The hydroponic experiment was conducted at room temperature from April to May 2025. Sorghum seedlings were grown under natural light conditions with an intensity of approximately 800–1000 μmol·m⁻²·s⁻¹. Environmental conditions were maintained at day/night temperatures of 26/22°C, a 10/14 h light/dark cycle, and a relative humidity of 60%–70%. Seeds of sorghum variety “Kangsi Gaoliang” were procured from Mengya Seed Sales Co., Ltd. (Hejian City, Hebei Province). Plump seeds were selected, surface-sterilized with a 5% NaClO solution for 20 min, and thoroughly rinsed with distilled water.

The ZnO NPs used in this study were synthesized using sea buckthorn leaf extract. The synthesized nanoparticles exhibited an average particle size of 200 nm and a zeta potential of −44.07 mV, demonstrating good stability. Informed by preliminary experiments and previous studies, ZnO NPs suspensions at concentrations of 50, 100, and 200 mg·L⁻¹ were utilized as seed priming agents. The sterilized seeds were soaked in these respective suspensions for 24 h, whereas the control group was soaked in distilled water. To simulate combined saline-alkali stress, mixed salt solutions containing NaCl, Na₂SO₄, Na₂CO₃, and NaHCO₃ were prepared at a molar ratios of 1:1:1:1, yielding final concentrations of 50, 100, and 150 mmol·L⁻¹.

For cultivation, filter papers were saturated with the prepared mixed salt solutions, while distilled water was used for the control group. The primed seeds were sown in Petri dishes lined with three layers of the saturated filter paper, with 5 g of seeds allocated per dish. The experimental design comprised a total of 16 treatments, each with three biological replicates. For daily maintenance, each Petri dish was supplemented with 5 mL of distilled water. Weekly, each dish received 5 mL of the corresponding mixed salt solution (or distilled water for the control) alongside Hoagland nutrient solution. Two weeks post-emergence, 10 mL of the respective ZnO NPs suspension was applied to each dish as a foliar spray. This foliar application was performed twice at a one-week interval. The seedlings were harvested four weeks post-emergence for subsequent experimental analysis.

The aboveground length (AL) of sorghum seedlings was measured using a tape measure, and the aboveground fresh weight (AFW) was determined with an electronic analytical balance. The aboveground tissues were then heated at 105°C for 30 min to deactivate enzymes, and subsequently oven-dried at 80°C for 72 h. Finally, the aboveground dry weight (ADW) was recorded using the analytical balance.

Chlorophyll a (Chl a) and b (Chl b) contents in sorghum leaves were determined following the methods described by Hiscox et al. [34] and Wellburn [35]. Briefly, 0.5 g of fresh leaf tissue from each treatment was cut into small segments and placed into stoppered tubes containing 10 mL of dimethyl sulfoxide (DMSO). The samples were then incubated in a water bath at 65°C for 4 h. After cooling to room temperature, the absorbance of the supernatant was measured at 645 nm and 663 nm to assess the concentrations of Chl a and Chl b, respectively.

For the antioxidant enzyme assays, 0.5 g of leaf tissue was first homogenized in an ice bath at 4°C with 10 mL of pre-chilled 0.05 M phosphate buffer (PBS, pH 7.8; prepared from Na₂HPO₄ and NaH₂PO₄ and supplemented with 1% EDTA-Na₂). The homogenate was then centrifuged at 10,000 rpm for 10 min [36], and the resulting supernatant was collected and stored at 4°C for the determination of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) activities. SOD activity was evaluated according to the method of Zhang et al. by measuring the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm [37]. POD activity was determined following the method of Samadi et al. by monitoring the oxidation rate of guaiacol at 470 nm in the presence of H₂O₂ [38]. CAT activity was assayed based on the method of Chance et al. by measuring the rate of decrease in H₂O₂ absorbance at 240 nm [39]. Finally, APX activity was assessed according to the method of Khaleghi et al. by tracking the decrease in absorbance at 290 nm resulting from the oxidation of ascorbic acid by H₂O₂ [40].

The ascorbic acid (AsA) content was determined following the protocol described by Huang et al. [41]. Briefly, fresh leaf samples (0.5 g) were homogenized in 5 mL of 5% PBS at 4°C. The homogenate was centrifuged at 22,000 rpm for 15 min, and the resulting supernatant was collected. AsA concentration was subsequently quantified by measuring the absorbance at 525 nm, which corresponds to the reaction product of AsA, tetraphenylbenzidine, and ferric chloride.

For the quantitative analysis of glutathione (GSH), the method established by Feng et al. was employed [42]. Leaf tissues (0.5 g) were homogenized in 5% trichloroacetic acid (TCA) at 4°C and centrifuged at 15,000 rpm for 15 min. The GSH content in the supernatant was then evaluated by monitoring the absorbance at 412 nm, reflecting the formation of a yellow compound generated from the reaction between GSH and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB).

The damage of plant lipid peroxidation was evaluated by determining the malondialdehyde (MDA) content, following the protocol outlined by Tanveer et al. [43]. Briefly, 0.5 g of fresh leaf tissue was homogenized in 5 mL of 0.05 M PBS (pH 7.8) at 4°C. The homogenate was then centrifuged at 15,000 rpm for 20 min, and the supernatant was collected. Subsequently, a 1.5 mL aliquot of the supernatant was mixed with 2.5 mL of a reaction solution containing 0.05 M PBS, 5% thiobarbituric acid (TBA), and 5% TCA. The resulting mixture was incubated in a boiling water bath (100°C) for 10 min. After cooling to room temperature, the mixture was centrifuged at 4800 rpm for 10 min. The absorbance of the final supernatant was measured at 532 nm and 600 nm to calculate the MDA content.

The Na⁺ and K⁺ contents were determined following the methods described by Barin et al. [44] and Yang et al. [45]. Briefly, dried sorghum seedlings were ground into a fine powder. A 0.5 g aliquot of the powdered sample was transferred into a polytetrafluoroethylene (PTFE) tube, and 10 mL of an HClO₄ and HNO₃ mixture (1:4, v/v) was added for microwave digestion. After digestion, the concentrations of Na⁺ and K⁺ were quantified using inductively coupled plasma mass spectrometry (ICP-MS), and the Na⁺/K⁺ ratio was subsequently calculated.

All experiments were performed in triplicate, and the results are expressed as the mean ± standard deviation (SD). Statistical analyses were conducted using SPSS 26.0 software. Prior to analysis, the data were assessed for normality and homogeneity of variance using the Shapiro-Wilk and Levene’s tests, respectively. Once the assumptions of normal distribution and equal variances were confirmed, the data were subjected to a one-way analysis of variance (ANOVA). Subsequently, Duncan’s multiple range test was employed for post hoc comparisons to identify statistically significant differences among the treatment groups (p < 0.05). In addition, correlation analysis was performed to evaluate the linear relationships among the various physiological indicators. Principal component analysis (PCA) was utilized for dimensionality reduction, providing a clearer visualization of the variations among different treatments. A two-way ANOVA was also conducted to determine the main effects of ZnO NPs and saline-alkali stress, as well as their interaction effects. Subsequently, response surface methodology (RSM) models were constructed using RStudio (version 2025.05.01) for an in-depth analysis of the interactive effects of ZnO NPs and saline-alkali stress on the physiological parameters, and to predict the optimal response ranges for both factors. Finally, all graphical representations were generated and processed using Origin 2025.

The effects of ZnO NPs on the growth of sorghum seedlings under saline-alkali stress are presented in Fig. 1. Across all treatment groups, the aboveground length of sorghum seedlings exhibited a distinct unimodal trend (initial increase followed by a subsequent decrease) with rising ZnO NPs concentrations (Fig. 1A). In the absence of saline-alkali stress, ZnO NPs at concentrations of 50–100 mg·L⁻¹ significantly promoted shoot elongation, reaching a maximum of 14.5 cm (a 34.3% increase compared to the control). However, this growth was markedly inhibited by 43.4% at a concentration of 200 mg·L⁻¹, suggesting a phytotoxic effect at higher dosages. Similar growth-promoting effects were observed under 50 and 100 mmol·L⁻¹ saline-alkali stress, where the aboveground lengths peaked at 17.3 cm and 15 cm, respectively, at a ZnO NPs concentration of 100 mg·L⁻¹, before decline at 200 mg·L⁻¹.

Consistent with the changes in aboveground length, the shoot fresh weight exhibited a similar biphasic trend, rising initially before declining at higher ZnO NPs dosages (Fig. 1B). Under non-stressed conditions, the application of 50 and 100 mg·L⁻¹ ZnO NPs significantly elevated the fresh weight by 24.4% and 46.2%, respectively (p < 0.05). Under low to moderate saline-alkali stress (50 and 100 mmol·L⁻¹), the 100 mg·L⁻¹ ZnO NPs treatment proved to be the most effective, yielding fresh weight increases of 29.6% and 24.8%, respectively. Notably, under severe stress conditions (200 mmol·L⁻¹), this same optimal concentration (100 mg·L⁻¹) demonstrated a pronounced mitigating effect, boosting the fresh weight by an impressive 61.6% compared to the control (p < 0.05).

Mirroring the trends observed in fresh weight, the shoot dry weight exhibited an initial increase followed by a decline as ZnO NPs concentrations rose (Fig. 1C). In the non-stressed control, the 100 mg·L⁻¹ ZnO NPs treatment maximized dry weight accumulation, reaching a peak of 0.169 g (a 92.4% increase). This specific dosage (100 mg·L⁻¹) consistently provided the most pronounced growth benefits as saline-alkali stress intensified. Specifically, it enhanced dry weight by 79.3% under mild stress (50 mmol·L⁻¹), and produced striking increases of 84.0% and 208.8% under moderate to severe stress conditions (100 and 150 mmol·L⁻¹, respectively). Collectively, these growth parameters indicate that an optimal dosage of ZnO NPs (approximately 100 mg·L⁻¹) effectively alleviates saline-alkali stress and promotes sorghum seedlings growth, whereas excessive concentrations induce phytotoxicity.

The effect of ZnO NPs on chlorophyll content of sorghum seedlings under saline-alkali stress is illustrated in Fig. 2. Specifically, the response of Chl a to varying ZnO NPs concentrations was highly dependent on the severity of the stress (Fig. 2A). Under non-stressed conditions, ZnO NP application had no significant impact on Chl a levels. However, as saline-alkali stress intensified, ZnO NPs exerted a clear positive regulatory effect. Under mild stress (50 mmol·L⁻¹), the 100 and 200 mg·L⁻¹ ZnO NPs treatments elevated Chl a content by 13.2% and 24.7%, respectively. As the stress escalated to 100 and 150 mmol·L⁻¹, all tested ZnO NPs concentrations significantly enhanced Chl a accumulation. Notably, the 100 mg·L⁻¹ treatment yielded the most pronounced improvements, increasing Chl a content by 35.2% and 34.3% under 100 and 150 mmol·L⁻¹ stress, respectively (p < 0.05).

The response of Chl b to ZnO NPs (Fig. 2B) presented a different pattern from that of Chl a. Under unstressed conditions, Chl b levels remained largely unaffected, with a significant alteration observed only at the highest ZnO NPs concentration (200 mg·L⁻¹). However, under mild saline-alkali stress (50 mmol·L⁻¹), the 100 mg·L⁻¹ ZnO NPs treatment significantly elevated Chl b content by 47.8% (p < 0.05). As the stress intensified to 100 and 150 mmol·L⁻¹, Chl b accumulation exhibited a progressive increase with rising ZnO NPs concentrations. Notably, the 200 mg·L⁻¹ treatment yielded the most substantial improvements, boosting Chl b by 135.8% and 92.9% under 100 and 150 mmol·L⁻¹ stress, respectively (p < 0.05). Collectively, these findings indicate that while ZnO NPs exert minimal influence on photosynthetic pigments in the absence of stress, they significantly enhance pigment accumulation under saline-alkali conditions. Specifically, the 100 mg·L⁻¹ dosage optimally promotes Chl a synthesis, whereas the 200 mg·L⁻¹ concentration exerts a more pronounced stimulatory effect on Chl b (p < 0.05).

The effects of ZnO NPs on the antioxidant enzyme activities of sorghum seedlings under saline-alkali stress are illustrated in Fig. 3. Overall, ZnO NPs application significantly modulated these enzyme activities across varying stress levels. In the unstressed control, the 100 mg·L⁻¹ ZnO NPs treatment induced a 55.3% increase in CAT activity (Fig. 3A). Notably, this stimulatory effect intensified progressively as the severity of saline-alkali stress escalated. Compared to the respective stress controls, applying 100 mg·L⁻¹ ZnO NPs dramatically enhanced CAT activity by 108.9%, 435.2%, and 449.7% under 50, 100, and 150 mmol·L⁻¹ stress conditions, respectively. Similarly, ZnO NPs significantly upregulated APX activity (Fig. 3B), with the most pronounced effects observed under moderate to severe stress. Specifically, the 100 mg·L⁻¹ ZnO NPs dosage boosted APX activity by 205% and 283.1% under 100 and 150 mmol·L⁻¹ saline-alkali conditions, respectively (p < 0.05).

POD and SOD activities corroborated the protective role of ZnO NPs. Under 50 mmol·L⁻¹ saline-alkali stress, the various ZnO NPs treatments increased POD activity by 40.3%, 81.2%, and 37.1%, respectively (Fig. 3C). This upregulation was exceptionally pronounced under severe stress (150 mmol·L⁻¹), where the corresponding ZnO NPs treatments triggered striking surges in POD activity of 354.7%, 775.1%, and 522.6% (p < 0.05). Similarly, SOD activity was significantly enhanced (Fig. 3D). Under 50 mmol·L⁻¹ stress, applications of 50 and 100 mg·L⁻¹ ZnO NPs elevated SOD activity by 36.5% and 96.6%, respectively. As the stress intensified to 100 and 150 mmol·L⁻¹, the 100 mg·L⁻¹ ZnO NPs dosage continued to exhibit the most potent stimulatory effect, boosting SOD activity, by 198.5% and 411.8%, respectively (p < 0.05). Collectively, these physiological characteristics reveal that while ZnO NPs exert no significant influence on antioxidant enzymes under non-stressed conditions, they initiate a robust, concentration-dependent defense mechanism under saline-alkali stress. Across all evaluated stress levels, the 100 mg·L⁻¹ ZnO NPs dosage consistently demonstrated optimal performance in maximizing antioxidant enzyme activities.

The impact of ZnO NPs on the non-enzymatic antioxidant system in saline-alkali stressed sorghum seedlings is presented in Fig. 4. Mirroring the enzymatic responses, GSH content exhibited a distinct dose-dependent, biphasic trend, initially rising and subsequently declining as ZnO NPs concentrations increased (Fig. 4A). Under unstressed control conditions, the 100 mg·L⁻¹ ZnO NPs treatment moderately increased GSH content by 24.6%. However, under 50 mmol·L⁻¹ saline-alkali stress, this same optimal dosage (100 mg·L⁻¹) significantly boosted GSH levels by 141.3%, while the 50 and 200 mg·L⁻¹ treatments also induced significant, albeit smaller, increases (p < 0.05). As the saline-alkali stress further intensified, the upregulatory efficacy of the 100 mg·L⁻¹ treatment became increasingly pronounced, culminating in a striking 446.5% elevation in GSH content under the severe150 mmol·L⁻¹ stress condition.

Regarding AsA content (Fig. 4B), the 200 mg·L⁻¹ ZnO NPs treatment induced a 33.8% reduction in the unstressed control group. Conversely, under 50 mmol·L⁻¹ saline-alkali stress, the application of 100 mg·L⁻¹ ZnO NPs significantly elevated AsA content by 136.9% (p < 0.05). Under more severe saline-alkali stress conditions, all tested ZnO NPs concentrations significantly enhanced AsA accumulation, with the 100 mg·L⁻¹ dosage consistently yielding the optimal response. In summary, applying an optimal dosage of ZnO NPs (100 mg·L⁻¹) ZnO NPs effectively upregulates the synthesis of critical non-enzymatic antioxidants (both GSH and ASA) to combat saline-alkali stress, whereas excessive concentrations may exert detrimental physiological effects (p < 0.05).

The effects of ZnO NPs on the Na⁺/K⁺ ratio and MDA content in sorghum seedlings under saline-alkali stress are presented in Fig. 5. In the non-stressed control group, ZnO NPs treatments did not significantly alter the Na⁺/K⁺ ratio (Fig. 5A), which remained stable at approximately 1 across all applied concentrations. However, as the severity of saline-alkali stress intensified, notable changes were observed, with ZnO NPs applications leading to a profound and significant reduction in the Na⁺/K⁺ ratio (p < 0.05). Specifically, under 50 mmol·L⁻¹ saline-alkali stress, the application of 100 mg·L⁻¹ ZnO NPs resulted in a striking 96.9% decrease in this ratio. This potent mitigating effect persisted at higher stress levels, where the same 100 mg·L⁻¹ dosage reduced the Na⁺/K⁺ ratio by 90.4% and 97.1% under 100 and 150 mmol·L⁻¹ saline-alkali stress, respectively.

MDA serves as a critical marker for lipid peroxidation, reflecting the extent of cell membrane damage. Under non-stressed control conditions, the highest concentration of ZnO NPs (200 mg·L⁻¹) induced a significant 74.7% increase in MDA content (Fig. 5B). Conversely, under 50 mmol·L⁻¹ saline-alkali stress, the 100 mg·L⁻¹ ZnO NPs application significantly decreased MDA levels by 46.3%, whereas the 200 mg·L⁻¹ treatment caused a slight, yet significant, accumulation of MDA (p < 0.05). As the severity of saline-alkali stress escalated to 100 and 150 mmol·L⁻¹, treatments with 50 and 100 mg·L⁻¹ ZnO NPs markedly alleviated MDA accumulation. Most notably, under the severe 150 mmol·L⁻¹ stress condition, the 100 mg·L⁻¹ dosage achieved a striking 85.2% reduction in MDA levels (p < 0.05). Collectively, these findings elucidate the physiological mechanisms by which appropriate ZnO NPs application (optimally at 100 mg·L⁻¹) mitigates saline-alkali-induced oxidative damage in sorghum seedlings, while demonstrating that excessive concentrations can act as a pro-oxidant, exacerbating membrane injury.

The Pearson correlation heatmap (Fig. 6) reveals significant physiological interrelationships in ZnO NPs-treated sorghum seedlings under saline-alkali stress. A pronounced synergistic network was identified among growth traits, photosynthetic pigments, and antioxidants. Growth indicators (AL, AFW, ADW) were strongly positively intercorrelated (r ≥ 0.91, p ≤ 0.001). Moreover, robust links emerged between photosynthetic pigments and antioxidant enzymes, notably Chl a with CAT/POD (r = 0.84/0.87) and Chl b with APX (r = 0.85). The antioxidant system displayed highly synchronized directional changes (p ≤ 0.001), evidenced by strong correlations among enzymes (e.g., CAT vs. POD, r = 0.92) and between enzymatic and non-enzymatic components (e.g., POD vs. GSH, r = 0.93; SOD vs. AsA, r = 0.83).

In contrast, stress markers displayed clear inverse relationships with plant health indicators. MDA negatively correlated with growth traits (AL, AFW, ADW; r = −0.65 to −0.71, p ≤ 0.01) and antioxidants (GSH, AsA; r = −0.50, −0.62, p ≤ 0.05). Concurrently, the Na⁺/K⁺ ratio exhibited negative correlations with AL, SOD, GSH, and AsA (p ≤ 0.05). Importantly, the Na⁺/K⁺ ratio correlated positively and strongly with MDA (r = 0.73, p ≤ 0.01). Together, these results demonstrate that elevated oxidative stress and disrupted ion homeostasis are consistently associated with diminished seedling growth and impaired antioxidant defenses.

To comprehensively evaluate the physiological response patterns of sorghum seedlings to saline-alkali stress under varying ZnO NPs treatments, a principal component analysis (PCA) was performed on the 13 evaluated physiological indicators (Table 1). Two principal components with eigenvalues greater than 1 were extracted (12.6933 and 4.0538, respectively). The first (PC1) and second (PC2) principal components accounted for 60.4% and 19.3% of the total variance, respectively. Together, they yielded a cumulative variance contribution of 79.7%, effectively capturing the majority of the dataset’s variability and satisfying the analytical requirements for dimensionality reduction. An analysis of the variable loadings revealed that PC1 was primarily driven by Chlb, APX, SOD, GSH, AsA, and the Na⁺/K⁺ ratio. Conversely, PC2 was largely dominated by the growth indices (AL, AFW and ADW), along with Chla, CAT, POD, and MDA.

Subsequently, a comprehensive evaluation score (F) was calculated for each treatment by weighing the principal component score (F₁ and F₂) against their respective variance contribution (F = F₁ × 60.4% + F₂ × 19.3%). All treatments were then ranked according to their resulting F values (Table 2). The analysis revealed that ZnO NPs applications generally improved the overall rankings of sorghum seedlings under saline-alkali stress. Most notably, the top three positions were exclusively occupied by the 100 mg·L⁻¹ ZnO NPs treatments. This quantitative assessment not only substantiates the positive regulatory role of ZnO NPs in promoting seedling growth but also confirms the 100 mg·L⁻¹ dosage as the optimal concentration for maximizing multidimensional physiological resilience. Furthermore, these comprehensive rankings are in perfect alignment with the empirical observations of seedling growth and physiological responses detailed above.

To comprehensively evaluate the coupling effects between ZnO NPs and saline-alkali stress, a RSM was applied to the 13 evaluated growth and physiological indices (Table 3). An initial two-way ANOVA (Table 4) revealed that the individual main effects of ZnO NPs and saline-alkali stress, as well as their interaction, exerted a highly significant influence on all tested parameters (p < 0.001). Three-dimensional response surface plots illustrating these interactive effects on AL, APX, MDA, and the Na⁺/K⁺ ratio are presented in Fig. 7. Based on the model optimization, the beneficial indicators AL and APX achieved their theoretical maximum at the coordinate points (ZnO NPs concentration, saline-alkali concentration) of (111.33, 77.84) and (105.04, 95.56), respectively. Conversely, the stress markers MDA and the Na⁺/K⁺ ratio were minimized at the coordinates (89.65, 50.32) and (98.16, 64.87), respectively.

Finally, by integrating these predictive models to map the combined distribution surface, an optimal overlapping parameter region was identified (Fig. 8). This rectangular region is bounded by a ZnO NPs range of 89.65–118.53 mg·L⁻¹ and a saline-alkali stress range of 50.04–96.56 mmol·L⁻¹. Consequently, the optimal application range of ZnO NPs for mitigating saline-alkali stress was mathematically pinpointed to 89.65–118.53 mg·L⁻¹. Notably, this theoretically derived optimal range perfectly aligns with the empirical phenotypic and physiological observations detailed previously.