fig. 7A, B). No significant difference in K⁺ accumulation was observed between wild barley, tolerant lines, and sensitive cultivars in either root or shoot tissues (Fig. 7C, D). This may be driven primarily by their superior capacity to restrict Na⁺ influx rather than by variations in K⁺ retention (Fig. 7E, F). These findings suggest that the enhanced salt tolerance of wild barley was partly attributable to its ability to maintain low cytosolic Na⁺ levels.

Fig. 7.

Na⁺ and K⁺ content and Na⁺/K.⁺ ratio in shoot (A, C, and E, respectively) and root (B, D, and F, respectively). Data are means ± SE (n = 3)

Confocal imaging in barley roots reveals differential Na⁺/K⁺ homeostasis and ROS accumulation under salt stress

Confocal imaging of barley roots using fluorescent probes enabled in situ visualization of ion distribution and ROS accumulation. Under salt stress, the salt-tolerant YNP5 and YNP7 exhibited relatively lower Na⁺ and higher K⁺ fluorescence than the sensitive varieties Gairdner and Franklin, while the wild barley maintained higher K⁺ fluorescence (Asante Potassium Green-2 AM) and lower Na⁺ fluorescence (CoroNa Green AM) in roots compared to the salt-tolerant cultivated barley (Fig. 8A-D). ROS fluorescence (H₂DCFDA) was lowest in wild barley, intermediate in tolerant lines, and highest in sensitive cultivars (Fig. 8E, F). Quantitative analysis of fluorescence intensity confirmed these visual patterns (Fig. 8G-I).

Fig. 8.

Distribution and quantification of root Na⁺ and K⁺ and ROS of six barley varieties under control and salt stress condition. Representative images indicate the distribution of K⁺ (A, B) and Na⁺ (C, D) and ROS accumulation (E, F). One out of six typical image is shown for each variety. Scale bar = 100 μm. The relative fluorescence intensity of K⁺ (G), Na⁺ (H) and ROS (I) was calculated. Data are means ± SE (n = 3)

Discussion

Previous research on the differences in salt tolerance between wild and cultivated barley is scarce and has mostly focused on single-trait measurements, Kiani-Pouya A et al. [32] revealed the salt tolerance of wild barley through stomatal analysis, while Garthwaite AJ et al. [33] have revealed the better salt tolerance of wild barley through studies on salt ions transport. Gharaghanipor et al. [34] used transcriptomics technology combined with physiological analyses to study the difference in salt tolerance between wild and cultivated barley, but more focused on the transcriptomics analysis. Few studies have employed multiple complementary indicators to investigate these differences, despite the fact that salinity stress elicits a complex array of responses and plant adaptation relies on the coordinated action of multiple protective mechanisms. Given this complexity, our study employed a multi-trait approach to evaluate salt tolerance among barley varieties. The results provide a comprehensive view of how wild barley achieves superior salt tolerance through the integration of enhanced stomatal regulation, ion homeostasis, and antioxidant defense.

Wild barley enhances photosynthetic capacity and Na ⁺ transport through stomatal regulation

Photosynthetic capacity is an important indicator of plant performance under environmental stress. As noted by Zhang et al. [35], stomata function as the primary sites for water and gas exchange between plants and their environment, facilitating the entry of CO₂ (a critical substrate for photosynthesis) into plant tissues. Most previous studies have shown that reduced stomatal aperture leads to decreased carbon assimilation and lower photosynthetic rates [36, 37]. Although stomatal closure may limit CO₂ influx to some extent, it also plays a protective role under salt stress. Yinaykumar et al. [38] revealed that salt-tolerant plants maintain higher chlorophyll content and photosynthetic rate through more precise stomatal regulation. In this study, wild barley maintained a relatively higher photosynthetic capacity and SPAD value despite exhibiting a lower stomatal aperture (Fig. 2A, 3B). Wild barley likely minimized transpirational water loss and associated physiological disturbances by reducing stomatal aperture, thereby preserving chloroplast structure and function. In addition to protecting photosynthetic structures, stomatal closure serves as a key mechanism to regulate ion transport. Michael F et al. [39] revealed that stomatal regulation can limit the transport of Na⁺ to leaves, thereby reducing damage to the leaves. Similarly, Niu et al. [40] demonstrated that stomatal closure acts as a crucial adaptive response to restrict Na⁺ accumulation in leaves, thereby maintaining chloroplast integrity, chlorophyll levels, and photosynthetic capacity under salt stress. In the current study, the wild barley’s Na⁺ content was much lower than that of both salt-tolerant and salt-sensitive barley in shoots and roots (Fig. 6A, C). Wild barley can maintain high photosynthetic capacity and low Na⁺ content through improved stomatal regulation.

Wild barley maintains ion homeostasis through enhanced sodium exclusion capacity

Salt stress induces long-term ionic toxicity in plants. Garthwaite AJ et al. [33] revealed that salt ions such as Na⁺ and Cl⁻ are taken up by the roots and subsequently translocated via the xylem to the shoots. The salt ions may cause series of damage through different mechanisms when accumulated in plants [41]. To counteract the detrimental effects of Na⁺ and other salt ions, plants have evolved a suite of adaptive strategies. Research has demonstrated that the regulation of sodium–potassium ion homeostasis within the cytoplasm is crucial for salt tolerance. This conclusion has been corroborated by studies investigating the functional roles of genes associated with sodium transport. In particular, HKT functions in sodium transport, the knockout lines of HvHKT1.4 negatively regulate the salt tolerance of barley [42]. In root tips, SOS1 mediates sodium extrusion, and its upregulation in Arabidopsis enhances this process, increasing salt tolerance [43]. Similarly, ZmHAK4 promotes sodium efflux from maize stems, and its overexpression enhances salinity tolerance [44]. In the present study, salt-tolerant cultivated barley showed lower root Na⁺/K⁺ ratios than salt-sensitive cultivated barley (Fig. 7A, B, E, F). Wild barley exhibited significantly lower shoot and root Na⁺ concentrations and Na⁺/K⁺ ratio than salt-tolerant cultivated barley (Fig. 7A, B, E, F), with no significant difference in K⁺ content. This observation revealed that wild barley has a more effective sodium ion exclusion mechanism to maintain internal ionic balance and reduce damage.

Higher ROS scavenging capacity confers greater salt tolerance to wild barley

Elevated Na⁺ concentrations directly inhibit photosynthesis, leading to over-reduction of the photosynthetic electron transport chain. This, in turn, causes electron leakage to molecular oxygen, resulting in the massive production of ¹O₂ (singlet oxygen) and O₂·⁻ (superoxide anion) [45, 46]. Concurrently, reduced CO₂ concentrations within chloroplasts promote photorespiration, a process that generates substantial amounts of hydrogen peroxide (H₂O₂) in mitochondria and peroxisomes [46]. In the present study, both H₂O₂ and MDA levels increased in the roots and shoots of all six barley varieties under salt stress. Notably, H₂O₂ and MDA levels were lower in salt-tolerant than in salt-sensitive cultivated varieties, with wild barley exhibiting the lowest levels overall (Fig. 4). Fluorescence staining further confirmed that wild barley sustained lower ROS accumulation in root tissues (Fig. 8E, F, I). These findings are consistent with those of Rahman H et al., who reported that salt-tolerant genotypes maintain reduced H₂O₂ and MDA levels under saline conditions [47]. To counteract the rapid accumulation of intracellular ROS, plants have evolved complex regulatory networks that upregulate antioxidant enzyme genes and enhance enzymatic activities, thereby maintaining ROS homeostasis. The PagSOD2a gene encodes the POD protein, and its overexpression has been shown to enhance the salt tolerance of poplar trees [48]. Pan et al. analyzed how ZmCAT1 is transcriptionally upregulated upon binding to ZmNAC84, thereby enhancing maize salt tolerance [49]. Previous studies have demonstrated that salt-tolerant plants exhibit higher antioxidant enzyme activities under salt stress [25, 47]. In this study, the salt-tolerant cultivated barley showed no significant differences of CAT and SOD compared to the salt-sensitive barley (Fig. 6C, D, E, F), while the wild barley showed higher activities of all the antioxidant enzymes than cultivated barley. Collectively, these results indicate that wild barley achieves enhanced salt tolerance by upregulating antioxidant enzyme activity to effectively scavenge excess ROS.

Comprehensive evaluation of salt tolerance indicators to develop salt-tolerant varieties

Wild barley (H. spontaneum) represents an untapped genetic reservoir for stress tolerance traits [23, 50]. The wild barley possesses multiple complementary mechanisms conferring salt tolerance: efficient stomatal regulation, superior ion homeostasis, and enhanced antioxidant defense. By comparison, salt-tolerant cultivated barley varieties YNP5 and YNP7 still have very limited abilities to regulate stomata, maintain ionic homeostasis, and scavenge reactive oxygen species. Future research should focus on the excellent physiological indicators of salt tolerance in wild barley, identify genes regulating these physiological traits in wild barley and integrate them into barley salt tolerance breeding programs through marker-assisted pyramiding selection approach.

Conclusions

In summary, the superior salt tolerance of wild barley is not conferred by any single mechanism but emerges from the integration of a series of physiological mechanisms. Wild barley exhibits more stringent ionic homeostasis (low Na⁺/K⁺ ratio), a potent antioxidant defense system (elevated activities of POD, CAT, and SOD, coupled with lower levels of MDA and H₂O₂), precise stomatal regulation, and sustained physiological performance (maintained photosynthesis). These findings indicate that wild barley combines multiple stress-adaptive traits to a degree not yet achieved in currently cultivated germplasm. Future efforts should focus on identifying the potential genes regulating these physiological traits in wild barley to enhance salt tolerance in cultivated barley.

Authors’ contributions

R G.X. and. L.B. Z conceived and designed the study; Y H. Z performed the experiments, analyzed the data and wrote the manuscript; C.C., X.Y. and J.Z. performed the experiments. L F.L., M N. Z and Y.H. assisted in data collection. C.L., B J.G. and F F.W. assisted in data processing and phenotype collection. All authors read and approved the final manuscript.

Funding

This work was supported by the Key Research and Development Program of the Xizang Autonomous Region (XZ202501ZY0029), the National Natural Science Foundation of China (32301871), the National Modern Agriculture Industry Technology System, China (CARS-05), the Priority, and Academic Program Development of Jiangsu Higher Education Institutions, China (PAPD).

Data availability

All datasets generated for this study are included in the article and figures.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.