Soil salinization has become a global problem affecting around one third of the cultivable area worldwide (Kazemi et al., 2022). It is estimated that more than 800 million hectares of land are affected by salinity (Zhao et al., 2023). It also poses serious threats to social resources, population, the environment, and global food security (Zhang C. et al., 2019). Salinity represents a major threat to agriculture, as it severely impacts plant growth and productivity. It has significantly reduced crop growth and yields in crops such as wheat (Triticum aestivum), maize (Zea mays), rice (Oryza sativa), and soybean (Glycine max), as reported previously (Kazemi et al., 2022). Salinity disrupts plant physiological mechanisms because of the higher availabilities of sodium (Na⁺) and chloride (Cl⁻) ions, leading to osmotic stress and reducing water availability in plant tissues (Rodríguez Coca et al., 2023; Zhao et al., 2023). Imbalances in nutrient ions, such as Na⁺ and potassium (K⁺), lead to the excessive production of reactive oxygen species (ROS) (Xiong et al., 2020), damaging cellular components through lipid peroxidation and nucleic acid degradation (Ahmad and Anjum, 2023; Sadia et al., 2023). Thus, plants begin to wilt, and a reduction in growth occurs. In addition, salinity affects plant health and soil quality by disrupting the root air–water balance and degrading the soil structure (El Sebai et al., 2016). In plants, salt stress reduces antioxidant enzymatic activities such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and suppresses plant immunity against biotic and abiotic stress (Ludwiczak et al., 2021). However, some studies report that salt stress could also upregulate alternative oxidase (AOX) defenses as a compensatory mechanism (Sousa et al., 2022; Spormann et al., 2024). Moreover, salinity stress adversely impacts phytohormones such as abscisic acid (ABA), gibberellins (GAs), salicylic acid (SA), and auxin production, as indicated previously (Duan et al., 2023). Currently, various strategies have been applied to reduce or completely mitigate salinity stress, including introducing new salt-tolerant genotypes, seed treatments, and biostimulant applications (Bouzroud et al., 2023). As an important biostimulator, ascorbic acid (ASA) is a water-soluble antioxidant naturally produced in plants (Hernández et al., 2023). Recently, some studies have indicated that it could alleviate various abiotic stresses, including salinity, by influencing ion homeostasis and maintaining physiological responses to salt stress (Zhang C. et al., 2019; Naz et al., 2016; El-Hawary et al., 2023). In addition, some investigations demonstrated that it could positively affect plant growth through variations in metabolic pathways, such as the Calvin cycle, tricarboxylic acid cycle, proline biosynthesis pathway, glycine betaine biosynthesis pathway, ascorbate-glutathione cycle, and salt overly sensitive pathway (Sadak et al., 2023; Hasanuzzaman et al., 2023). These pathways are closely associated with photosynthesis, antioxidant defense mechanisms, and ion homeostasis, and their enhancement enables plant to better tolerate salinity stress, while maintaining normal growth as indicated by Akram et al. (2017). In addition, several studies have reported that ASA is an efficient and cost-effective approach for mitigating salt stress in crops such as wheat, maize, and rice (Zhang G. et al., 2019). However, its role in quinoa remains unexplored, particularly during the seedling stage, a critical phase for establishing crop resilience under stress. Understanding the effects of ASA at this early growth stage is essential, as seedling vigor strongly influences overall plant development, salt tolerance, and yield potential under saline conditions.

Quinoa (Chenopodium quinoa Willd.) is a pseudo-cereal crop belonging to the Amaranthaceae family. It is highly nutritious and usually grown for protein-rich, gluten-free seeds (Keutgen and Pawelzik, 2009). It can be used in salads, porridges, and flour-based products for human nutrition. The leaves are used as a animals feed and for medicinal purposes such as diabetes control, by pharmacologists. However, cultivation, genetic diversity, and biochemical composition depend on the area and quinoa variety. Although quinoa is recognized for its tolerance to environmental stresses. However, salinity still significantly affects its growth and chemical composition (Song et al., 2019). The seedling stage is particularly sensitive to salt stress, and early damage can impair plant development and productivity. To address this, the present study aimed to investigate the effects of exogenous ASA application on quinoa seedlings under such conditions. The objective of the study was to evaluate how ASA modulates key physiological, biochemical and growth-related parameters under salt stress. Specifically, we examined changes in germination, biomass accumulation, photosynthetic traits, ion balance, antioxidant activity, and osmotic adjustment to better understand the mechanisms through which ASA supports stress resilience at this critical stage. Investigating the role of ASA in early growth could enhance crop resilience and improve agriculture sustainability under saline conditions.

Salt stress inhibits seed germination and growth by reducing water and osmotic potential, adversely affecting the embryo and sprout (Otie, 2022; Begum et al., 2022). It also impairs seed germination and development in essential crops (Otie, 2022; Chakma et al., 2019; Chauhan, 2022). The results of this study indicate that the observed decreases in GP, GE, GRI, SP, and SVI can be attributed to osmotic stress and ion toxicity, disrupting water absorption and altering the nutrient balance, thus reducing seedling development and germination, as noted previously by Mittal et al. (2018). These findings align with the conclusion of Chen HAKM. et al. (2021), who highlighted the harmful effect of salt stress on seed germination and seedling development. To counteract these effects, ASA was applied at 0.1 µM and 0.5 µM concentrations, selected based on earlier research showing they effectively mitigated salt-induce stress. Notably, the lower ASA dose markedly improved SVI and GRI ( Figures 6c, e ), suggesting its role in enhancing early seedling vigor by regulating osmotic balance and reducing ROS-induced damage. Previous research on tomatoes (Solanum lycopersicum) and mustard (Brassica rapa) under salinity stress has shown that both exogenous and endogenous ASA application enhances germination parameters (Mittal et al., 2018; Chen et al., 2023). In these studies, the improvement in germination is attributable to the role of ASA as an antioxidant, mitigating oxidative damage and enhancing osmotic balance. The results of this study further corroborate the findings of Zhang G. et al. (2019) and Chan et al. (2013), which showed that exogenous ASA promotes cell growth, root development, and seedling growth while mitigating the detrimental impacts of salinity on seed germination of wheat and okra (Abelmoschus esculentus L.). However, the concentration and duration of ASA application are critical, as optimal levels improve germination, while improper dosages may induce toxicity (Sarkar et al., 2023). The experimental analysis revealed that a lower ASA dose is particularly effective in improving quinoa seed germination and seedling development ( Tables 3 , 4 ), probably by mitigating oxidative stress and enhancing antioxidant defenses.

Salt stress hampers plant development and growth by disrupting osmotic balance and inducing ion toxicity (Sardar, 2023; Gupta and Huang, 2014). A plethora of studies have confirmed that salt stress significantly hampers plant growth, productivity, and yield by inducing Na⁺ toxicity (Biswas et al., 2023; Sardar, 2023). It also disrupts photosynthesis by causing stomatal closure, which reduces intercellular CO₂ concentrations, and interferes with the transport of essential ions (Najafi Zilaie et al., 2023). Similarly, the results of this study indicate that salt stress significantly reduced RGR, FW, DW, R/S, LN, PH, and LA. These effects could be attributed to disruptions in osmotic balance, impaired water uptake, and ion toxicity, all hindering quinoa growth. The reduction in FW and DW ( Figures 2c, d ), underscores the inhibitory impact of salt stress on biomass accumulation, primarily due to impaired water uptake and disrupted nutrient homeostasis as can be seen in RWC results ( Figure 2b ). The reduction is closely associated with osmotic stress, which disrupts metabolic processes, impacting cell growth, division, and the efficiency of photosynthesis (Chen HAKM. et al., 2021). Understanding the mechanisms behind these physiological disruptions is key for developing effective agronomic strategies to enhance salt tolerance. However, several studies such as El-Afry et al. (2018), and Azeem (2023) have demonstrated that applying ASA improves plant growth, photosynthesis, stomatal conductance, oxidative defense, and the levels of photosynthetic pigments. The improvement in RGR and PH ( Figures 2a, e ) in our study suggests that ASA significantly supports cell expansion and elongation under stress conditions. In addition, the increase in LN and large LA in ASA-treated plants ( Figures 2f, g ) indicate improved photosynthetic efficiency and carbon assimilation. Interestingly, the expectation that growth parameters such as FW, DW per plant, and LN of ASA-treated plants would be lower than CK plants under salt stress, was not supported as shown in Figure 2 . This could be due to the antioxidant properties of ASA, which may have alleviated the negative impacts of salt stress. Additionally, the applied salt concentration (200 mM) may not have been high enough to induce severe toxicity in the quinoa cultivar Longli No.1, which is known for its tolerance to salinity levels between 200–400 mM (Yang et al., 2017). Therefore, the observed slower growth in salt treated plants compared to CK may still reflect a mild salt stress effect, particularly in the absence of ASA. In contrast, ASA treated plants likely benefited from enhanced antioxidant defense, improved nutrient uptake, and photosynthetic performance, resulting in better biomass accumulation even under 200 mM salt. These findings suggest that the combination of a salt-tolerant and ASA application may partially overcome growth limitations typically associated with salt stress. Moreover, the observed interaction between salt stress and ASA treatment highlights the complex and adaptive physiological responses of quinoa under high-salinity conditions. The findings are consistent with previous reports and further support the hypothesis that ASA enhances quinoa growth by facilitating photosynthesis, ion homeostasis, biochemical adjustments and growth under salt stress (El-Sayed and El-Sayed, 2013).

Leaf gas exchange, photosynthetic pigments, and chlorophyll a fluorescence are key indicators of plant health and stress tolerance (Nouman and Aziz, 2022; Bernier Brillon et al., 2023). These parameters are often reduced in saline environments because of disrupted chlorophyll biosynthesis, impaired metabolic processes, and damage to the photosynthetic apparatus caused by ion toxicity and osmotic stress (Swapna, 2003; Askari, 2023). In the current study, salt stress significantly reduced chlorophyll contents, which could be attributed to disrupted pigment biosynthesis and instability. Similarly, in accordance with our results, salt stress negatively impacts the chlorophyll content in tomatoes, carotenoids in milk thistle (Silybum marianum), stomatal conductance in common bean (Phaseolus vulgaris), and the Fv/Fm ratio in sweet peppers, all reflecting significant impairments associated with salt stress (El-Sayed and El-Sayed, 2013; Ekmekçi and Karaman, 2012; Gaafar, 2020; El-Beltagi, 2022). Recent studies on broccoli (Brassica oleracea), sweet pepper, and rice have shown that applying ASA enhances photosynthetic efficiency by improving the performance of PSII, pigment contents, electron transport, and chloroplast membrane protection (MacDonald et al., 2022; El-Beltagi, 2022; Chen HAKM. et al., 2021). El-Sayed and El-Sayed (2013) reported that exogenous ASA stimulates the enzymes involved in chlorophyll and carotenoid biosynthesis, leading to higher photosynthetic pigment levels under salt stress. Similarly, our results support the aforementioned findings, as higher chlorophyll levels were maintained in ASA-treated plants under salt stress ( Figures 4a–c ). Furthermore, a study on chicory (Cichorium intybus L.) found that ASA increased P_N and g_s (Sadia et al., 2023). In our results, P _N and g_s also improved, indicating enhanced stomatal regulation and CO₂ assimilation. Likewise, the increase in WUE suggests that ASA treated plants maintained better water use efficiency under stress conditions. Chen et al. (2023) demonstrated that exogenous ASA mitigates the inhibition of Fv/Fm, PSII quantum yield, and NPQ values under salt stress. The same results were found in our study, especially the improvement in NPQ, ( Figure 4f ), suggesting that ASA enhanced energy dissipation mechanisms, which is crucial for preventing photodamage and dissipating excess energy under salinity stress. Also, qP and ETR ( Figures 4e, g ) showed that ASA enhanced electron transport efficiency, supporting sustained photosynthetic activity under stress conditions. Furthermore, Henschel et al. (2023) found that applying ASA increased the levels of soluble proteins, sugars, and proline, as well as the transpiration rate, all of which could contribute to improved photosynthetic efficiency. Soluble sugar content was markedly increased in our study, indicating enhanced osmotic adjustment and energy reserves under salinity conditions. Our results also indicate that ASA induced sugar accumulation varies with treatment intensity, suggesting a threshold effect in osmotic regulation. Similarly, proline accumulation was significantly higher in ASA combined treatments, suggesting its role in osmotic balance and ROS scavenging ( Figure 6b ). These effects are attributable to the higher priming ability of ASA to enhance antioxidant defenses, maintain cellular function, and protect the photosynthetic apparatus from stress-induced damage (Koleva-Valkova and Harizanova, 2019). The findings support these conclusions, likely because of the antioxidant properties of ASA. The results align with previous studies on wheat, sorghum (Sorghum bicolor), chicory, and marigold (Tagetes spp) (Mohammed Ibrahim Elsiddig et al., 2022; Ishaq et al., 2021; Sadia et al., 2023; Azizi et al., 2021). However, the results of this study contradict those of Zonouri et al. (2014), who found no notable impact of exogenous ASA on the pigments involved in photosynthesis under high salinity conditions.

Exogenous ASA has been found to improve physiological functions and boost non-enzymatic and enzymatic antioxidant pathways under saline stress (Alamri et al., 2018; Ekmekçi et al., 2021). In this study, the ASA treatment significantly enhanced antioxidant enzymes activities (POD, CAT, and SOD) and reduced MDA levels under salt stress. The adverse impact of salt-induced stress on quinoa could be attributable to an increase in ROS production that causes oxidative damage and lipid peroxidation (Hasaneen et al., 2010; Shah et al., 2020). However, ASA mitigated these adverse effects by scavenging ROS, regulating the redox balance, and activating antioxidant enzymes. Foliar application of ASA at 5 mM significantly mitigated the negative effects of salt stress and increased enzymatic antioxidants i.e., SOD (22.3%), POD (34.1%) and CAT (39%) in pea (Pisum sativum L.) plants (Kanwal et al., 2024). The same results were observed in our findings, where ASA treatments, especially ASA combined with salt stress, induced the strongest antioxidant response in quinoa ( Figures 9a, c ). Additionally, a significant declined in MDA content at low ASA concentrations underscores its role in reducing lipid peroxidation under salt stress. Similarly, ASA application effectively protected okra seedlings by enhancing growth, photosynthetic pigments, and antioxidant activities while reducing the hydrogen peroxide and lipid peroxidation under salt stress (Zhang C. et al., 2019). In barley (Hordeum vulgare), ASA enhanced antioxidant production and reduced lipid peroxidation due to its capacity to neutralize ROS and protect cellular components from oxidative damage (Talaat et al., 2023). Furthermore, Naz et al. (2013) reported that ASA reduces oxidative stress by synergizing with other antioxidants to regulate key enzymes, thereby managing oxidative damage and maintaining cellular balance. These findings aligned with the results from wheat (El-Hawary et al., 2023), rice (Che Radziah et al., 2016), sugarcane (El-Hawary et al., 2023), and sorghum (Mohammed Ibrahim Elsiddig et al., 2022), where exogenous ASA treatments significantly enhanced SOD, POD, and CAT activities while decreasing MDA levels. Although ASA levels were not quantified in this study, it is acknowledged that measuring endogenous ASA content could have provided further insight into its direct role as a non-enzymatic oxidant in ROS detoxification. In addition to understanding its distribution within plant tissues, such data would allow a more precise evaluation of its active participation in oxidative stress regulation. Future studies should consider this aspect to strengthen the mechanistic understanding of ASA role under salt stress.

Excess salt ions hinder the uptake of essential nutrients and induce osmotic stress in plants (Alamri et al., 2018; Zhang C. et al., 2019). The results showed reduced levels of essential ions and elevated Na⁺ levels in both leaves and roots, inducing a nutrient imbalance, as indicated by higher Na⁺/K⁺ and Na⁺/Ca²⁺ ratios. This imbalance may be due to the competitive uptake of Na⁺ over these essential ions under salt stress. Consistent with Riaz (2020), high Na⁺ levels disrupt the availability of K⁺, Ca²⁺, Mg²⁺, and SO₄ ^2- for plants. However, ASA treatments alleviated these adverse effects by reducing Na⁺ accumulation and restoring the balance of essential ions especially K⁺ and Ca²⁺, in both leaves and roots as shown in our results ( Figures 7b, c , 8b, c ). This restoration of ion homeostasis an important finding, as it highlights the potential role of ASA enhancing ion selectivity and transport under stressful conditions such as high salinity levels. Especially, notable is ASA’s ability to enhance K⁺ uptake, as observed in alfalfa (Medicago sativa L.) (Chen Z. et al., 2021). Similarly, ASA protects wheat plants from salt stress by improving K⁺ uptake (Zhang G. et al., 2019). Moreover, ASA’s ability restore of Ca²⁺ levels is crucial, as Ca²⁺ supports cell wall integrity and stress signaling under salinity stress (Zhang G. et al., 2019). Similar benefits have been observed in rice (Che Radziah et al., 2016), rapeseed (Brassica campestris cv) (Rainhan et al., 2023), and peanut (Arachis hypogea L.) (Oliveira et al., 2022), where ASA improves ion transport and selectivity while alleviating salt-induced ion imbalances via its antioxidant properties. In our results, ASA demonstrated a dual role, as it not only reduced Na⁺ toxicity, but also boosted the uptake and retention of essential ions, as mentioned above. This dual mechanism combined ASA’s antioxidant properties offers an integrated strategy to mitigate salt-induced inhibition in quinoa. Thus, the alleviation of quinoa growth inhibition under salt-induced stress can be linked to improved ion balance facilitated by ASA, particularly at a concentration of 0.1 μM. The applied ASA concentration and mode of exogenous application influence plant growth, stress tolerance, and overall resilience, as illustrated in Figure 12 . However, this study focused on a single salt concentration (200 mM), and future research should explore a broader range, including 300, 400, and 500 mM, to test ASA in quinoa and other crops. Additionally, the effects of ASA throughout the full quinoa life cycle, from germination to harvest, should be examined, since this study only focused on the seedling stage. Moreover, this study considered a single growth season, so long-term studies across multiple seasons are needed to determine the sustainability and effectiveness of ASA under various soil conditions. Finally, although ASA improved ion homeostasis and osmotic adjustment, its effects on other critical growth parameters, such as root development and yield formation, require further investigation.

In the current study, salt stress inhibited seed germination, growth traits, photosynthetic pigments, chlorophyll a fluorescence, ion balance, and antioxidant activities in quinoa. However, ASA treatments at (0.1, 0.5 µM) significantly mitigated these adverse effects, with 0.1 µM exhibiting the most pronounced improvements. At this optimal concentration, ASA enhanced seed germination indices (SIV and GRI) by regulating osmotic balance and reducing ROS-induced damage. It also maintained photosynthetic pigments and improved PH, LN, and LA, suggesting its important role in promoting cell expansion, elongation, and photosynthetic efficiency. Moreover, ASA enhanced NPQ, ETR, and qP, indicating its role in stabilizing photosynthetic machinery under salt stress. Additionally, ASA-mediated upregulation of proline and soluble sugar biosynthesis contributed to osmotic adjustment and stress tolerance. Furthermore, ASA facilitated ion homeostasis and enhanced photosynthetic efficiency, further reinforcing its potential as a stress-mitigating agent. Statistically, the results indicated that the 0.1 µM ASA mitigated the adverse effects of salinity-induced stress, thus improving growth parameters, photosynthetic efficiency, chlorophyll content, and ion balance. Overall, this study offers important insights into the potential application of ASA as a stress-mitigating agent for quinoa cultivation and other crops subjected to salt stress. The findings suggest that ASA plays a critical role in enhancing germination, growth, and stress tolerance during the sensitive stages of quinoa development under salt stress. These results encourage further investigation into the role of ASA during the sensitive stages of crop growth and yield performance under varying salinity and climatic conditions. Based on these findings, it is recommended to incorporate ASA as a biostimulant in agricultural practices for quinoa exposed to saline stress. Future research should focus on long-term field studies to assess the practical application of ASA for quinoa under different saline-alkaline conditions. In addition, investigations into the molecular mechanisms underlying its protective effects will provide deeper insights into its mechanism of action.