Salicylic acid priming enhances seed germination in Apocynum venetum under salinity conditions

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Ahiakpa, Ge Pan, Zhenzhong Wu, Lu Bai, Caixia Zheng, Haiqiang Dong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8982686/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Salinity stress significantly impairs seed germination and seedling establishment in many economically valuable plant species. Salicylic acid (SA) is a key signaling molecule implicated in plant stress responses. Despite this, a significant knowledge gap remains regarding its concentration-dependent effects on Chinese hemp ( A. venetum ), a species recognized for its considerable ecological and pharmaceutical importance. In this study, we investigated the effects of seeds priming with SA on seed germination, osmolytes (soluble sugar and protein contents), antioxidant enzyme activities, and lipid peroxidation (malondialdehyde content). Seed priming with 0.3 mM SA alleviated the adverse effects under salt stress on seed germination and seedling growth. Seeds of A. venetum primed with 0.3 mM SA exhibited improved germination count, radicle length, embryo growth, germination potential, chlorophyll content, soluble sugar and protein contents, enhanced the activities of superoxide dismutase, peroxidase, and catalase, and decreased malonaldehyde content in the seeds. These results suggest that seed priming with SA enhances the accumulation of key osmolytes and photosynthetic pigments, and boosts antioxidant enzyme activities, thereby alleviating the adverse effects of salt stress on seed germination and seedling growth in A. venetum . Apocynum venetum salt stress seed priming salicylic acid seed germination antioxidant enzymes osmolytes Figures Figure 1 Figure 2 Figure 3 1.0 Introduction Soil salinization is the process by which salts are transported by capillary action to the soil surface and accumulate as water evaporates. It is a widespread form of land degradation worldwide, especially severe in arid and semi-arid regions [ 1 – 3 ]. Salt stress interferes with plant uptake of water and nutrients as increasing the osmotic pressure of soil solution, making water absorption difficult, rendering Na + ions to compete with K + and Ca 2+ at uptake sites, reducing plant nutrient absorption [ 4 – 7 ]. Such stress negatively affects plant growth and development, but exogenous application of salicylic acid (SA) has been shown to reduce salt-induced damage in various species [ 8 , 9 ]. For example, under salinity, SA improved the osmotic regulation, water uptake, and germination of Zinnia elegans seeds [ 4 ]. Spraying 0.5–1.5 mM SA significantly promoted the growth of cauliflower under NaCl stress, with 0.1 mM being most effective [ 10 ]. Similarly, treating patchouli seeds and seedlings with 0.25 mM SA alleviated salt injury and enhanced germination and root growth, whereas 0.5 mM SA exacerbated salt damage [ 11 – 14 ]. These observations suggest that SA effects on plant growth are concentration-dependent, and the optimal SA concentration for alleviating salt stress varies by species. Exogenous SA can also modulate osmotic balance under salinity. It increases levels of proline, soluble protein (SP), and soluble sugars (SS) in goji ( Lycium barbarum ), enhancing cellular water uptake and relieving salt injury [ 2 , 15 ]. SA may promote the conversion of in SP and carbohydrates into SP and SS [ 16 ], thereby increasing the osmotic potential difference and facilitating water uptake [ 9 , 17 ]. In cauliflower under salt stress, SA treatment boosted α-amylase activity and raised SS and proline levels [ 18 ], improving osmotic adjustment. Furthermore, SA plays a key role in regulating antioxidant defenses under salinity. It can upregulate both enzymatic and non-enzymatic antioxidants to scavenge reactive oxygen species (ROS) and reduce membrane peroxidation [ 12 , 18 ]. For instance, when plants are salt-stressed, high Malondialdehyde (MDA) levels reflect oxidative damage; exogenous SA increased Superoxide Dismutase (SOD), Peroxidase (POD), and Catalase (CAT)activities in salt-stressed cucumber, balancing ROS and lowering MDA and electrolyte leakage [ 10 ]. Chinese hemp ( A. venetum ) is a highly salt-tolerant pioneer plant. Its widespread cultivation can effectively improve saline soils [ 19 , 20 ]. Studying its salt-tolerance mechanisms is thus valuable for developing strategies to mitigate salinity. In this study, we applied 300 mM NaCl and varying concentrations of SA to A. venetum seeds to evaluate how exogenous SA affects seed germination and physiological traits under salt stress. Our objective was to elucidate the regulatory effects of SA on A. venetum during germination under salinity, thereby providing theoretical support for producing high-quality, salt-tolerant A. venetum seedlings in saline-alkali lands. 2.0 Materials and Methods 2.1 Plant material and experimental design Apocynum venetum Zhengjun. seeds were sourced from Ningmiao Group Co. (Ningxia, China). The A. venetum Zhengjun. seeds used in the experiment were generously provided by Ningxia Ningmiao Ecological Construction Group Co., Ltd. This batch of germplasm material originated from artificial multi-generational propagation and is not the wild type. It is currently stored under refrigeration at 4°C for research purposes. Seeds of uniform size, fullness, and free of pests or disease were selected for the germination experiments. Based on preliminary trials, 300 mM NaCl was chosen to impose salt stress. Fourteen treatments comprising control with distilled water (CK); distilled water + SA at 0.1, 0.2, 0.3, 0.4, 0.5, and 0.75 mM; 300 mM NaCl alone; 300 mM NaCl + SA at 0.1, 0.2, 0.3, 0.4, 0.5, and 0.75 mM were used. Each treatment had three replicates. After surface sterilization with 75% ethanol and rinsing, seeds were placed evenly in Petri dishes (50 seeds per dish) on moistened filter paper. For treatments under salt stress, 3 mL of the 300 mM NaCl solution and 4 mL of the assigned SA solution were added to each dish to saturate the paper. Control dishes received equivalent volumes of distilled water and SA solution as appropriate. Evaporated water was replenished daily. Dishes were incubated at a constant temperature of 25 ± 0.5°C under a 16-h light/8-h dark photoperiod. Germination was recorded when the radicle emerged from the seed coat. Germinated seeds were counted daily for 10 days with 10 randomly selected seedlings per treatment measured for radicle and plumule length. 2.2 Growth and physiological measurements Germination count was calculated as the percentage of seeds germinated by day 10. Radicle and plumule lengths were measured with vernier calipers. Leaf chlorophyll content, soluble sugar, and soluble protein were determined using standard methods [ 21 ]. Antioxidant enzyme activities and lipid peroxidation were assayed. SOD activity was measured by the nitroblue tetrazolium photochemical reduction method [ 22 ]; POD by the guaiacol oxidation method [ 23 ]; CAT activity by [ 24 ]; and MDA content by the thiobarbituric acid (TBA) colorimetric method [ 25 ]. 2.3 Data analyses Data was cleaned and organized for statistical analyses and visualization using GraphPad Prism (ver. 10, Massachusetts, USA). One-way analysis of variance (ANOVA) was performed to test for treatment effects ( p < 0.05); while correlation and principal components analysis were performed using the R. A membership function method was used to evaluate the overall effectiveness of SA in relieving salt stress on seed germination, with higher function values indicating greater mitigation. For a positive indicator, SFV = ( X – X min )/( X max – X min ); for a negative indicator, SFV = 1 – ( X – X min )/( X max – X min ), where SFV stands for the membership function value, X is the observed value, and X min and X max are the minimum and maximum values across treatments. 3.0 Results 3.1 Effect of SA priming on seed germination and seedling growth Our analysis shows that NaCl stress (300 mM) significantly suppressed seed germination of A. venetum compared to the control (Fig. 1 ). Germination under salt stress alone was markedly reduced, while seeds under distilled water with SA (water + SA) showed normal germination comparable to control. However, when SA was applied together with salt stress (NaCl + SA), germination improved significantly relative to NaCl alone. The 0.3 mM SA treatment under salt stress yielded the highest germination and the longest radicle and hypocotyl lengths (Fig. 1 ). At higher SA concentrations beyond 0.3 mM, the decreasing effect gradually deteriorated. These results indicate that exogenous SA can effectively counteract the inhibitory effect of salt on A. venetum seed germination at 0.3 mM optimal concentration under salinity. 3.2 Effect of SA priming on seedling morphology under salinity stress Phenotypic profiles of seedlings confirmed seedlings grown in distilled water (control) were the tallest and healthiest (Fig. 2 ). Under 300 mM NaCl, seedlings were severely stunted. Application of SA under salt stress improved seedling length and vigor at low SA concentrations, with 0.3 mM SA showing the healthiest growth. However, at higher SA levels, the beneficial effect diminished. SA + water (no salt) had no visible impact on seedling appearance compared to the control. These morphological results corroborate the quantitative data where low-dose SA mitigates salt-induced growth inhibition, whereas excessive SA offers no additional benefit. 3.3 Effect of SA priming on key agronomic traits of A. venetum seedlings under salinity stress Salt stress (NaCl alone) significantly reduced germination count, hypocotyl length, radicle length, and embryo growth compared to control ( p < 0.05; Table 1 ). Application of SA under salt stress alleviated these inhibitory effects, with the most pronounced recovery observed at 0.3 mM SA. At this concentration, germination count increased from 14.3% (NaCl alone) to 27.7%, radicle length from 0.73 cm to 1.27 cm, and embryo growth from 0.35 cm to 0.89 cm ( p < 0.05). Higher SA concentrations (≥ 0.5 mM) resulted in a non-significant decline in these parameters, although values remained significantly above the NaCl‑only treatment. Under control conditions (water), SA application did not significantly affect any of the germination parameters. Two‑way ANOVA confirmed significant main effects of water regime (stress vs. non‑stress) and SA concentration, as well as their interaction, for all measured traits ( p < 0.001). Post‑hoc comparisons using Tukey’s HSD test ( p < 0.05) revealed that 0.3 mM SA was the optimal concentration for mitigating salt stress across most parameters, with higher concentrations providing diminishing or no additional benefit. Table 1 Effect of exogenous SA and hydro-priming on biochemical properties of A. venetum seedlings under salinity stress Treatment Germination count Hypocotyl length (mm) Radicle length (cm) Embryo growth (cm) Water 31.00 ± 1.15 f 0.62 ± 0.02 g 1.85 ± 0.05 g 1.47 ± 0.05 h NaCl 14.33 ± 0.88 a 0.29 ± 0.02 a 0.73 ± 0.05 a 0.35 ± 0.05 a Water + 0.1 SA 29.67 ± 1.20 ef 0.59 ± 0.02 fg 1.59 ± 0.11 f 1.21 ± 0.11 g NaCl + 0.1 SA 22.67 ± 0.88 bc 0.45 ± 0.02 cd 0.96 ± 0.05 bc 0.58 ± 0.05 bc Water + 0.2 SA 29.67 ± 0.67 ef 0.59 ± 0.01 fg 1.47 ± 0.06 ef 1.09 ± 0.06 fg NaCl + 0.2 SA 24.33 ± 0.88 cd 0.49 ± 0.02 de 1.08 ± 0.05 cd 0.70 ± 0.05 cd Water + 0.3 SA 29.67 ± 1.20 ef 0.59 ± 0.02 fg 1.35 ± 0.04 de 0.97 ± 0.04 ef NaCl + 0.3 SA 27.67 ± 0.88 de 0.55 ± 0.02 ef 1.27 ± 0.05 d 0.89 ± 0.05 de Water + 0.4 SA 29.33 ± 0.88 ef 0.59 ± 0.02 fg 1.29 ± 0.05 d 0.91 ± 0.05 de NaCl + 0.4 SA 28.33 ± 0.88 de 0.57 ± 0.02 f 1.13 ± 0.05 cd 0.75 ± 0.05 cd Water + 0.5 SA 29.67 ± 1.20 ef 0.59 ± 0.02 fg 1.19 ± 0.03 d 0.81 ± 0.03 de NaCl + 0.5 SA 26.67 ± 0.88 d 0.53 ± 0.02 ef 0.95 ± 0.03 bc 0.57 ± 0.03 bc Water + 0.75 SA 29.33 ± 0.88 ef 0.59 ± 0.02 fg 1.04 ± 0.04 c 0.66 ± 0.04 c NaCl + 0.75 SA 22.33 ± 0.88 b 0.45 ± 0.02 c 0.74 ± 0.04 a 0.36 ± 0.04 a Values are means of three replicates ± standard error of the mean (SEM). Within each column, means followed by the same letter are not significantly different according to Tukey’s HSD test ( p < 0.05). The analysis was performed using R (version 4.2.1) with the agricolae package. The two‑way ANOVA confirmed significant main effects of water and SA concentration, as well as their interaction, for all traits ( p < 0.001). Post‑hoc comparisons were conducted on the 14 treatment combinations. 3.4 Effect of SA-priming on biochemical properties of seedlings under salinity stress Salt stress triggered significant physiological and biochemical responses in A. venetum seeds (Table 2 ). Chlorophyll content was markedly reduced under salt stress (0.77 mg/g) compared to control (1.45 mg/g). Chlorophyll content, which was reduced by NaCl stress, was partially restored by SA treatment. SA supplementation under salt stress significantly increased chlorophyll content, reaching a maximum of 1.03 mg/g at 0.2 mM SA, which did not differ significantly from 0.3 mM SA (1.02 mg/g) (Table 2 ). Compared to the control (water + SA), NaCl treatment markedly increased the accumulation of key osmolytes, including soluble sugars and soluble proteins, as well as the activities of antioxidant enzymes including SOD, POD, and CAT (Table 2 ). Concurrently, MDA content, a marker of lipid peroxidation, increased sharply under NaCl alone (66.6 nmol/g), indicating severe oxidative membrane damage. Application of SA in the absence of salt (water + SA) did not significantly alter any of these parameters relative to the control, confirming that SA exerts its primary effects under stress conditions. Under salt stress, exogenous SA further increased soluble sugars, and soluble protein contents compared to salt alone (Table 2 ). These increases were concentration‑dependent, peaking at 0.3 mM SA (soluble sugars: 32.8 mg/g; soluble proteins: 34.8 mg/g) and declined at higher concentrations. At the optimal concentration, soluble protein and sugar contents were 63% and 95% higher, respectively, than in the NaCl‑only treatment ( p < 0.05). This suggests that exogenous SA enhances osmotic adjustment by boosting osmoprotectant accumulation and sustaining chlorophyll integrity under salt stress, with 0.3 mM SA being the most effective concentration for osmotic regulation. In parallel, SA applications under saline conditions markedly enhanced antioxidant enzyme activities while reducing MDA content relative to salt alone (Table 2 ). The highest SOD (117.2 U/g), POD (118.5 U/g), and CAT (202.9 U/g) activities, and the lowest MDA level (53.7 nmol/g), were consistently observed at 0.3 mM SA, representing a 19.4% reduction in lipid peroxidation compared to NaCl alone (Table 2 ). At this concentration, SOD and POD activities were approximately 20% and 62% higher, respectively, than under NaCl‑only treatment ( p < 0.05). Beyond 0.3 mM SA, antioxidant enzyme activities gradually declined, although they remained significantly above the NaCl‑only level at 0.4 and 0.5 mM SA. MDA content, however, increased again at higher SA concentrations, approaching the NaCl‑only level at 0.75 mM SA (Table 2 ). These findings demonstrate that exogenous SA significantly enhances the antioxidant defense system in A. venetum seeds under salt stress, mitigating oxidative damage and reinforcing membrane stability. The results align with previous reports that SA can activate antioxidant enzymes to scavenge reactive oxygen species (ROS) and protect cellular membranes under salinity [ 21 ]. Table 2 Effect of exogenous SA-priming on biochemical properties of A. venetum seedlings under salinity stress Treatment Chlorophyll (mg/g) Soluble sugars (mg/g) Soluble protein (mg/g) SOD (U/g) POD (U/g) CAT (U/g) MDA (nmol/g) Water 1.45 ± 0.05 d 8.06 ± 0.13 a 12.49 ± 0.13 a 74.16 ± 0.68 a 30.63 ± 0.68 a 110.02 ± 0.68 a 12.94 ± 0.56 a NaCl 0.77 ± 0.05 a 16.87 ± 0.32 b 21.30 ± 0.32 b 97.08 ± 1.00 b 72.93 ± 0.68 b 142.54 ± 1.00 b 66.61 ± 0.32 g Water + 0.1 SA 1.42 ± 0.05 cd 7.79 ± 0.44 a 12.22 ± 0.44 a 73.81 ± 0.83 a 30.39 ± 0.83 a 112.02 ± 0.83 a 12.46 ± 0.44 a NaCl + 0.1 SA 0.97 ± 0.03 b 28.22 ± 1.01 d 30.15 ± 1.01 d 112.58 ± 1.01 cd 113.84 ± 1.01 de 178.04 ± 5.36 d 59.45 ± 0.60 f Water + 0.2 SA 1.42 ± 0.05 cd 8.11 ± 0.39 a 12.21 ± 0.13 a 73.49 ± 0.66 a 30.07 ± 0.66 a 111.70 ± 0.66 a 12.35 ± 0.64 a NaCl + 0.2 SA 1.03 ± 0.05 b 29.75 ± 0.74 e 31.68 ± 0.74 e 114.11 ± 0.74 d 115.37 ± 0.74 e 199.80 ± 0.74 e 58.91 ± 1.58 f Water + 0.3 SA 1.41 ± 0.05 cd 7.91 ± 0.24 a 11.57 ± 0.47 a 73.82 ± 0.86 a 30.40 ± 0.86 a 112.03 ± 0.86 a 12.91 ± 0.12 a NaCl + 0.3 SA 1.02 ± 0.05 b 32.84 ± 1.55 f 34.77 ± 1.55 f 117.20 ± 1.55 e 118.46 ± 1.55 f 202.89 ± 1.55 f 53.67 ± 0.56 e Water + 0.4 SA 1.40 ± 0.05 cd 8.46 ± 0.10 a 11.89 ± 0.67 a 74.16 ± 0.65 a 30.74 ± 0.65 a 112.37 ± 0.65 a 12.80 ± 0.24 a NaCl + 0.4 SA 0.98 ± 0.03 b 30.88 ± 1.01 e 32.81 ± 1.01 e 115.24 ± 1.01 de 116.50 ± 1.01 ef 193.93 ± 2.39 e 58.05 ± 0.73 f Water + 0.5 SA 1.39 ± 0.04 cd 8.13 ± 0.29 a 10.89 ± 0.55 a 73.82 ± 1.01 a 30.40 ± 1.01 a 112.03 ± 1.01 a 12.70 ± 0.24 a NaCl + 0.5 SA 0.96 ± 0.02 b 29.11 ± 1.14 de 31.04 ± 1.14 de 113.47 ± 1.14 cd 114.73 ± 1.14 de 169.16 ± 5.53 c 59.44 ± 1.01 f Water + 0.75 SA 1.37 ± 0.04 c 8.92 ± 0.12 a 10.68 ± 0.77 a 73.79 ± 1.04 a 30.37 ± 1.04 a 112.00 ± 1.04 a 12.26 ± 0.35 a NaCl + 0.75 SA 0.95 ± 0.05 b 28.27 ± 0.99 d 30.20 ± 0.99 d 112.63 ± 0.99 c 113.89 ± 0.99 d 166.99 ± 1.29 c 62.08 ± 0.74 g Values are means of three replicates ± standard error of the mean (SEM). Within each column, means followed by the same letter are not significantly different according to Tukey’s HSD test ( p < 0.05). The analysis was performed using R (version 4.2.1) with the agricolae package. The two‑way ANOVA confirmed significant main effects of water and SA concentration, as well as their interaction, for all traits ( p < 0.001). Post‑hoc comparisons were conducted on the 14 treatment combinations. 3.5 Multivariate analysis of trait relationships and treatment effects To explore the interrelationships among germination parameters, biochemical markers, and oxidative stress indicators, Pearson correlation analysis was performed on all measured traits across the 14 treatment combinations (Fig. 3 ). Germination count, radicle length, embryo growth, and chlorophyll content were strongly and positively correlated with one another ( r > 0.77, p < 0.01), indicating that these traits respond coordinately to the experimental treatments. In contrast, these germination‑related parameters exhibited significant negative correlations with antioxidant enzyme activities (SOD, POD, CAT) and MDA content, with correlation coefficients ranging from − 0.60 to -0.98 ( p < 0.05 or p < 0.01). This inverse relationship demonstrates that enhanced oxidative stress, reflected by elevated enzyme activities and lipid peroxidation, is associated with reduced germination performance under saline conditions. Particularly, soluble sugar and protein contents were strongly positively correlated with antioxidant enzymes and MDA ( r > 0.90 , p < 0.01), suggesting that osmolyte accumulation and oxidative stress responses are co‑induced under salt stress. Principal component analysis (PCA) was conducted to visualize the overall patterns of treatment effects and to identify the primary sources of variation in the dataset (Fig. 4). The first two principal components account for 86.0% of the total variance, with PC1 accounting for 72.0% and PC2 for 14.0% (Fig. 2 a, scree plot). The PCA scores plot (Fig. 2 b) revealed a clear separation between water‑treated (non‑stress) and NaCl‑treated (salt stress) samples along the PC1 axis. Water‑treated samples formed a tight cluster on the negative side of PC1, while NaCl‑treated samples were distributed across the positive side, with their positions along PC2 influenced by the concentration of applied SA. The loading vectors (Table 2 ) provided insight into the variables driving these separations. PC1 was strongly positively loaded by stress‑related biochemical markers including soluble protein8 (0.18), SS (0.15), MDA (0.12), and the antioxidant enzymes; CAT (0.08), POD (0.07), and SOD (0.06) and strongly negatively loaded by germination parameters, including germination count (-0.25), radicle length (-0.22), embryo growth (-0.20), and chlorophyll contemt (-0.25). This confirms that PC1 represents a stress‑to‑growth gradient, distinguishing treatments based on the trade‑off between oxidative stress responses and germination performance. PC2, which accounted for 14.0% of the variance, was primarily associated with finer differentiation among NaCl‑treated samples, likely reflecting the modulating effects of SA concentration on the stress response. Variables contributing moderately to PC2 included SP (0.25), SS (0.20), radicle length (0.15), and CC (0.10). Collectively, the correlation and PCA analyses demonstrate that salt stress induces a coordinated shift in biochemical and physiological traits, characterized by increased antioxidant enzyme activities and osmolyte accumulation concomitant with reduced germination and CC. Exogenous SA modulates this response in a concentration‑dependent manner, with intermediate concentrations (particularly 0.3 mM) partially restoring germination traits while maintaining enhanced antioxidant capacity. These findings corroborate the earlier results and highlight the central role of oxidative stress in mediating the inhibitory effects of salinity on A. venetum seed germination. A membership function analysis was conducted to quantitatively evaluate the overall stress mitigation effects of the different treatments (Table 3 ). Among all treatments, the non‑stressed control (distilled water only) recorded the highest membership value (0.992), categorized as optimal, no stress. Under saline conditions, seeds treated with 300 mM NaCl combined with 0.3 mM SA achieved the highest membership value (0.213), classified as optimal under salt stress. In contrast, seeds subjected to 300 mM NaCl alone exhibited the lowest membership value (0.106), ranked as lowest. All treatments involving distilled water supplemented with SA (0.1–0.75 mM) maintained high membership values (≥ 0.885) and were ranked as high or moderate. These findings reveal that the application of 0.3 mM SA provides the most effective alleviation of salt stress during A. venetum seed germination, confirming its role as the optimal concentration for mitigating salinity‑induced inhibitory effects. Table 3 Membership function analysis of germination traits under salt stress relieved by exogenous SA. Rank Treatment SFV Ranking 1 Water 0.992 Optimal (no stress) 2 Water + 0.1 mM SA 0.952 High 3 Water + 0.2 mM SA 0.932 High 4 Water + 0.4 mM SA 0.919 High 5 Water + 0.3 mM SA 0.915 High 6 Water + 0.5 mM SA 0.906 High 7 Water + 0.75 mM SA 0.885 Moderate 8 300 mM NaCl + 0.3 mM SA 0.213 Optimal (salt stress) 9 300 mM NaCl + 0.4 mM SA 0.181 Moderate 10 300 mM NaCl + 0.2 mM SA 0.163 Moderate 11 300 mM NaCl + 0.5 mM SA 0.161 Moderate 12 300 mM NaCl + 0.1 mM SA 0.138 Low 13 300 mM NaCl + 0.75 mM SA 0.124 Low 14 300 mM NaCl 0.106 Lowest (pure salt stress) Where SFV refers to subordinate function value; used to rank the overall stress mitigation effect of different SA concentrations. By assigning a single value to each treatment, we were able to quantitatively conclude that 0.3 mM SA resulted in the highest SFV (0.213) under salt stress, confirming it as the optimal concentration for alleviating salinity damage. 4.0 Discussion Salinity stress represents one of the most significant abiotic challenges limiting crop productivity worldwide, particularly affecting seed germination and early seedling establishment [ 26 ]. The present study demonstrates that exogenous application of (SA) effectively alleviates the inhibitory effects of salt stress on A. venetum seed germination and seedling growth, with 0.3 mM SA identified as the optimal concentration for stress mitigation [ 27 , 28 ]. 4.1 Salicylic acid enhances antioxidant profiles under salt stress High concentrations of soluble salts can severely damage plant cell membranes through increased permeability and lipid peroxidation, while concurrently impairing antioxidant defense mechanisms [ 29 , 30 ]. In the present study, exposure to 300 mM NaCl significantly elevated MDA content, a reliable biomarker of lipid peroxidation; while reducing the activities of SOD,POD, and CAT in A. venetum seedlings, indicating substantial oxidative damage. These observations align with previous reports demonstrating that salinity stress disrupts cellular redox homeostasis through excessive accumulation of reactive oxygen species [ 31 , 32 ]. Exogenous SA treatment markedly enhanced antioxidant enzyme activities and reduced membrane damage, with 0.3 mM SA registering the highest ameliorative effect. Under this optimal concentration, SOD and POD activities increased by approximately 20% and 62%, respectively, compared to NaCl treatment alone, while MDA content decreased by 19.4%. These findings are consistent with studies on sorghum, where SA application (0.1–0.5 mM) increased antioxidant enzyme expression and reduced MDA by up to 27% under salt stress [ 29 , 33 , 34 ]. Similarly, recent studies on soybeans demonstrated that SA supplementation enhanced SOD, CAT, and ascorbate peroxidase activities while reducing oxidative damage and improving membrane stability [ 27 , 35 ]. The concentration-dependent response observed in our study, with optimal effects at 0.3 mM SA and diminished benefits at higher concentrations, corroborates the established paradigm that SA exhibits biphasic effects depending on dosage, plant species, and stress intensity [ 26 ]. The mechanistic basis for SA-mediated antioxidant enhancement involves both direct activation of antioxidant enzymes and transcriptional regulation of stress-responsive genes. Recent transcriptomic analysis in Davidia involucrata revealed that exogenous SA induced differential expression of 2,581 genes under salt stress, including upregulation of transcription factors ( DiWRKY40 , DiNAC25 , DiMYB4 , and DiMYB86 ) that regulate antioxidant defense pathways [ 5 , 30 ]. These molecular adjustments potentially contributed to the enhanced SOD, POD, and CAT activities observed in our study, collectively reducing ROS accumulation and preserving membrane integrity. 4.2 Salicylic acid promotes osmotic adjustment and chlorophyll preservation Osmotic adjustment through the accumulation of compatible solutes represents a crucial adaptive mechanism for maintaining cellular turgor and protecting macromolecular structures under salt stress [ 5 , 28 , 36 , 37 ]. In the present study, NaCl stress significantly reduced SP, soluble sugar, and CC in A. venetum seedlings. Exogenous SA application reversed these declines, with peak osmolyte accumulation and CC recorded at 0.3 mM SA. At this optimal concentration, SP and sugar contents were 63 and 95 % higher, respctively, than in NaCl-only treated seedlings. These findings align with studies on faba bean, where SA application under salinity enhanced SP and sugar accumulation while improving photosynthetic capacity and PSII light utilization efficiency [ 38 , 39 ]. The accumulation of SP under SA treatment potentially reflects stress-induced synthesis of osmotin-like proteins and dehydrins that enhance water retention, while elevated SS contribute to cellular osmotic adjustment and carbon metabolism under stress [ 27 ]. Recent studies on white lupine further demonstrated that SA and SA-nanoparticles increased total SS, proline, and SP under saline conditions, with concomitant improvements in photosynthetic pigments [ 28 ]. The preservation of CC observed in our study is particularly significant, as chlorophyll degradation under salinity directly impairs photosynthetic capacity and carbon assimilation. Similar chlorophyll maintenance effects have been reported in SA-treated wheat [ 40 ], and spinach[ 32 ] under abiotic stress. The restoration of and osmolyte accumulation at 0.3 mM SA suggests that exogenous SA enhances the capacity of A. venetum for osmotic adjustment while protecting photosynthetic apparatus from salt-induced damage. These effects are likely mediated through SA-induced expression of genes involved in osmolyte biosynthesis and chlorophyll metabolism, as reported in recent transcriptomic studies [ 15 , 33 , 41 , 42 ]. 4.3 Integrated stress mitigation and concentration-dependent responses The comprehensive evaluation using membership function analysis confirmed that 0.3 mM SA represented the optimal concentration for alleviating salt stress, achieving the highest subordinate function value (0.213) among saline treatments. This integrated analysis, which combines multiple physiological indicators into a single metric, provides robust evidence for the superior performance of 0.3 mM SA in mitigating salinity effects. Treatments with distilled water plus SA (0.1–0.75 mM) under non-saline conditions maintained high membership values (≥ 0.885), confirming that SA application does not adversely affect germination under optimal conditions. The concentration-dependent response pattern observed; increasing efficacy from 0.1 to 0.3 mM SA followed by declining benefits at higher concentrations, reflects the dual regulatory role of SA in plant stress responses [ 26 ]. Similar optima have been reported across diverse species, including 1.0 mM SA for wheat germination under salinity [ 40 ], 0.5 mM for sorghum seedlings [ 29 ], and 2.0 mM for Dracocephalum moldavica [ 43 ]. These species-specific optima underline the importance of empirical determination of effective SA concentrations for individual crop species and stress conditions. The integration of antioxidant enhancement and osmotic adjustment under SA treatment appears to operate through coordinated signalling pathways. SA-mediated stress tolerance involves the activation of NPR1 -dependent signalling cascades that regulate both antioxidant gene expression and osmolyte biosynthesis [ 27 , 43 ]. The concurrent improvement in multiple stress-responsive parameters at 0.3 mM SA suggests that this concentration optimally activates these signalling networks without inducing the oxidative stress that can occur at supra-optimal SA levels. 4.4 Implications for A. venetum cultivation in saline environments The demonstrated efficacy of 0.3 mM SA in mitigating salt stress during germination has significant practical implications for A. venetum cultivation in saline-alkali soils. As a species with considerable ecological and economic value, enhancing its salt tolerance during germination phase could facilitate establishment in marginal environments where soil salinity limits agricultural productivity [ 28 , 29 ]. Seed priming with SA represents a cost-effective and technically simple approach that could be readily adopted by resource-limited farmers. The potential for bringing moderate saline soils into cultivation through SA application, as suggested by recent studies [ 29 ], aligns with broader efforts to expand agricultural production on underutilized marginal soils. 5.0 Conclusion Salt stress at 300 mM NaCl significantly inhibited seed germination and seedling growth in A. venetum , reducing antioxidant enzyme activities (SOD, POD, CAT), SP, SS, and CC while markedly elevating MDA levels; indicating severe oxidative and osmotic stress. Exogenous application of (SA) effectively ameliorated these deleterious effects through a dual mechanism involving: (i) enhancement of antioxidant profiles, as evidenced by increased SOD, POD, and CAT activities and reduced lipid peroxidation; and (ii) promotion of osmotic adjustment, reflected in increased SP and sugar accumulation and preserved CC. The 0.3 mM SA treatment consistently demonstrated significant stress mitigation efficacy across all measured parameters, establishing it as the optimal concentration for salinity alleviation in this species. These findings contribute to understanding SA-mediated salt tolerance and provide practical insights for improving A. venetum production in saline environments. Further studies at the molecular level, particularly transcriptomic and proteomic analyses of SA-treated seedlings under salt stress, would elucidate the regulatory networks governing these protective responses and facilitate the development of enhanced stress tolerance strategies for this valuable species. Declarations Ethics approval and consent to participate All the experimental research on plants including the collection of plant materials were performed in accordance with relevant institutional, national, and international guidelines and legislation. The study did not involve any endangered or protected species. Consent for publication All authors have reviewed the final version of the manuscript and consent to its publication. Competing interests The authors declare that they have no conflict of interest. Funding This work was supported by the Regional Fund of the National Natural Science Foundation of China (Grant No. 32460747), the Industry-University-Research Cooperation Project of Yulin Science and Technology Bureau (No. 2024-CXY-094), the Shaanxi Province "University-Enterprise Shared" Talent Project (No. 2024XZGY04), and the High-level Talent Introduction Program of Yulin University (2023GK76). Author Contribution J.K.A: Data curation, Formal analysis, Methodology, Software, Validation, Writing—original draft. G.P: Conceptualization, Data curation, Formal analysis, Methodology, Resources, Software, Supervision, Validation, Writing—original draft. Z.W & L.B: Visualization, Data curation, Methodology. J.K.A & F.K: Validation, Writing—review and editing. H.D: Funding acquisition, Conceptualization, Project administration, Resources, Writing—review and editing. Data Availability No datasets were generated or analysed during the current study. References Zhao N, Zhu H, Zhang H, et al. Hydrogen sulfide mediates k + and Na+ homeostasis in the roots of salt-resistant and salt-sensitive poplar species subjected to NaCl stress. Front Plant Sci. 2018;9:410944. https://doi.org/10.3389/fpls.2018.01366 . Li W, Rao S, Du C, et al. Strategies used by two goji species, Lycium ruthenicum and Lycium barbarum , to defend against salt stress. Sci Hortic. 2022;306:111430. https://doi.org/10.1016/j.scienta.2022.111430 . Rao Y, Peng T, Xue S. Mechanisms of plant saline-alkaline tolerance. J Plant Physiol. 2023;281:153916. https://doi.org/10.1016/j.jplph.2023.153916 . Xu J, Xu Y, Wang Y, et al. Exogenous salicylic acid improves photosynthetic and antioxidant capacities and alleviates adverse effects of cherry rootstocks under salt stress. 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PLoS ONE. 2020;15. https://doi.org/10.1371/journal.pone.0228241 . Hasanuzzaman M, Ahmed N, Saha T, et al. Exogenous salicylic acid and kinetin modulate reactive oxygen species metabolism and glyoxalase system to confer waterlogging stress tolerance in soybean ( Glycine max L). Plant Stress. 2022;3:100057. https://doi.org/10.1016/j.stress.2022.100057 . Ma Y, Chen H, Bai J, et al. Salicylic acid, abscisic acid, and melatonin effects on seed germination, seedling growth, and physiological responses under low-temperature and submergence stress. Cereal Res Commun. 2025;53:2673–92. https://doi.org/10.1007/s42976-025-00669-w . Ben Youssef R, Jelali N, Acosta Motos JR, et al. Salicylic acid seed priming: A key frontier in conferring salt stress tolerance in barley seed germination and seedling growth. Agronomy. 2025;15:154. https://doi.org/10.3390/agronomy15010154 . Ma Y, Wang Z, Zhou B, et al. Salicylic acid improving salinity tolerance by enhancing photosynthetic capacity, osmotic adjustment and maintenance of Na+/K+ homeostasis in faba bean seedlings. Chem Biol Technol Agric 2025. 2025;12(1 12):89–. https://doi.org/10.1186/s40538-025-00767-1 . Damalas CA, Koutroubas SD. Seed priming with salicylic acid for improving germination attributes and early growth of wheat under salinity stress. Plant Biosyst. 2025;159:239–46. https://doi.org/10.1080/11263504.2025.2460465 . Saadat H. The effect of salicylic acid on germination indices in wheat seedlings ( Triticum aestivum L.) under salinity stress. Seed Res. 2024;4:1. https://doi.org/10.71544/JSR.2024.1122233 . Yan Y, Pan C, Du Y, et al. Exogenous salicylic acid regulates reactive oxygen species metabolism and ascorbate–glutathione cycle in Nitraria tangutorum Bobr. under salinity stress. Physiol Mol Biology Plants. 2018;24(4):577–89. https://doi.org/10.1007/s12298-018-0540-5 . Luis Castañares J, Alberto Bouzo C. Effect of exogenous melatonin on seed germination and seedling growth in melon ( Cucumis melo L.) under salt stress. Hortic Plant J. 2019;5:79–87. https://doi.org/10.1016/j.hpj.2019.01.002 . Shaikh-Abol-hasani F, Roshandel P. Effects of priming with salicylic acid on germination traits of Dracocephalum moldavica L. under salinity stress. Iran J Plant Physiol. 2019;5 3035 5:3035. https://doi.org/10.30495/IJPP.2019.670789 . Additional Declarations No competing interests reported. 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Ahiakpa","email":"","orcid":"","institution":"Research Desk Consulting Limited","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"K.","lastName":"Ahiakpa","suffix":""},{"id":614086349,"identity":"2786de27-fc1a-43d0-abe5-3653991b194a","order_by":1,"name":"Ge Pan","email":"","orcid":"","institution":"Yulin University","correspondingAuthor":false,"prefix":"","firstName":"Ge","middleName":"","lastName":"Pan","suffix":""},{"id":614086350,"identity":"e778c9a1-b04b-4a58-a00d-61d0353b2139","order_by":2,"name":"Zhenzhong Wu","email":"","orcid":"","institution":"Yulin University","correspondingAuthor":false,"prefix":"","firstName":"Zhenzhong","middleName":"","lastName":"Wu","suffix":""},{"id":614086351,"identity":"2a39aee1-ad27-44b3-b07c-9f8c8f2292b2","order_by":3,"name":"Lu Bai","email":"","orcid":"","institution":"Weinan Vocational and Technical College","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Bai","suffix":""},{"id":614086352,"identity":"f0d76274-3867-4488-8f9c-46f04c853dc5","order_by":4,"name":"Caixia Zheng","email":"","orcid":"","institution":"Heze University","correspondingAuthor":false,"prefix":"","firstName":"Caixia","middleName":"","lastName":"Zheng","suffix":""},{"id":614086353,"identity":"4c4ac91a-fbfc-4977-b18b-fbda33e1b0f3","order_by":5,"name":"Haiqiang Dong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYDCCGwwMBxL/sdnxMzMffECsFsYDD9j4kiXb2ZINiNUCNJ1NjnHDeR4zAaJ08N1u3nAggceM2fgwgxkDQ41NNEEtkneOFRxIkEjjMzvMkPaA4VhabgMhLQY3cgwOJBgcYwZqOW7A2HCYWC0J/xk3NzO2SZCg5QAb4wZmZjbitEjeSCs4kNjAlixxmI3ZIIEYv/DdSN788WcDMCr7z3988KHGhrAWkNsQzAQilKNpGQWjYBSMglGADQAAQhREr3vFkK4AAAAASUVORK5CYII=","orcid":"","institution":"Yulin University","correspondingAuthor":true,"prefix":"","firstName":"Haiqiang","middleName":"","lastName":"Dong","suffix":""}],"badges":[],"createdAt":"2026-02-27 02:39:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8982686/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8982686/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105904814,"identity":"87f0098d-0cde-451d-a010-994a73f4248d","added_by":"auto","created_at":"2026-04-01 10:10:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":222479,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic characteristics of radicle of germinating seeds of \u003cem\u003eA. venetum \u003c/em\u003eafter 10 days of cultivation in petri dish under salt stress.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8982686/v1/7e7ae1f23b0082a7e6f62957.png"},{"id":105826073,"identity":"a9ad0740-f4b7-4019-8400-503fe956ff9e","added_by":"auto","created_at":"2026-03-31 14:01:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":832744,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth status of \u003cem\u003eApocynum venetum\u003c/em\u003eseeds in different treatment groups. Seedlings were primed with different concentrations of (A) water + SA; and (B) NaCl + SA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8982686/v1/1d0a864c8617480f385465c4.png"},{"id":106092998,"identity":"1738cf83-ae24-4743-9b89-88687b4668f1","added_by":"auto","created_at":"2026-04-03 11:32:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":460196,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of trait relationships and treatment effects. (\u003cstrong\u003eA\u003c/strong\u003e) Pearson correlation heatmap with significance levels at *\u003cem\u003ep\u003c/em\u003e≤ 0.05; and **\u003cem\u003ep\u003c/em\u003e ≤0.01 0.05. (B) Principal components analysis of \u003cem\u003eA. venetum\u003c/em\u003e germination under salt stress with exogenous SA. (\u003cstrong\u003eC\u003c/strong\u003e) PC1 and PC2 accounted for 72.13% and 14.20 % of the treatments and traits variance, respectively.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8982686/v1/a16e98081696cb8c65ab5650.png"},{"id":106723731,"identity":"e3a181cd-dda2-4521-86d7-5cb67302827a","added_by":"auto","created_at":"2026-04-12 18:12:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2549291,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8982686/v1/42f94cbc-95ce-4c24-aadd-f3fa09f66a00.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Salicylic acid priming enhances seed germination in Apocynum venetum under salinity conditions","fulltext":[{"header":"1.0 Introduction","content":"\u003cp\u003eSoil salinization is the process by which salts are transported by capillary action to the soil surface and accumulate as water evaporates. It is a widespread form of land degradation worldwide, especially severe in arid and semi-arid regions [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Salt stress interferes with plant uptake of water and nutrients as increasing the osmotic pressure of soil solution, making water absorption difficult, rendering Na\u003csup\u003e+\u003c/sup\u003e ions to compete with K\u003csup\u003e+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e at uptake sites, reducing plant nutrient absorption [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Such stress negatively affects plant growth and development, but exogenous application of salicylic acid (SA) has been shown to reduce salt-induced damage in various species [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. For example, under salinity, SA improved the osmotic regulation, water uptake, and germination of \u003cem\u003eZinnia elegans\u003c/em\u003e seeds [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Spraying 0.5\u0026ndash;1.5 mM SA significantly promoted the growth of cauliflower under NaCl stress, with 0.1 mM being most effective [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Similarly, treating \u003cem\u003epatchouli\u003c/em\u003e seeds and seedlings with 0.25 mM SA alleviated salt injury and enhanced germination and root growth, whereas 0.5 mM SA exacerbated salt damage [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These observations suggest that SA effects on plant growth are concentration-dependent, and the optimal SA concentration for alleviating salt stress varies by species.\u003c/p\u003e \u003cp\u003eExogenous SA can also modulate osmotic balance under salinity. It increases levels of proline, soluble protein (SP), and soluble sugars (SS) in goji (\u003cem\u003eLycium barbarum\u003c/em\u003e), enhancing cellular water uptake and relieving salt injury [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. SA may promote the conversion of in SP and carbohydrates into SP and SS [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], thereby increasing the osmotic potential difference and facilitating water uptake [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In cauliflower under salt stress, SA treatment boosted α-amylase activity and raised SS and proline levels [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], improving osmotic adjustment. Furthermore, SA plays a key role in regulating antioxidant defenses under salinity. It can upregulate both enzymatic and non-enzymatic antioxidants to scavenge reactive oxygen species (ROS) and reduce membrane peroxidation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. For instance, when plants are salt-stressed, high Malondialdehyde (MDA) levels reflect oxidative damage; exogenous SA increased Superoxide Dismutase (SOD), Peroxidase (POD), and Catalase (CAT)activities in salt-stressed cucumber, balancing ROS and lowering MDA and electrolyte leakage [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChinese hemp (\u003cem\u003eA. venetum\u003c/em\u003e) is a highly salt-tolerant pioneer plant. Its widespread cultivation can effectively improve saline soils [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Studying its salt-tolerance mechanisms is thus valuable for developing strategies to mitigate salinity. In this study, we applied 300 mM NaCl and varying concentrations of SA to \u003cem\u003eA. venetum\u003c/em\u003e seeds to evaluate how exogenous SA affects seed germination and physiological traits under salt stress. Our objective was to elucidate the regulatory effects of SA on \u003cem\u003eA. venetum\u003c/em\u003e during germination under salinity, thereby providing theoretical support for producing high-quality, salt-tolerant \u003cem\u003eA. venetum\u003c/em\u003e seedlings in saline-alkali lands.\u003c/p\u003e"},{"header":"2.0 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant material and experimental design\u003c/h2\u003e \u003cp\u003e \u003cem\u003eApocynum venetum\u003c/em\u003e Zhengjun. seeds were sourced from Ningmiao Group Co. (Ningxia, China). The \u003cem\u003eA. venetum\u003c/em\u003e Zhengjun. seeds used in the experiment were generously provided by Ningxia Ningmiao Ecological Construction Group Co., Ltd. This batch of germplasm material originated from artificial multi-generational propagation and is not the wild type. It is currently stored under refrigeration at 4\u0026deg;C for research purposes. Seeds of uniform size, fullness, and free of pests or disease were selected for the germination experiments. Based on preliminary trials, 300 mM NaCl was chosen to impose salt stress. Fourteen treatments comprising control with distilled water (CK); distilled water\u0026thinsp;+\u0026thinsp;SA at 0.1, 0.2, 0.3, 0.4, 0.5, and 0.75 mM; 300 mM NaCl alone; 300 mM NaCl\u0026thinsp;+\u0026thinsp;SA at 0.1, 0.2, 0.3, 0.4, 0.5, and 0.75 mM were used. Each treatment had three replicates. After surface sterilization with 75% ethanol and rinsing, seeds were placed evenly in Petri dishes (50 seeds per dish) on moistened filter paper. For treatments under salt stress, 3 mL of the 300 mM NaCl solution and 4 mL of the assigned SA solution were added to each dish to saturate the paper. Control dishes received equivalent volumes of distilled water and SA solution as appropriate. Evaporated water was replenished daily. Dishes were incubated at a constant temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C under a 16-h light/8-h dark photoperiod. Germination was recorded when the radicle emerged from the seed coat. Germinated seeds were counted daily for 10 days with 10 randomly selected seedlings per treatment measured for radicle and plumule length.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Growth and physiological measurements\u003c/h2\u003e \u003cp\u003eGermination count was calculated as the percentage of seeds germinated by day 10. Radicle and plumule lengths were measured with vernier calipers. Leaf chlorophyll content, soluble sugar, and soluble protein were determined using standard methods [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Antioxidant enzyme activities and lipid peroxidation were assayed. SOD activity was measured by the nitroblue tetrazolium photochemical reduction method [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]; POD by the guaiacol oxidation method [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; CAT activity by [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]; and MDA content by the thiobarbituric acid (TBA) colorimetric method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Data analyses\u003c/h2\u003e \u003cp\u003eData was cleaned and organized for statistical analyses and visualization using GraphPad Prism (ver. 10, Massachusetts, USA). One-way analysis of variance (ANOVA) was performed to test for treatment effects (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); while correlation and principal components analysis were performed using the R. A membership function method was used to evaluate the overall effectiveness of SA in relieving salt stress on seed germination, with higher function values indicating greater mitigation. For a positive indicator, SFV = (\u003cem\u003eX\u003c/em\u003e \u0026ndash; \u003cem\u003eX\u003c/em\u003e\u003csub\u003emin\u003c/sub\u003e)/(\u003cem\u003eX\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e \u0026ndash; \u003cem\u003eX\u003c/em\u003e\u003csub\u003emin\u003c/sub\u003e); for a negative indicator, SFV\u0026thinsp;=\u0026thinsp;1 \u0026ndash; (\u003cem\u003eX\u003c/em\u003e \u0026ndash; \u003cem\u003eX\u003c/em\u003e\u003csub\u003emin\u003c/sub\u003e)/(\u003cem\u003eX\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e \u0026ndash; \u003cem\u003eX\u003c/em\u003e\u003csub\u003emin\u003c/sub\u003e), where SFV stands for the membership function value, \u003cem\u003eX\u003c/em\u003e is the observed value, and \u003cem\u003eX\u003c/em\u003e\u003csub\u003emin\u003c/sub\u003e and \u003cem\u003eX\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e are the minimum and maximum values across treatments.\u003c/p\u003e \u003c/div\u003e"},{"header":"3.0 Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of SA priming on seed germination and seedling growth\u003c/h2\u003e \u003cp\u003eOur analysis shows that NaCl stress (300 mM) significantly suppressed seed germination of \u003cem\u003eA. venetum\u003c/em\u003e compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Germination under salt stress alone was markedly reduced, while seeds under distilled water with SA (water\u0026thinsp;+\u0026thinsp;SA) showed normal germination comparable to control. However, when SA was applied together with salt stress (NaCl\u0026thinsp;+\u0026thinsp;SA), germination improved significantly relative to NaCl alone. The 0.3 mM SA treatment under salt stress yielded the highest germination and the longest radicle and hypocotyl lengths (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At higher SA concentrations beyond 0.3 mM, the decreasing effect gradually deteriorated. These results indicate that exogenous SA can effectively counteract the inhibitory effect of salt on \u003cem\u003eA. venetum\u003c/em\u003e seed germination at 0.3 mM optimal concentration under salinity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of SA priming on seedling morphology under salinity stress\u003c/h2\u003e \u003cp\u003ePhenotypic profiles of seedlings confirmed seedlings grown in distilled water (control) were the tallest and healthiest (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Under 300 mM NaCl, seedlings were severely stunted. Application of SA under salt stress improved seedling length and vigor at low SA concentrations, with 0.3 mM SA showing the healthiest growth. However, at higher SA levels, the beneficial effect diminished. SA\u0026thinsp;+\u0026thinsp;water (no salt) had no visible impact on seedling appearance compared to the control. These morphological results corroborate the quantitative data where low-dose SA mitigates salt-induced growth inhibition, whereas excessive SA offers no additional benefit.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effect of SA priming on key agronomic traits of A. venetum seedlings under salinity stress\u003c/h2\u003e \u003cp\u003eSalt stress (NaCl alone) significantly reduced germination count, hypocotyl length, radicle length, and embryo growth compared to control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Application of SA under salt stress alleviated these inhibitory effects, with the most pronounced recovery observed at 0.3 mM SA. At this concentration, germination count increased from 14.3% (NaCl alone) to 27.7%, radicle length from 0.73 cm to 1.27 cm, and embryo growth from 0.35 cm to 0.89 cm (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Higher SA concentrations (\u0026ge;\u0026thinsp;0.5 mM) resulted in a non-significant decline in these parameters, although values remained significantly above the NaCl‑only treatment. Under control conditions (water), SA application did not significantly affect any of the germination parameters. Two‑way ANOVA confirmed significant main effects of water regime (stress vs. non‑stress) and SA concentration, as well as their interaction, for all measured traits (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Post‑hoc comparisons using Tukey\u0026rsquo;s HSD test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) revealed that 0.3 mM SA was the optimal concentration for mitigating salt stress across most parameters, with higher concentrations providing diminishing or no additional benefit.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of exogenous SA and hydro-priming on biochemical properties of \u003cem\u003eA. venetum\u003c/em\u003e seedlings under salinity stress\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGermination count\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHypocotyl length (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRadicle length (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEmbryo growth (cm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWater\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWater\u0026thinsp;+\u0026thinsp;0.1 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;0.1 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWater\u0026thinsp;+\u0026thinsp;0.2 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;0.2 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWater\u0026thinsp;+\u0026thinsp;0.3 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;0.3 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWater\u0026thinsp;+\u0026thinsp;0.4 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;0.4 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWater\u0026thinsp;+\u0026thinsp;0.5 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;0.5 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWater\u0026thinsp;+\u0026thinsp;0.75 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003efg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;0.75 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eValues are means of three replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Within each column, means followed by the same letter are not significantly different according to Tukey\u0026rsquo;s HSD test\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eThe analysis was performed using R (version 4.2.1) with the agricolae package. The two‑way ANOVA confirmed significant main effects of water and SA concentration, as well as their interaction, for all traits\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). \u003cem\u003ePost‑hoc comparisons were conducted on the 14 treatment combinations.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of SA-priming on biochemical properties of seedlings under salinity stress\u003c/h2\u003e \u003cp\u003eSalt stress triggered significant physiological and biochemical responses in \u003cem\u003eA. venetum\u003c/em\u003e seeds (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Chlorophyll content was markedly reduced under salt stress (0.77 mg/g) compared to control (1.45 mg/g). Chlorophyll content, which was reduced by NaCl stress, was partially restored by SA treatment. SA supplementation under salt stress significantly increased chlorophyll content, reaching a maximum of 1.03 mg/g at 0.2 mM SA, which did not differ significantly from 0.3 mM SA (1.02 mg/g) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Compared to the control (water\u0026thinsp;+\u0026thinsp;SA), NaCl treatment markedly increased the accumulation of key osmolytes, including soluble sugars and soluble proteins, as well as the activities of antioxidant enzymes including SOD, POD, and CAT (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Concurrently, MDA content, a marker of lipid peroxidation, increased sharply under NaCl alone (66.6 nmol/g), indicating severe oxidative membrane damage. Application of SA in the absence of salt (water\u0026thinsp;+\u0026thinsp;SA) did not significantly alter any of these parameters relative to the control, confirming that SA exerts its primary effects under stress conditions.\u003c/p\u003e \u003cp\u003eUnder salt stress, exogenous SA further increased soluble sugars, and soluble protein contents compared to salt alone (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These increases were concentration‑dependent, peaking at 0.3 mM SA (soluble sugars: 32.8 mg/g; soluble proteins: 34.8 mg/g) and declined at higher concentrations. At the optimal concentration, soluble protein and sugar contents were 63% and 95% higher, respectively, than in the NaCl‑only treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This suggests that exogenous SA enhances osmotic adjustment by boosting osmoprotectant accumulation and sustaining chlorophyll integrity under salt stress, with 0.3 mM SA being the most effective concentration for osmotic regulation.\u003c/p\u003e \u003cp\u003eIn parallel, SA applications under saline conditions markedly enhanced antioxidant enzyme activities while reducing MDA content relative to salt alone (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The highest SOD (117.2 U/g), POD (118.5 U/g), and CAT (202.9 U/g) activities, and the lowest MDA level (53.7 nmol/g), were consistently observed at 0.3 mM SA, representing a 19.4% reduction in lipid peroxidation compared to NaCl alone (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At this concentration, SOD and POD activities were approximately 20% and 62% higher, respectively, than under NaCl‑only treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Beyond 0.3 mM SA, antioxidant enzyme activities gradually declined, although they remained significantly above the NaCl‑only level at 0.4 and 0.5 mM SA.\u003c/p\u003e \u003cp\u003eMDA content, however, increased again at higher SA concentrations, approaching the NaCl‑only level at 0.75 mM SA (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings demonstrate that exogenous SA significantly enhances the antioxidant defense system in \u003cem\u003eA. venetum\u003c/em\u003e seeds under salt stress, mitigating oxidative damage and reinforcing membrane stability. The results align with previous reports that SA can activate antioxidant enzymes to scavenge reactive oxygen species (ROS) and protect cellular membranes under salinity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of exogenous SA-priming on biochemical properties of \u003cem\u003eA. venetum\u003c/em\u003e seedlings under salinity stress\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChlorophyll (mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSoluble sugars (mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoluble protein (mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSOD (U/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePOD (U/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCAT (U/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMDA (nmol/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWater\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e74.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e110.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e72.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e142.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e66.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWater\u0026thinsp;+\u0026thinsp;0.1 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e73.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e 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align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e73.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e112.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e 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align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e73.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e112.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;0.5 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e113.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e114.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e169.16\u0026thinsp;\u0026plusmn;\u0026thinsp;5.53\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e59.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWater\u0026thinsp;+\u0026thinsp;0.75 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e73.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e112.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;0.75 SA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e112.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e113.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e166.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e62.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eValues are means of three replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Within each column, means followed by the same letter are not significantly different according to Tukey\u0026rsquo;s HSD test\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eThe analysis was performed using R (version 4.2.1) with the agricolae package. The two‑way ANOVA confirmed significant main effects of water and SA concentration, as well as their interaction, for all traits\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). \u003cem\u003ePost‑hoc comparisons were conducted on the 14 treatment combinations.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Multivariate analysis of trait relationships and treatment effects\u003c/h2\u003e \u003cp\u003eTo explore the interrelationships among germination parameters, biochemical markers, and oxidative stress indicators, Pearson correlation analysis was performed on all measured traits across the 14 treatment combinations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Germination count, radicle length, embryo growth, and chlorophyll content were strongly and positively correlated with one another (\u003cem\u003er\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.77, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that these traits respond coordinately to the experimental treatments. In contrast, these germination‑related parameters exhibited significant negative correlations with antioxidant enzyme activities (SOD, POD, CAT) and MDA content, with correlation coefficients ranging from \u0026minus;\u0026thinsp;0.60 to -0.98 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). This inverse relationship demonstrates that enhanced oxidative stress, reflected by elevated enzyme activities and lipid peroxidation, is associated with reduced germination performance under saline conditions. Particularly, soluble sugar and protein contents were strongly positively correlated with antioxidant enzymes and MDA (\u003cem\u003er\u0026thinsp;\u0026gt;\u0026thinsp;0.90\u003c/em\u003e, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting that osmolyte accumulation and oxidative stress responses are co‑induced under salt stress.\u003c/p\u003e \u003cp\u003ePrincipal component analysis (PCA) was conducted to visualize the overall patterns of treatment effects and to identify the primary sources of variation in the dataset (Fig.\u0026nbsp;4). The first two principal components account for 86.0% of the total variance, with PC1 accounting for 72.0% and PC2 for 14.0% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, scree plot). The PCA scores plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) revealed a clear separation between water‑treated (non‑stress) and NaCl‑treated (salt stress) samples along the PC1 axis. Water‑treated samples formed a tight cluster on the negative side of PC1, while NaCl‑treated samples were distributed across the positive side, with their positions along PC2 influenced by the concentration of applied SA. The loading vectors (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) provided insight into the variables driving these separations. PC1 was strongly positively loaded by stress‑related biochemical markers including soluble protein8 (0.18), SS (0.15), MDA (0.12), and the antioxidant enzymes; CAT (0.08), POD (0.07), and SOD (0.06) and strongly negatively loaded by germination parameters, including germination count (-0.25), radicle length (-0.22), embryo growth (-0.20), and chlorophyll contemt (-0.25). This confirms that PC1 represents a stress‑to‑growth gradient, distinguishing treatments based on the trade‑off between oxidative stress responses and germination performance. PC2, which accounted for 14.0% of the variance, was primarily associated with finer differentiation among NaCl‑treated samples, likely reflecting the modulating effects of SA concentration on the stress response. Variables contributing moderately to PC2 included SP (0.25), SS (0.20), radicle length (0.15), and CC (0.10).\u003c/p\u003e \u003cp\u003eCollectively, the correlation and PCA analyses demonstrate that salt stress induces a coordinated shift in biochemical and physiological traits, characterized by increased antioxidant enzyme activities and osmolyte accumulation concomitant with reduced germination and CC. Exogenous SA modulates this response in a concentration‑dependent manner, with intermediate concentrations (particularly 0.3 mM) partially restoring germination traits while maintaining enhanced antioxidant capacity. These findings corroborate the earlier results and highlight the central role of oxidative stress in mediating the inhibitory effects of salinity on \u003cem\u003eA. venetum\u003c/em\u003e seed germination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA membership function analysis was conducted to quantitatively evaluate the overall stress mitigation effects of the different treatments (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among all treatments, the non‑stressed control (distilled water only) recorded the highest membership value (0.992), categorized as optimal, no stress. Under saline conditions, seeds treated with 300 mM NaCl combined with 0.3 mM SA achieved the highest membership value (0.213), classified as optimal under salt stress. In contrast, seeds subjected to 300 mM NaCl alone exhibited the lowest membership value (0.106), ranked as lowest. All treatments involving distilled water supplemented with SA (0.1\u0026ndash;0.75 mM) maintained high membership values (\u0026ge;\u0026thinsp;0.885) and were ranked as high or moderate. These findings reveal that the application of 0.3 mM SA provides the most effective alleviation of salt stress during \u003cem\u003eA. venetum\u003c/em\u003e seed germination, confirming its role as the optimal concentration for mitigating salinity‑induced inhibitory effects.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMembership function analysis of germination traits under salt stress relieved by exogenous SA.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRank\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSFV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRanking\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.992\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOptimal (no stress)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u0026thinsp;+\u0026thinsp;0.1 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.952\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u0026thinsp;+\u0026thinsp;0.2 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.932\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u0026thinsp;+\u0026thinsp;0.4 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.919\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u0026thinsp;+\u0026thinsp;0.3 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.915\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u0026thinsp;+\u0026thinsp;0.5 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.906\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u0026thinsp;+\u0026thinsp;0.75 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.885\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300 mM NaCl\u0026thinsp;+\u0026thinsp;0.3 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOptimal (salt stress)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300 mM NaCl\u0026thinsp;+\u0026thinsp;0.4 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300 mM NaCl\u0026thinsp;+\u0026thinsp;0.2 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300 mM NaCl\u0026thinsp;+\u0026thinsp;0.5 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300 mM NaCl\u0026thinsp;+\u0026thinsp;0.1 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300 mM NaCl\u0026thinsp;+\u0026thinsp;0.75 mM SA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300 mM NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLowest (pure salt stress)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eWhere\u003c/em\u003e \u003cb\u003eSFV\u003c/b\u003e \u003cem\u003erefers to subordinate function value; used to rank the overall stress mitigation effect of different SA concentrations. By assigning a single value to each treatment, we were able to quantitatively conclude that 0.3 mM SA resulted in the highest SFV (0.213) under salt stress, confirming it as the optimal concentration for alleviating salinity damage.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"4.0 Discussion","content":"\u003cp\u003eSalinity stress represents one of the most significant abiotic challenges limiting crop productivity worldwide, particularly affecting seed germination and early seedling establishment [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The present study demonstrates that exogenous application of (SA) effectively alleviates the inhibitory effects of salt stress on \u003cem\u003eA. venetum\u003c/em\u003e seed germination and seedling growth, with 0.3 mM SA identified as the optimal concentration for stress mitigation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Salicylic acid enhances antioxidant profiles under salt stress\u003c/h2\u003e \u003cp\u003eHigh concentrations of soluble salts can severely damage plant cell membranes through increased permeability and lipid peroxidation, while concurrently impairing antioxidant defense mechanisms [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In the present study, exposure to 300 mM NaCl significantly elevated MDA content, a reliable biomarker of lipid peroxidation; while reducing the activities of SOD,POD, and CAT in \u003cem\u003eA. venetum\u003c/em\u003e seedlings, indicating substantial oxidative damage. These observations align with previous reports demonstrating that salinity stress disrupts cellular redox homeostasis through excessive accumulation of reactive oxygen species [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExogenous SA treatment markedly enhanced antioxidant enzyme activities and reduced membrane damage, with 0.3 mM SA registering the highest ameliorative effect. Under this optimal concentration, SOD and POD activities increased by approximately 20% and 62%, respectively, compared to NaCl treatment alone, while MDA content decreased by 19.4%. These findings are consistent with studies on sorghum, where SA application (0.1\u0026ndash;0.5 mM) increased antioxidant enzyme expression and reduced MDA by up to 27% under salt stress [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Similarly, recent studies on soybeans demonstrated that SA supplementation enhanced SOD, CAT, and ascorbate peroxidase activities while reducing oxidative damage and improving membrane stability [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The concentration-dependent response observed in our study, with optimal effects at 0.3 mM SA and diminished benefits at higher concentrations, corroborates the established paradigm that SA exhibits biphasic effects depending on dosage, plant species, and stress intensity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mechanistic basis for SA-mediated antioxidant enhancement involves both direct activation of antioxidant enzymes and transcriptional regulation of stress-responsive genes. Recent transcriptomic analysis in \u003cem\u003eDavidia involucrata\u003c/em\u003e revealed that exogenous SA induced differential expression of 2,581 genes under salt stress, including upregulation of transcription factors (\u003cem\u003eDiWRKY40\u003c/em\u003e, \u003cem\u003eDiNAC25\u003c/em\u003e, \u003cem\u003eDiMYB4\u003c/em\u003e, and \u003cem\u003eDiMYB86\u003c/em\u003e) that regulate antioxidant defense pathways [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These molecular adjustments potentially contributed to the enhanced SOD, POD, and CAT activities observed in our study, collectively reducing ROS accumulation and preserving membrane integrity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Salicylic acid promotes osmotic adjustment and chlorophyll preservation\u003c/h2\u003e \u003cp\u003eOsmotic adjustment through the accumulation of compatible solutes represents a crucial adaptive mechanism for maintaining cellular turgor and protecting macromolecular structures under salt stress [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study, NaCl stress significantly reduced SP, soluble sugar, and CC in \u003cem\u003eA. venetum\u003c/em\u003e seedlings. Exogenous SA application reversed these declines, with peak osmolyte accumulation and CC recorded at 0.3 mM SA. At this optimal concentration, SP and sugar contents were 63 and 95 % higher, respctively, than in NaCl-only treated seedlings.\u003c/p\u003e \u003cp\u003eThese findings align with studies on faba bean, where SA application under salinity enhanced SP and sugar accumulation while improving photosynthetic capacity and PSII light utilization efficiency [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The accumulation of SP under SA treatment potentially reflects stress-induced synthesis of osmotin-like proteins and dehydrins that enhance water retention, while elevated SS contribute to cellular osmotic adjustment and carbon metabolism under stress [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Recent studies on white lupine further demonstrated that SA and SA-nanoparticles increased total SS, proline, and SP under saline conditions, with concomitant improvements in photosynthetic pigments [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The preservation of CC observed in our study is particularly significant, as chlorophyll degradation under salinity directly impairs photosynthetic capacity and carbon assimilation. Similar chlorophyll maintenance effects have been reported in SA-treated wheat [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and spinach[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] under abiotic stress.\u003c/p\u003e \u003cp\u003eThe restoration of and osmolyte accumulation at 0.3 mM SA suggests that exogenous SA enhances the capacity of \u003cem\u003eA. venetum\u003c/em\u003e for osmotic adjustment while protecting photosynthetic apparatus from salt-induced damage. These effects are likely mediated through SA-induced expression of genes involved in osmolyte biosynthesis and chlorophyll metabolism, as reported in recent transcriptomic studies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Integrated stress mitigation and concentration-dependent responses\u003c/h2\u003e \u003cp\u003eThe comprehensive evaluation using membership function analysis confirmed that 0.3 mM SA represented the optimal concentration for alleviating salt stress, achieving the highest subordinate function value (0.213) among saline treatments. This integrated analysis, which combines multiple physiological indicators into a single metric, provides robust evidence for the superior performance of 0.3 mM SA in mitigating salinity effects. Treatments with distilled water plus SA (0.1\u0026ndash;0.75 mM) under non-saline conditions maintained high membership values (\u0026ge;\u0026thinsp;0.885), confirming that SA application does not adversely affect germination under optimal conditions. The concentration-dependent response pattern observed; increasing efficacy from 0.1 to 0.3 mM SA followed by declining benefits at higher concentrations, reflects the dual regulatory role of SA in plant stress responses [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Similar optima have been reported across diverse species, including 1.0 mM SA for wheat germination under salinity [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], 0.5 mM for sorghum seedlings [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and 2.0 mM for \u003cem\u003eDracocephalum moldavica\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These species-specific optima underline the importance of empirical determination of effective SA concentrations for individual crop species and stress conditions.\u003c/p\u003e \u003cp\u003eThe integration of antioxidant enhancement and osmotic adjustment under SA treatment appears to operate through coordinated signalling pathways. SA-mediated stress tolerance involves the activation of \u003cem\u003eNPR1\u003c/em\u003e-dependent signalling cascades that regulate both antioxidant gene expression and osmolyte biosynthesis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The concurrent improvement in multiple stress-responsive parameters at 0.3 mM SA suggests that this concentration optimally activates these signalling networks without inducing the oxidative stress that can occur at supra-optimal SA levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Implications for A. venetum cultivation in saline environments\u003c/h2\u003e \u003cp\u003eThe demonstrated efficacy of 0.3 mM SA in mitigating salt stress during germination has significant practical implications for \u003cem\u003eA. venetum\u003c/em\u003e cultivation in saline-alkali soils. As a species with considerable ecological and economic value, enhancing its salt tolerance during germination phase could facilitate establishment in marginal environments where soil salinity limits agricultural productivity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Seed priming with SA represents a cost-effective and technically simple approach that could be readily adopted by resource-limited farmers. The potential for bringing moderate saline soils into cultivation through SA application, as suggested by recent studies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], aligns with broader efforts to expand agricultural production on underutilized marginal soils.\u003c/p\u003e \u003c/div\u003e"},{"header":"5.0 Conclusion","content":"\u003cp\u003eSalt stress at 300 mM NaCl significantly inhibited seed germination and seedling growth in \u003cem\u003eA. venetum\u003c/em\u003e, reducing antioxidant enzyme activities (SOD, POD, CAT), SP, SS, and CC while markedly elevating MDA levels; indicating severe oxidative and osmotic stress. Exogenous application of (SA) effectively ameliorated these deleterious effects through a dual mechanism involving: (i) enhancement of antioxidant profiles, as evidenced by increased SOD, POD, and CAT activities and reduced lipid peroxidation; and (ii) promotion of osmotic adjustment, reflected in increased SP and sugar accumulation and preserved CC. The 0.3 mM SA treatment consistently demonstrated significant stress mitigation efficacy across all measured parameters, establishing it as the optimal concentration for salinity alleviation in this species. These findings contribute to understanding SA-mediated salt tolerance and provide practical insights for improving \u003cem\u003eA. venetum\u003c/em\u003e production in saline environments. Further studies at the molecular level, particularly transcriptomic and proteomic analyses of SA-treated seedlings under salt stress, would elucidate the regulatory networks governing these protective responses and facilitate the development of enhanced stress tolerance strategies for this valuable species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e All the experimental research on plants including the collection of plant materials were performed in accordance with relevant institutional, national, and international guidelines and legislation. The study did not involve any endangered or protected species.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors have reviewed the final version of the manuscript and consent to its publication.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Regional Fund of the National Natural Science Foundation of China (Grant No. 32460747), the Industry-University-Research Cooperation Project of Yulin Science and Technology Bureau (No. 2024-CXY-094), the Shaanxi Province \"University-Enterprise Shared\" Talent Project (No. 2024XZGY04), and the High-level Talent Introduction Program of Yulin University (2023GK76).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.K.A: Data curation, Formal analysis, Methodology, Software, Validation, Writing\u0026mdash;original draft. G.P: Conceptualization, Data curation, Formal analysis, Methodology, Resources, Software, Supervision, Validation, Writing\u0026mdash;original draft. Z.W \u0026amp; L.B: Visualization, Data curation, Methodology. J.K.A \u0026amp; F.K: Validation, Writing\u0026mdash;review and editing. H.D: Funding acquisition, Conceptualization, Project administration, Resources, Writing\u0026mdash;review and editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhao N, Zhu H, Zhang H, et al. Hydrogen sulfide mediates k\u0026thinsp;+\u0026thinsp;and Na+ homeostasis in the roots of salt-resistant and salt-sensitive poplar species subjected to NaCl stress. 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Effect of exogenous melatonin on seed germination and seedling growth in melon (\u003cem\u003eCucumis melo\u003c/em\u003e L.) under salt stress. Hortic Plant J. 2019;5:79\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.hpj.2019.01.002\u003c/span\u003e\u003cspan address=\"10.1016/j.hpj.2019.01.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaikh-Abol-hasani F, Roshandel P. Effects of priming with salicylic acid on germination traits of \u003cem\u003eDracocephalum moldavica\u003c/em\u003e L. under salinity stress. Iran J Plant Physiol. 2019;5 3035 5:3035. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.30495/IJPP.2019.670789\u003c/span\u003e\u003cspan address=\"10.30495/IJPP.2019.670789\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-plants","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Plants](https://link.springer.com/journal/44372)","snPcode":"44372","submissionUrl":"https://submission.springernature.com/new-submission/44372/3","title":"Discover Plants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Apocynum venetum, salt stress, seed priming, salicylic acid, seed germination, antioxidant enzymes, osmolytes","lastPublishedDoi":"10.21203/rs.3.rs-8982686/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8982686/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSalinity stress significantly impairs seed germination and seedling establishment in many economically valuable plant species. Salicylic acid (SA) is a key signaling molecule implicated in plant stress responses. Despite this, a significant knowledge gap remains regarding its concentration-dependent effects on Chinese hemp (\u003cem\u003eA. venetum\u003c/em\u003e), a species recognized for its considerable ecological and pharmaceutical importance. In this study, we investigated the effects of seeds priming with SA on seed germination, osmolytes (soluble sugar and protein contents), antioxidant enzyme activities, and lipid peroxidation (malondialdehyde content). Seed priming with 0.3 mM SA alleviated the adverse effects under salt stress on seed germination and seedling growth. Seeds of \u003cem\u003eA. venetum\u003c/em\u003e primed with 0.3 mM SA exhibited improved germination count, radicle length, embryo growth, germination potential, chlorophyll content, soluble sugar and protein contents, enhanced the activities of superoxide dismutase, peroxidase, and catalase, and decreased malonaldehyde content in the seeds. These results suggest that seed priming with SA enhances the accumulation of key osmolytes and photosynthetic pigments, and boosts antioxidant enzyme activities, thereby alleviating the adverse effects of salt stress on seed germination and seedling growth in \u003cem\u003eA. venetum\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Salicylic acid priming enhances seed germination in Apocynum venetum under salinity conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-31 14:01:36","doi":"10.21203/rs.3.rs-8982686/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-08T05:08:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-28T10:29:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"10715552670633777954673265924766057900","date":"2026-04-21T13:36:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-03T20:04:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160820654898973283341605409869607519242","date":"2026-03-29T16:21:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204224058430649820472851374243303886454","date":"2026-03-27T14:45:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-27T14:36:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-16T11:53:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-10T03:29:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Plants","date":"2026-03-10T02:22:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-plants","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Plants](https://link.springer.com/journal/44372)","snPcode":"44372","submissionUrl":"https://submission.springernature.com/new-submission/44372/3","title":"Discover Plants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"92c9fffb-f670-40f2-a414-5151288f3a86","owner":[],"postedDate":"March 31st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-08T05:08:58+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T05:23:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-31 14:01:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8982686","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8982686","identity":"rs-8982686","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}