Impact of Melatonin on Oxidative Enzymes and Soluble Metabolites in Salt-Alkali Stressed Poplar (Populus spp.): A Comparative Study of Pretreatment and Post-Treatment Effects

Impact of Melatonin on Oxidative Enzymes and Soluble Metabolites in Salt-Alkali Stressed Poplar (Populus spp.): A Comparative Study of Pretreatment and Post-Treatment Effects | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impact of Melatonin on Oxidative Enzymes and Soluble Metabolites in Salt-Alkali Stressed Poplar (Populus spp.): A Comparative Study of Pretreatment and Post-Treatment Effects Jiefei Nai, Wanpeng He, Tieming Ma, Xidong Han, Zhenxing Luo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8293236/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Melatonin plays a crucial role in modulating plant stress responses; however, its potential for mitigating salt-alkali stress remains incompletely understood. The present study evaluates the efficacy of exogenous melatonin in alleviating moderate salt-alkali stress (120 µM) in poplar ( Populus spp .) seedlings, investigating both pre- and post-stress treatments (0–1000 mmol·L⁻¹). Physiological and morphological parameters including chlorophyll content, antioxidant enzyme activities, and osmolyte accumulation were analyzed to assess stress responses. Under salt-alkali stress, seedlings exhibited elevated stress markers and osmolyte levels, indicating activated stress responses. More importantly, melatonin at a concentration of 200 mmol·L⁻¹ was identified as the most effective in mitigating stress effects, significantly enhancing antioxidant enzyme activities such as superoxide dismutase (SOD) and catalase (CAT), while restoring chlorophyll content and reducing oxidative damage markers like malondialdehyde (MDA). Additionally, it contributed to the regulation of osmotic regulators in the leaves, indicating improved cellular stability under stress conditions. Notably, post-stress application of melatonin required slightly higher concentrations to achieve comparable levels of recovery compared to pre-treatment, underscoring the critical influence of application timing on its efficacy. These findings highlight the valuable insights into the strategic use of melatonin for stress mitigation and provide a foundation for molecular breeding efforts aimed at developing salt-alkali-tolerant poplar varieties. Melatonin Salt-alkali stress oxidative enzymes soluble metabolites Poplar plant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Soil salinization, exacerbated by human activities and climate change, poses a critical threat to global agriculture. Recent studies indicate that over half of the world’s arable land is at risk of salinization and desertification (Tarolli et al., 2024 ) with the FAO reporting more than 1.4 billion hectares affected by salinity, alkalinity, or their combined effects (FAO, 2023). These conditions generate saline-alkali stress, a synergistic phenomenon where salt and alkali stresses coexist, inflicting greater harm on plants than either stress alone. Saline-alkali stress damages plants through four primary mechanisms: osmotic stress oxidative stress (Xu et al., 2022 ), osmotic stress (Hongna et al., 2021 ), ion toxicity (Yuan et al., 2021), and high pH stress (Wang et al., 2021 ). Elevated sodium ion (Na⁺) concentrations and alkaline pH disrupt cellular functions, impairing water uptake and metabolic processes (Zhang et al., 2023 ; Zhao et al., 2020 ). High salt levels increase soil osmotic pressure, inducing “physiological drought”, a state where plants cannot absorb water despite its presence, leading to cellular dehydration, wilting, and even death (Wei et al., 2024 ). Moreover, saline-alkali conditions destabilize redox balance in plant cells, triggering excessive reactive oxygen species (ROS) production (Zhang et al., 2024 ). ROS overaccumulation damages cellular structures, exacerbating oxidative stress and further compromising plant health. These multifaceted stressors collectively reduce agricultural productivity, underscoring the urgency of developing adaptive strategies to mitigate soil degradation and enhance crop resilience. Melatonin (MEL) acts as a potent antioxidant in plants, effectively mitigating oxidative damage by neutralizing ROS and maintaining cellular redox balance (Khan et al., 2022 ). However, under stress conditions, excessive accumulation of ROS can disrupt chlorophyll (Chl), impair light absorption, and reduce photosynthetic efficiency, while lipid peroxidation produces toxic compounds such as malondialdehyde (MDA), which can hinder growth in species like poplar (Harfouche et al., 2014 ). To counter these detrimental effects, plants activate various defense mechanisms, including increased activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as the enhanced production of osmolytes like soluble sugars (sSUG) and proteins (sPRO) to alleviate oxidative and osmotic stress (Qi et al., 2020 ; Zhao et al., 2019 ). Poplar ( Populus spp.), a keystone genus in the Salicaceae family, is a high-value domesticated tree cultivated globally for woody biomass production, with plantations spanning ~ 31.4 million hectares. Comprising over 100 species classified such as Tacamahaca and Aigeiros (Müller et al., 2020 ), poplar is prized for its rapid growth, stress resilience, and ecological versatility, making it vital for industrial timber and reforestation (Guo et al., 2023 ). However, its sensitivity to soil conditions, particularly salt-alkali stress, poses a major threat to agricultural and forestry productivity. This stress destabilizes ion homeostasis, damages cellular membranes, and disrupts photosynthesis and metabolic processes, leading to stunted growth or mortality (Mei et al., 2023 ). While melatonin (MEL) has been shown to enhance oxidative enzymes and soluble metabolites in poplar trees under salt stress, its effectiveness in addressing the more complex and ecologically significant combined salt-alkali stress remains largely unexplored. To date, few studies have examined the effects of melatonin on the hybrid poplar Populus cathayana × canadensis ‘Xinlin 1’ under combined salt-alkali stress (Li et al., 2024 ; Song et al., 2022 ). However, further research is essential to fully understand the effects of melatonin before and after exposure to salt and alkali stress. The objective of this study is to improve our understanding of the impact of exogenous melatonin on essential physiological and biochemical markers, such as Chl content, MDA, sSUG, sPRO, and antioxive enzymes (SOD, POD, CAT) when subjected to salt-alkali stress. The findings will guide strategies for cultivating poplars in saline-alkali conditions, ultimately supporting sustainable timber production and environmental restoration efforts. 2. Materials and Methods In this study, we selected Populus davidiana × P. bolleana 'Baicheng Shanxinyang No. 1,' developed by the Baicheng Forestry Research Institute in Jilin Province, China. A pot experiment was conducted. First, a layer of non-woven fabric was placed at the bottom of each plastic pot. Next, sterilized peat soil and vermiculite were mixed in a 3:1 ratio to create the potting substrate. The height and upper diameter of the plastic pots were both 20 cm, with the substrate level with the upper edge. Seedlings taller than 25 cm were selected. Every six pots were grouped and placed in the same tray. Melatonin was dissolved in alcohol and distilled water, creating six gradients (0, 200, 400, 600, 800, 1000 µM). A 120 mM/L NaCl and Na2CO3 salt solution and a nutrient solution were prepared using distilled water. In this experiment, 0 µM was designated as the control group (CK). Six concentrations of exogenous melatonin solution were sprayed onto the leaves using an agricultural lithium battery high-pressure cyclone sprayer (Dv0.5 < 500 µm). During the experiment, the group subjected to saline-alkali stress followed by melatonin treatment received 100 mL of the 120 mM/L NaCl and Na 2 CO 3 combined stress solution and nutrient solution for each sample. After 48 hours, an air atomizing nozzle was used to spray six concentrations of exogenous melatonin solution onto the leaves, ensuring coverage of the leaf tips. This process was repeated every three days. For the group treated with melatonin before saline-alkali stress, six concentrations of exogenous melatonin solution were sprayed on the leaves, followed by the application of 100 mL of the 120 mM/L NaCl and Na2CO3 combined stress solution and nutrient solution after 24 hours, with the process repeated every three days. As illustrated in the figure: 2.1 Determination of Physiological Indexes In the experiment, we utilized kits provided by Beijing BOX Biotechnology Co., Ltd. (Beijing, China), following the kit instructions to measure the contents of SOD, POD, CAT, MDA, Pro, soluble sugars, soluble proteins, and chlorophyll using an enzyme reader (Infinite F50) (Tecan: Mannedorf, Switzerland). The conductivity of the samples was measured with a DDSJ-308F conductivity meter (Rex Electric Chemical, Shanghai, China). 2.2 Data Analysis Statistical analyses were performed using Microsoft Excel 2017 and SPSS 23 software. All data are presented as mean ± standard error (SE). To assess significant differences among the indicators, we applied two-way ANOVA with a significance level set at 0.05, and the analysis was conducted using ggplot2 in R. 3. Results 3.1 Effects of saline-alkali stress on Poplar plant without melatonin treatment The experiment examined the effects of mixed salt-alkali stress on annual potted poplar cuttings by applying treatments of 0, 40, 80, 120, and 160 mmol·L⁻¹ NaCl and Na₂CO₃. Specifically, the plant growth indicators have been suppressed, as illustrated in Fig. 2 A-D. However, there was an increase in leaf area and height at a dose of 160 mmol·L⁻¹, suggesting a potential reallocation of resources in response to partial mortality (Fig. 3 A, D). Under non-stress conditions, exogenous melatonin did not affect growth (Fig. 3 A). However, escalating stress levels began to inhibit plant height, leaf length, and leaf area at 120 mmol·L⁻¹, while also causes for a increasing the peak of physiological parameters such as antioxidant enzymes, chlorophyll, malondialdehyde, soluble sugars, soluble proteins, and electrical conductivity measured in the second apical leaf (Fig. 4 A-H.). Notably, the antioxidant enzyme peaked at 120 mmol·L⁻¹ before sharply declining, while chlorophyll content consistently decreased. The synthesized data indicated that the optimal concentration threshold for poplar is 120 mmol·L⁻¹ (used as subsequent evaluation), beyond which stressful conditions lead to plant growth suppression and negatively affect physiological and biochemical defenses. 3.2 Effect of melatonin pretreatment on saline-alkali stressed Poplar plant Poplar plants were pretreated with melatonin at concentrations of 0, 200, 400, 600, 800, or 1000 µmol·L⁻¹ for 24 hours, followed by exposure to 120 mmol·L⁻¹ mixed saline-alkali stress. Morphological and physiological parameters were measured every two days, starting one day post-stress induction. As shown in Figs. 5 and 6 , melatonin application significantly increased leaf number under stress compared to the stress-only control, with the most obvious effect observed at 400 µmol·L⁻¹ during the T1 and T2 stages (10 and 14 days). This concentration optimally alleviated stress impacts, while efficacy declined at higher doses (1000 µmol·L⁻¹). In contrast, melatonin pretreatment did not statistically alter ground diameter, plant height, or leaf area. These findings suggest that low melatonin concentrations enhance saline-alkali stress resistance in poplar, primarily through modulating leaf development rather than overall growth metrics. Notably, as illustrated in Figs. 7 D and E, the activities of SOD and CAT exhibited a biphasic response to melatonin concentration. These activities increased at lower concentrations, reached a peak at 200 µmol·L⁻¹, and then decreased at higher concentrations (≥ 400 µmol·L⁻¹). In contrast, POD activity initially decreased at melatonin concentrations below 200 µmol·L⁻¹ but increased at higher concentrations (Fig. 7 C), potentially reflecting its dual role in stress adaptation and alternative signaling pathways. Furthermore, the dynamic changes in soluble metabolites, including sugars and proteins, across the experimental groups at different time points are depicted in Fig. 7 F and G. Melatonin's influence on osmolyte synthesis was found to be concentration-dependent, exhibiting a biphasic effect: lower concentrations (200 µmol·L⁻¹) suppressed the accumulation of soluble sugars and proteins, while higher concentrations (≥ 400 µmol·L⁻¹) significantly enhanced their production. This suggests that melatonin's role in osmoregulation is dose-specific, likely mediated by its dual function as both a signaling molecule and a stress modulator. the 200 µmol·L⁻¹ melatonin pretreatment emerged as the optimal concentration for rescued the plant growth to stressed. To further evaluate these effects, we systematically analyzed MDA and chlorophyll dynamics across the experimental groups, as shown in Fig. 7 B. Specifically, MDA levels decreased progressively with increasing melatonin dosage, indicating that exogenous melatonin effectively preserved membrane integrity by reducing ROS-induced lipid degradation. In addition, chlorophyll content, an essential indicator of photosynthetic capacity, also displayed a biphasic response. While no statistically significant changes in chlorophyll levels were observed during pretreatment (Fig. 7 A), the concentration of 200 µmol·L⁻¹ was noted for its optimal stress-alleviating effect. However, at concentrations exceeding 200 µmol·L⁻¹, chlorophyll levels declined, indicating potential phytotoxicity or resource reallocation resulting from excessively high melatonin doses. 3.3. Effect of melatonin post-treatment on saline-alkali stressed Poplar plant Following exposure to 120 mmol·L⁻¹ mixed saline-alkali stress, the species were treated with melatonin at concentrations of 0, 200, 400, 600, 800, and 1000µmol•L-1, and morphological and physiological parameters were analyzed. Phenotypic analyses revealed pronounced stress-induced reductions in leaf number and leaf area (Figs. 8 and 9 ), indicative of arrested growth. Melatonin treatment, however, counteracted these effects in a dose-dependent manner. Plants treated with 400–1000 µmol·L⁻¹ melatonin exhibited significant increases in leaf number and expanded leaf area compared to untreated controls, with these parameters showing similar stress-mitigating effects across this concentration range. Notably, 200 µmol·L⁻¹ melatonin uniquely reduced leaf number relative to controls despite improving leaf area. Notably, Fig. 10 illustrates the temporal variations in antioxidant enzyme activities (SOD, CAT, and POD) across experimental groups under different melatonin treatments. Over the treatment period, SOD and CAT activities exhibited a progressive decline in all groups. However, melatonin application at 200 µM significantly attenuated this reduction, with SOD and CAT levels remaining markedly higher than those in the untreated control group. Specifically, SOD activity peaked during the T1 phase (10 days), while CAT activity reached its maximum value of 1034.18 U/g in the T2 phase (14 days) under the 200 µM treatment (Fig. 10 C, E). In contrast to SOD and CAT, peroxidase (POD), Notably, the 200 µM melatonin group displayed comparatively lower POD levels than other melatonin-treated groups. Meanwhile, higher melatonin concentrations (600–1000 µM) showed no statistically significant differences in POD activity relative to the control group (Fig. 10 D). We also analyzed temporal changes in MDA and chlorophyll content across experimental groups, as shown in Fig. 10 A, B. MDA levels, a biomarker of oxidative lipid damage, exhibited a biphasic trend in all groups, initially rising before declining post-treatment. Notably, the 1000 µM melatonin group demonstrated the most pronounced stress-alleviating effect, with MDA accumulation reduced to the lowest observed levels at 200µM. In parallel, chlorophyll content exhibited a distinct dose-dependent pattern. Under 200 µM melatonin treatment, chlorophyll concentrations reached their minimum values; however, a progressive recovery was noted at higher doses (400–1000 µM). This recovery was particularly evident during the T1 phase (10 days), where chlorophyll levels rebounded significantly. Furthermore, we analyzed the osmotic regulators, as Fig. 10 F, G illustrates the temporal dynamics of osmotic regulator content (soluble sugars and proteins) across experimental groups exposed to varying concentrations of melatonin. Soluble sugar levels reached their lowest point on day 25 across all melatonin-treated groups, with the 200 µM group recording the minimum value (3.61 mg g⁻¹). Similarly, under the 200 µM melatonin treatment, soluble protein content dropped to minimal levels of 821.5 µg g⁻¹ and 658.2 µg g⁻¹ on days 14 and 18, respectively. Importantly, melatonin concentration had a dose-dependent effect on osmotic regulator accumulation. The 200 µM and 400 µM groups consistently showed significantly lower average soluble sugar and slightly reduced protein levels compared to the control group. Conversely, higher melatonin doses (800–1000 µM) resulted in moderately increased osmotic regulator content relative to the control, although these differences were less obvious. 4. Discussion 4.1. Melatonin impacts on plants under salt-alkali stress conditions Among numerous abiotic stresses, salt-alkali is one of the most common environmental factors that restrict plant growth and development. Under salt-alkali stress, the concentration of Na + ions in plants increases, resulting in an imbalance between Na + and K+, which harms the plants’ functionality (Khan et al., 2022 ). Melatonin, a pleiotropic signaling molecule, has garnered significant attention in plant physiology for its dual capacity to modulate growth dynamics and enhance resilience to abiotic stress (Zhang et al., 2022 ). This study systematically evaluates the role of melatonin in mitigating salt-alkali stress in poplar ( Populus spp.), focusing on its regulatory effects on oxidative enzyme activity and soluble metabolite profiles. To delineate melatonin’s stress-alleviating potential, we employed a dual-phase experimental design: (1) initial screening of salt-alkali stress intensity using various concentrations (0, 40, 80, 120, 160 mmol·L⁻¹), and (2) subsequent application of melatonin at varying doses (0, 200, 400, 600, 800, 1000 µmol·L⁻¹) under pre- and post-treatment schedules. The initial stress response identified 120 mmol·L⁻¹ as the threshold salt-alkali concentration inducing significant physiological disruption, which was then adopted as the baseline stress condition, aligning with previous studies (Song et al., 2022 ). This concentration was subsequently applied to assess melatonin’s efficacy in alleviating stress when administered either before or after exposure to stress. The dual experimental approach comparing stress-only conditions with melatonin-augmented interventions provides a comprehensive understanding of how melatonin influences stress adaptation in poplar. Salt-alkali stress primarily damages plants by disrupting cell membrane integrity and inducing ROS accumulation, which plants counteract via antioxidant enzyme systems such as SOD, CAT, and POD. In this study, melatonin pretreatment enhanced salt-alkali tolerance in poplar seedlings by controlling antioxidant enzyme activity. In particular, under stress conditions, SOD and CAT activities increased, with further amplification following melatonin application. However, enzyme responses demonstrated concentration-dependent biphasic behavior: SOD and CAT activities peaked at 200 µM melatonin but declined at higher concentrations (≥ 400 µM), whereas POD activity initially decreased at 200 µM before rebounding at elevated doses, highlighting its multifaceted role in stress signaling. These findings align with previous studies demonstrating that exogenous melatonin at 100 µM boosts SOD and CAT activities in stressed poplar and apple plants, mitigating oxidative damage by scavenging ROS and stabilizing membranes (Song et al., 2022 ; Xian et al., 2024 ). Notably, while post-treatment melatonin slightly increased CAT and POD activities, SOD activity remained unchanged at lower doses, and all enzymes declined at higher concentrations, emphasizing the critical role of dosage optimization. Similarly, managing melatonin as a pretreatment notably improves the salinity tolerance of poplar seedlings. This enhancement is achieved through efficiently scavenging of ROS and improvement of cellular membrane stability, thereby effectively reducing oxidative damage caused by salt (Song et al., 2022 ). Interestingly, a recent study on the transcriptome and metabolome shown of poplar seedlings has demonstrated that melatonin affects genes and metabolites associated with stress tolerance, with findings indicating that lower concentrations (100 µM) are supported by recent multi-omics studies (Duan et al., 2022 ; Li et al., 2024 ). Furthermore, prior research has shown that melatonin treatment significantly enhances the salt and drought tolerance of rice plants. This effect is realized by strengthening antioxidant defense mechanisms and upregulating stress-responsive genes, including OsSOS , OsNHX , OsHSF , and OsDREB , in rice (Khan et al., 2024 ). Notably, we strongly suggest prioritizing the identification of regulatory genes underlying stress response pathways in poplar plants subjected to abiotic stressors. Similarly, our results align with earlier research indicating that low levels of exogenous melatonin can significantly enhance antioxidant enzyme activity in Triticum aestivum and Pennisetum glaucum when exposed to salt or drought stress (Awan et al., 2024 ; Zhang et al., 2022 ). These findings collectively highlight melatonin’s potential as a priming agent to enhance stress tolerance, though its concentration-specific effects require further mechanistic investigation. In another, melatonin pre-treatment application induced distinct morphological responses in poplar seedlings exposed to salt-alkali stress. Plants treated with 400 µmol·L⁻¹ melatonin showed significant increases in leaf number and expanded leaf area compared to untreated controls, demonstrating consistent stress-mitigating effects across this concentration range. Interestingly, pretreatment with 200 µmol·L⁻¹ melatonin resulted in a decrease in leaf number compared to controls, despite causing an increase in leaf area. This indicates a dose-dependent variation in physiological responses. Additionally, most recent studies have shown that low concentrations of pretreatment melatonin (100 µmol·L⁻¹) can promote plant growth and help alleviate the negative impacts of saline-alkali stress on tomato plants (Dou et al., 2025 ). Furthermore, an early study on cotton ( Gossypium hirsutum ) demonstrated that the combined stress of salt and drought results in a significant reduction in plant growth and chlorophyll content (Ibrahim et al., 2019 ). In contrast, the post-treatment application of melatonin reversed these benefits; under stress conditions, leaf number significantly increased compared to controls exposed only to stress. The most notable alleviation was observed at 400 µmol·L⁻¹ across different growth stages, while higher concentrations, such as 1000 µmol·L⁻¹, exhibited reduced effectiveness. Notably, melatonin pretreatment did not statistically alter ground diameter, plant height, or overall leaf area, though subtle similarities between pre- and post-treatment conditions were observed. Seedlings subjected to saline-alkaline stress exhibited increased plant height, stem diameter, leaf number, and leaf area, implying that elevated melatonin concentrations may be required post-stress to fully mitigate damage, as stress conditions likely increase physiological demands. This differential response highlights the nuanced role of melatonin dosage and timing in modulating morphological adaptations, with optimal stress alleviation achieved at intermediate concentrations (400 µmol·L⁻¹), while excessive doses may disrupt beneficial effects. The observed trends suggest that melatonin’s efficacy in enhancing salt-alkali tolerance depends on balancing its regulatory influence on growth parameters with its capacity to counteract stress-induced oxidative and ionic imbalances. This study revealed the dose-dependent effects of melatonin on osmoregulation in poplar seedlings under salt-alkali stress. Lower melatonin concentrations (200 µmol·L⁻¹) suppressed the accumulation of soluble sugars and proteins during pretreatment, while higher concentrations (≥ 400 µmol·L⁻¹) significantly enhanced their production, underscoring melatonin’s dual role as both a signaling molecule and a stress modulator in osmoregulation. Notably, the 200 µmol·L⁻¹ pretreatment emerged as the optimal dose for improving stress resilience despite its inhibitory effect on osmolyte levels. A similar pattern was observed after salt-alkali stress induction: seedlings treated with 200 µM or 400 µM melatonin exhibited consistently lower soluble sugar levels and slightly reduced protein content compared to untreated controls. This aligns with previous findings by Song et al. ( 2022 ), which demonstrated that low concentrations of exogenous melatonin can significantly alter osmoregulation activity in plants. Saline-alkaline stress triggers initial root-derived stress signals that disrupt aboveground plant growth by degrading photosynthetic pigments, accelerating leaf senescence, and reducing photosynthetic capacity. Exogenous melatonin application mitigates these effects by slowing chlorophyll decline and preserving membrane integrity in poplar plants (Song et al., 2022 ). In this study, we confirmed that melatonin pretreatment significantly reduced MDA levels, a marker of oxidative membrane damage, with the lowest MDA observed at 200 µmol·L⁻¹. A recent study has demonstrated that pretreatment with melatonin reduces MDA content in rice plants under salt stress (Ubaidillah et al., 2024 ). Conversely, post-stress melatonin supplementation showed concentration-dependent efficacy: MDA levels decreased progressively with higher doses, with the 1000 µM treatment group showing the most significant alleviation of oxidative stress. In parallel, pretreatment with melatonin did not statistically alter chlorophyll levels; however, post-treatment application under stress revealed a dose-dependent recovery. At 200 µM, chlorophyll concentrations dropped to minimal levels, yet higher doses (400–1000 µM) induced progressive recovery, particularly during the T2 phase (14 days), where chlorophyll rebounded significantly. This suggests that post-stress conditions required higher melatonin concentrations to counteract severe oxidative damage as stress intensifies physiological demands. Notably, melatonin’s ability to stabilize membranes and scavenge free radicals aligns with previous findings (Dou et al., 2025 ; Li et al., 2024 ; Song et al., 2022 ), where lower concentrations (100 µmol·L⁻¹) alleviated stress by reducing cellular damage. Our results highlight melatonin’s dual role in osmoregulation and antioxidant defense, demonstrating optimal efficacy at 400–1000 µM post-stress. This concentration enhances the synthesis of photosynthetic pigments and enhances resilience by counteracting chlorophyll degradation and oxidative damage. These findings emphasize the significance of dosage and timing in melatonin’s protective function, indicating its potential as a targeted intervention for enhancing plant tolerance under saline-alkaline stress. Conclusion Overall, our findings demonstrate that exogenous melatonin application is a promising strategy for enhancing salt-alkali stress tolerance in poplar seedlings. More importantly, it primarily contributes to improving plant growth, chlorophyll synthesis, antioxidant enzyme activity (SOD, CAT), and osmoregulatory capacity, collectively improving ROS scavenging capacity and significantly restoring the growth and development of salt-alkali-stressed poplar seedlings by enhancing physiological and morphological resilience. While pre-melatonin application serves as a cost-effective strategy to improve stress resistance, post-treatment supplementation has also proven effective in alleviating the effects of stress, further enhancing tolerance, and reducing agricultural losses. These dual-phase benefits highlight melatonin’s versatility as a protective agent, employing both preventive and reparative mechanisms to counteract stress-induced damage, as illustrated in the proposed model (Fig. 11 ). Declarations Author Contributions: Conceptualization, Z.N. and X.Z.; methodology, Z.N. and X.Z.; validation, Z.N. and X.Z.; formal analysis, J.N, X.Z., W.H., T.M., X.H., Z.L., X.L., X.L., J.S.; data curation, J.N, and X.Z.; writing original draft preparation, J.N, X.H., X.L., and J.S.; writing review and editing, J.N, W.H., T.M., Z.L., X.L., J.S.; visualization, X.Z.; project administration, X.Z.; funding acquisition X.Z. All authors have read and agreed to the published version of the manuscript. Funding: We are grateful to the researchers who are contributing to this field. This paper was funded by the Scientific Research Project of the Education Department of Jilin Province (JJKH20250597KJ). Ethics approval and consent to participate: Not applicable. 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The black flag indicates the day the samples were treated with melatonin (this illustration applies to all melatonin gradients); the green flag indicates the day the samples were treated with NaCl and Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e; the red flag indicates the day the samples were collected.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/b126feb44f67481ce33bca74.png"},{"id":99215777,"identity":"0afd9c93-1de8-4757-8277-7ce1856b3445","added_by":"auto","created_at":"2025-12-30 08:56:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108406,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different salt and alkali treatments on the growth characteristics of poplar plants. \u003cstrong\u003eA.\u003c/strong\u003e plant height \u003cstrong\u003eB.\u003c/strong\u003e ground diameter \u003cstrong\u003eC.\u003c/strong\u003e number of leaves \u003cstrong\u003eD.\u003c/strong\u003e leaf area\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/4968893dd57b4a751f07bc49.png"},{"id":99215799,"identity":"4d20b420-3dbf-4404-8d56-6989cada7cad","added_by":"auto","created_at":"2025-12-30 08:56:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":398636,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different salt and alkali treatments on the morphology of Poplar plants.\u003cbr\u003e\nA, B, C, D, and E represent 0, 40, 80, 120, and 160mmol.L-1Nacl-NaCO\u003csub\u003e3\u003c/sub\u003e mixed salt-alkali treatment respectively\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/f7503995aa923c013a1a71a9.png"},{"id":99215822,"identity":"040d34a2-dd57-4d6a-b8fb-4e7a8589e4a4","added_by":"auto","created_at":"2025-12-30 08:56:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70721,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of salt-alkali treatment with different concentrations on physiological indexes of Poplar plants: \u003cstrong\u003eA.\u003c/strong\u003e electrical conductivity; \u003cstrong\u003eB.\u003c/strong\u003e Chlorophyll content; \u003cstrong\u003eC.\u003c/strong\u003e malondialdehyde content; \u003cstrong\u003eD.\u003c/strong\u003e superoxide dismutase content; \u003cstrong\u003eE.\u003c/strong\u003e peroxidase content; \u003cstrong\u003eF.\u003c/strong\u003e Catalase content; \u003cstrong\u003eG.\u003c/strong\u003e soluble protein content; \u003cstrong\u003eH.\u003c/strong\u003e soluble protein content. T1 means 10 days, T2 means 14 days, T3 means 18 days, T4 means 22 days, T5 means 26 days. The data is represented as the mean of three biological replicates, the error bar represents the standard deviation, and the lowercase letter \"abc\" indicates that the\u003cbr\u003e\ndata has a significant difference between P \u0026lt; 0.05, passing the \u003cem\u003eT-test.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/0d91a7e4b422d28992fef36c.png"},{"id":99215791,"identity":"e9878c4b-1fc0-42aa-9e25-b60493675107","added_by":"auto","created_at":"2025-12-30 08:56:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":108822,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of melatonin pretreatment on morphological indexes of Poplar plants under\u003cbr\u003e\nsaline-alkali stress \u003cstrong\u003eA\u003c/strong\u003e. plant height \u003cstrong\u003eB. \u003c/strong\u003eground diameter \u003cstrong\u003eC. \u003c/strong\u003enumber of leaves \u003cstrong\u003eD\u003c/strong\u003e. \u0026nbsp;leaf area. The data is represented as the mean of three biological replicates, the error bar represents the standard deviation, and the lowercase letter \"abc\" indicates that the data has a significant difference between P \u0026lt; 0.05, passing the \u003cem\u003eT-test\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/c9b37bbfc1c5e118974bae50.png"},{"id":99215784,"identity":"c6ddceae-de85-4197-b12a-491b3de08787","added_by":"auto","created_at":"2025-12-30 08:56:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":275291,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different melatonin pretreatments on the morphology traits of poplar plant under saline-alkali stress. A, B, C, D, E and F represent 0, 200, 400, 600, 800 and 1000 µmol·L⁻¹ melatonin pretreatment respectively\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/45618923f01cef5133fa77be.png"},{"id":99215808,"identity":"fbabdf30-d694-433d-8c14-9f4c0f71d7d4","added_by":"auto","created_at":"2025-12-30 08:56:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":67532,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different concentrations of melatonin pretreatment on physiological\u003cbr\u003e\ncharacteristics. \u003cstrong\u003eA.\u003c/strong\u003e chlorophyll content \u003cstrong\u003eB.\u003c/strong\u003e malondialdehyde content \u003cstrong\u003eC.\u003c/strong\u003e peroxidase content \u003cstrong\u003eD.\u003c/strong\u003e \u003cbr\u003e\nsuperoxide dismutase content \u003cstrong\u003eE. \u003c/strong\u003ecatalase content \u003cstrong\u003eF.\u003c/strong\u003e soluble sugar content. \u003cstrong\u003eG.\u003c/strong\u003e soluble protein content. T1 means 10 days, T2 means 14 days, T3 means 18 days, T4 means\u003cbr\u003e\n22 days, T5 means 26 days. The data is represented as the mean of three biological replicates, the error bar represents the standard deviation, and the lowercase letter \"abc\" indicates that the data has a significant difference between P \u0026lt; 0.05, passing the \u003cem\u003eT-test.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/ea6dcbd2e32d0e273ef5541a.png"},{"id":99317835,"identity":"e37a083f-bb37-4194-b6e7-b562c33001e7","added_by":"auto","created_at":"2025-12-31 16:30:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":130471,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different concentrations of melatonin on the growth characteristics of Aspen after salt and alkali stress. \u003cstrong\u003eA.\u003c/strong\u003e plant height \u003cstrong\u003eB. \u003c/strong\u003eground diameter \u003cstrong\u003eC. \u003c/strong\u003enumber of leaves \u003cstrong\u003eD. \u003c/strong\u003eleaf area. The data is represented as the mean of three biological replicates, the error bar represents the\u003cbr\u003e\nstandard deviation, and the lowercase letter \"abc\" indicates that the data has a significant difference between P \u0026lt; 0.05, passing the \u003cem\u003eT-test\u003c/em\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/836ce0b850ede15870e25958.png"},{"id":99215766,"identity":"d927835e-6468-4c7b-b0b6-3934bd189077","added_by":"auto","created_at":"2025-12-30 08:56:06","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":254570,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different concentrations of melatonin on the morphology of Poplar plant under saline-alkali stress. A, B, C, D, E and F represent 0, 200, 400, 600, 800 and 1000 µmol·L⁻¹ melatonin treatment, respectively\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/5425b9643ef93f48d4b3263b.png"},{"id":99215809,"identity":"81851c3f-46df-43e4-b6fb-44485e40fde3","added_by":"auto","created_at":"2025-12-30 08:56:16","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":76352,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different concentrations of melatonin treatment on physiological characteristics of Poplar plant after salt and alkali stress \u003cstrong\u003eA.\u003c/strong\u003e chlorophyll content \u003cstrong\u003eB.\u003c/strong\u003e malondialdehyde content \u003cstrong\u003eC.\u003c/strong\u003e peroxidase content \u003cstrong\u003eD.\u003c/strong\u003e superoxide dismutase content \u003cstrong\u003eE. \u003c/strong\u003ecatalase content \u003cstrong\u003eF.\u003c/strong\u003e soluble sugar content. \u003cstrong\u003eG.\u003c/strong\u003e soluble protein content. T1 means 10 days, T2 means 14 days, T3 means 18 days, T4 means 22 days, and T5 means 26 days. The data is represented as the mean of three biological replicates, the error bar represents the standard deviation, and the lowercase letter \"abc\" indicates that the data has a significant difference between P \u0026lt; 0.05, passing the \u003cem\u003eT-test\u003c/em\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/64dbb06056913ec5ec8780b8.png"},{"id":99215807,"identity":"a93795ca-8e59-479b-8c09-e73cad238d05","added_by":"auto","created_at":"2025-12-30 08:56:15","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":205126,"visible":true,"origin":"","legend":"\u003cp\u003eProposed model for the response of poplar plants to melatonin under salt-alkali stress.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/ee22adbcab1e1cfbe28597d9.png"},{"id":103504539,"identity":"fe20a887-5814-4b71-9c24-5a61df44e122","added_by":"auto","created_at":"2026-02-26 13:20:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2503021,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/e893cab1-7e71-4475-9f96-0c26e64b254f.pdf"},{"id":99318084,"identity":"4ba65c38-bb01-499f-bfff-a8748f251686","added_by":"auto","created_at":"2025-12-31 16:31:27","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":422855,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8293236/v1/98ac3fa1d1571a82c0f2c5c8.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eImpact of Melatonin on Oxidative Enzymes and Soluble Metabolites in Salt-Alkali Stressed Poplar (Populus spp.): A Comparative Study of Pretreatment and Post-Treatment Effects\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSoil salinization, exacerbated by human activities and climate change, poses a critical threat to global agriculture. Recent studies indicate that over half of the world\u0026rsquo;s arable land is at risk of salinization and desertification (Tarolli et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) with the FAO reporting more than 1.4\u0026nbsp;billion hectares affected by salinity, alkalinity, or their combined effects (FAO, 2023). These conditions generate saline-alkali stress, a synergistic phenomenon where salt and alkali stresses coexist, inflicting greater harm on plants than either stress alone. Saline-alkali stress damages plants through four primary mechanisms: osmotic stress oxidative stress (Xu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), osmotic stress (Hongna et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), ion toxicity (Yuan et al., 2021), and high pH stress (Wang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Elevated sodium ion (Na⁺) concentrations and alkaline pH disrupt cellular functions, impairing water uptake and metabolic processes (Zhang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). High salt levels increase soil osmotic pressure, inducing \u0026ldquo;physiological drought\u0026rdquo;, a state where plants cannot absorb water despite its presence, leading to cellular dehydration, wilting, and even death (Wei et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, saline-alkali conditions destabilize redox balance in plant cells, triggering excessive reactive oxygen species (ROS) production (Zhang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). ROS overaccumulation damages cellular structures, exacerbating oxidative stress and further compromising plant health. These multifaceted stressors collectively reduce agricultural productivity, underscoring the urgency of developing adaptive strategies to mitigate soil degradation and enhance crop resilience.\u003c/p\u003e \u003cp\u003eMelatonin (MEL) acts as a potent antioxidant in plants, effectively mitigating oxidative damage by neutralizing ROS and maintaining cellular redox balance (Khan et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, under stress conditions, excessive accumulation of ROS can disrupt chlorophyll (Chl), impair light absorption, and reduce photosynthetic efficiency, while lipid peroxidation produces toxic compounds such as malondialdehyde (MDA), which can hinder growth in species like poplar (Harfouche et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). To counter these detrimental effects, plants activate various defense mechanisms, including increased activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as the enhanced production of osmolytes like soluble sugars (sSUG) and proteins (sPRO) to alleviate oxidative and osmotic stress (Qi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePoplar (\u003cem\u003ePopulus\u003c/em\u003e spp.), a keystone genus in the \u003cem\u003eSalicaceae\u003c/em\u003e family, is a high-value domesticated tree cultivated globally for woody biomass production, with plantations spanning\u0026thinsp;~\u0026thinsp;31.4\u0026nbsp;million hectares. Comprising over 100 species classified such as \u003cem\u003eTacamahaca\u003c/em\u003e and \u003cem\u003eAigeiros\u003c/em\u003e (M\u0026uuml;ller et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), poplar is prized for its rapid growth, stress resilience, and ecological versatility, making it vital for industrial timber and reforestation (Guo et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, its sensitivity to soil conditions, particularly salt-alkali stress, poses a major threat to agricultural and forestry productivity. This stress destabilizes ion homeostasis, damages cellular membranes, and disrupts photosynthesis and metabolic processes, leading to stunted growth or mortality (Mei et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile melatonin (MEL) has been shown to enhance oxidative enzymes and soluble metabolites in poplar trees under salt stress, its effectiveness in addressing the more complex and ecologically significant combined salt-alkali stress remains largely unexplored. To date, few studies have examined the effects of melatonin on the hybrid poplar Populus \u003cem\u003ecathayana \u0026times; canadensis\u003c/em\u003e \u0026lsquo;Xinlin 1\u0026rsquo; under combined salt-alkali stress (Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, further research is essential to fully understand the effects of melatonin before and after exposure to salt and alkali stress. The objective of this study is to improve our understanding of the impact of exogenous melatonin on essential physiological and biochemical markers, such as Chl content, MDA, sSUG, sPRO, and antioxive enzymes (SOD, POD, CAT) when subjected to salt-alkali stress. The findings will guide strategies for cultivating poplars in saline-alkali conditions, ultimately supporting sustainable timber production and environmental restoration efforts.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eIn this study, we selected Populus davidiana \u0026times; P. bolleana 'Baicheng Shanxinyang No. 1,' developed by the Baicheng Forestry Research Institute in Jilin Province, China.\u003c/p\u003e \u003cp\u003eA pot experiment was conducted. First, a layer of non-woven fabric was placed at the bottom of each plastic pot. Next, sterilized peat soil and vermiculite were mixed in a 3:1 ratio to create the potting substrate. The height and upper diameter of the plastic pots were both 20 cm, with the substrate level with the upper edge. Seedlings taller than 25 cm were selected. Every six pots were grouped and placed in the same tray. Melatonin was dissolved in alcohol and distilled water, creating six gradients (0, 200, 400, 600, 800, 1000 \u0026micro;M). A 120 mM/L NaCl and Na2CO3 salt solution and a nutrient solution were prepared using distilled water. In this experiment, 0 \u0026micro;M was designated as the control group (CK). Six concentrations of exogenous melatonin solution were sprayed onto the leaves using an agricultural lithium battery high-pressure cyclone sprayer (Dv0.5\u0026thinsp;\u0026lt;\u0026thinsp;500 \u0026micro;m).\u003c/p\u003e \u003cp\u003eDuring the experiment, the group subjected to saline-alkali stress followed by melatonin treatment received 100 mL of the 120 mM/L NaCl and Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e combined stress solution and nutrient solution for each sample. After 48 hours, an air atomizing nozzle was used to spray six concentrations of exogenous melatonin solution onto the leaves, ensuring coverage of the leaf tips. This process was repeated every three days. For the group treated with melatonin before saline-alkali stress, six concentrations of exogenous melatonin solution were sprayed on the leaves, followed by the application of 100 mL of the 120 mM/L NaCl and Na2CO3 combined stress solution and nutrient solution after 24 hours, with the process repeated every three days. As illustrated in the figure:\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Determination of Physiological Indexes\u003c/h2\u003e \u003cp\u003eIn the experiment, we utilized kits provided by Beijing BOX Biotechnology Co., Ltd. (Beijing, China), following the kit instructions to measure the contents of SOD, POD, CAT, MDA, Pro, soluble sugars, soluble proteins, and chlorophyll using an enzyme reader (Infinite F50) (Tecan: Mannedorf, Switzerland). The conductivity of the samples was measured with a DDSJ-308F conductivity meter (Rex Electric Chemical, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Data Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Microsoft Excel 2017 and SPSS 23 software. All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). To assess significant differences among the indicators, we applied two-way ANOVA with a significance level set at 0.05, and the analysis was conducted using ggplot2 in R.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Effects of saline-alkali stress on Poplar plant without melatonin treatment\u003c/h2\u003e\n \u003cp\u003eThe experiment examined the effects of mixed salt-alkali stress on annual potted poplar cuttings by applying treatments of 0, 40, 80, 120, and 160 mmol\u0026middot;L⁻\u0026sup1; NaCl and Na₂CO₃. Specifically, the plant growth indicators have been suppressed, as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-D. However, there was an increase in leaf area and height at a dose of 160 mmol\u0026middot;L⁻\u0026sup1;, suggesting a potential reallocation of resources in response to partial mortality (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, D). Under non-stress conditions, exogenous melatonin did not affect growth (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, escalating stress levels began to inhibit plant height, leaf length, and leaf area at 120 mmol\u0026middot;L⁻\u0026sup1;, while also causes for a increasing the peak of physiological parameters such as antioxidant enzymes, chlorophyll, malondialdehyde, soluble sugars, soluble proteins, and electrical conductivity measured in the second apical leaf (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-H.). Notably, the antioxidant enzyme peaked at 120 mmol\u0026middot;L⁻\u0026sup1; before sharply declining, while chlorophyll content consistently decreased. The synthesized data indicated that the optimal concentration threshold for poplar is 120 mmol\u0026middot;L⁻\u0026sup1; (used as subsequent evaluation), beyond which stressful conditions lead to plant growth suppression and negatively affect physiological and biochemical defenses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Effect of melatonin pretreatment on saline-alkali stressed Poplar plant\u003c/h2\u003e\n \u003cp\u003ePoplar plants were pretreated with melatonin at concentrations of 0, 200, 400, 600, 800, or 1000 \u0026micro;mol\u0026middot;L⁻\u0026sup1; for 24 hours, followed by exposure to 120 mmol\u0026middot;L⁻\u0026sup1; mixed saline-alkali stress. Morphological and physiological parameters were measured every two days, starting one day post-stress induction. As shown in Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, melatonin application significantly increased leaf number under stress compared to the stress-only control, with the most obvious effect observed at 400 \u0026micro;mol\u0026middot;L⁻\u0026sup1; during the T1 and T2 stages (10 and 14 days). This concentration optimally alleviated stress impacts, while efficacy declined at higher doses (1000 \u0026micro;mol\u0026middot;L⁻\u0026sup1;). In contrast, melatonin pretreatment did not statistically alter ground diameter, plant height, or leaf area. These findings suggest that low melatonin concentrations enhance saline-alkali stress resistance in poplar, primarily through modulating leaf development rather than overall growth metrics.\u003c/p\u003e\n \u003cp\u003eNotably, as illustrated in Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD and E, the activities of SOD and CAT exhibited a biphasic response to melatonin concentration. These activities increased at lower concentrations, reached a peak at 200 \u0026micro;mol\u0026middot;L⁻\u0026sup1;, and then decreased at higher concentrations (\u0026ge;\u0026thinsp;400 \u0026micro;mol\u0026middot;L⁻\u0026sup1;). In contrast, POD activity initially decreased at melatonin concentrations below 200 \u0026micro;mol\u0026middot;L⁻\u0026sup1; but increased at higher concentrations (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC), potentially reflecting its dual role in stress adaptation and alternative signaling pathways.\u003c/p\u003e\n \u003cp\u003eFurthermore, the dynamic changes in soluble metabolites, including sugars and proteins, across the experimental groups at different time points are depicted in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF and G. Melatonin\u0026apos;s influence on osmolyte synthesis was found to be concentration-dependent, exhibiting a biphasic effect: lower concentrations (200 \u0026micro;mol\u0026middot;L⁻\u0026sup1;) suppressed the accumulation of soluble sugars and proteins, while higher concentrations (\u0026ge;\u0026thinsp;400 \u0026micro;mol\u0026middot;L⁻\u0026sup1;) significantly enhanced their production. This suggests that melatonin\u0026apos;s role in osmoregulation is dose-specific, likely mediated by its dual function as both a signaling molecule and a stress modulator. the 200 \u0026micro;mol\u0026middot;L⁻\u0026sup1; melatonin pretreatment emerged as the optimal concentration for rescued the plant growth to stressed.\u003c/p\u003e\n \u003cp\u003eTo further evaluate these effects, we systematically analyzed MDA and chlorophyll dynamics across the experimental groups, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB. Specifically, MDA levels decreased progressively with increasing melatonin dosage, indicating that exogenous melatonin effectively preserved membrane integrity by reducing ROS-induced lipid degradation. In addition, chlorophyll content, an essential indicator of photosynthetic capacity, also displayed a biphasic response. While no statistically significant changes in chlorophyll levels were observed during pretreatment (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA), the concentration of 200 \u0026micro;mol\u0026middot;L⁻\u0026sup1; was noted for its optimal stress-alleviating effect. However, at concentrations exceeding 200 \u0026micro;mol\u0026middot;L⁻\u0026sup1;, chlorophyll levels declined, indicating potential phytotoxicity or resource reallocation resulting from excessively high melatonin doses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Effect of melatonin post-treatment on saline-alkali stressed Poplar plant\u003c/h2\u003e\n \u003cp\u003eFollowing exposure to 120 mmol\u0026middot;L⁻\u0026sup1; mixed saline-alkali stress, the species were treated with melatonin at concentrations of 0, 200, 400, 600, 800, and 1000\u0026micro;mol\u0026bull;L-1, and morphological and physiological parameters were analyzed. Phenotypic analyses revealed pronounced stress-induced reductions in leaf number and leaf area (Figs. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e), indicative of arrested growth. Melatonin treatment, however, counteracted these effects in a dose-dependent manner. Plants treated with 400\u0026ndash;1000 \u0026micro;mol\u0026middot;L⁻\u0026sup1; melatonin exhibited significant increases in leaf number and expanded leaf area compared to untreated controls, with these parameters showing similar stress-mitigating effects across this concentration range. Notably, 200 \u0026micro;mol\u0026middot;L⁻\u0026sup1; melatonin uniquely reduced leaf number relative to controls despite improving leaf area.\u003c/p\u003e\n \u003cp\u003eNotably, Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e illustrates the temporal variations in antioxidant enzyme activities (SOD, CAT, and POD) across experimental groups under different melatonin treatments. Over the treatment period, SOD and CAT activities exhibited a progressive decline in all groups. However, melatonin application at 200 \u0026micro;M significantly attenuated this reduction, with SOD and CAT levels remaining markedly higher than those in the untreated control group. Specifically, SOD activity peaked during the T1 phase (10 days), while CAT activity reached its maximum value of 1034.18 U/g in the T2 phase (14 days) under the 200 \u0026micro;M treatment (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eC, E). In contrast to SOD and CAT, peroxidase (POD), Notably, the 200 \u0026micro;M melatonin group displayed comparatively lower POD levels than other melatonin-treated groups. Meanwhile, higher melatonin concentrations (600\u0026ndash;1000 \u0026micro;M) showed no statistically significant differences in POD activity relative to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003eWe also analyzed temporal changes in MDA and chlorophyll content across experimental groups, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA, B. MDA levels, a biomarker of oxidative lipid damage, exhibited a biphasic trend in all groups, initially rising before declining post-treatment. Notably, the 1000 \u0026micro;M melatonin group demonstrated the most pronounced stress-alleviating effect, with MDA accumulation reduced to the lowest observed levels at 200\u0026micro;M. In parallel, chlorophyll content exhibited a distinct dose-dependent pattern. Under 200 \u0026micro;M melatonin treatment, chlorophyll concentrations reached their minimum values; however, a progressive recovery was noted at higher doses (400\u0026ndash;1000 \u0026micro;M). This recovery was particularly evident during the T1 phase (10 days), where chlorophyll levels rebounded significantly.\u003c/p\u003e\n \u003cp\u003eFurthermore, we analyzed the osmotic regulators, as Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eF, G illustrates the temporal dynamics of osmotic regulator content (soluble sugars and proteins) across experimental groups exposed to varying concentrations of melatonin. Soluble sugar levels reached their lowest point on day 25 across all melatonin-treated groups, with the 200 \u0026micro;M group recording the minimum value (3.61 mg g⁻\u0026sup1;). Similarly, under the 200 \u0026micro;M melatonin treatment, soluble protein content dropped to minimal levels of 821.5 \u0026micro;g g⁻\u0026sup1; and 658.2 \u0026micro;g g⁻\u0026sup1; on days 14 and 18, respectively. Importantly, melatonin concentration had a dose-dependent effect on osmotic regulator accumulation. The 200 \u0026micro;M and 400 \u0026micro;M groups consistently showed significantly lower average soluble sugar and slightly reduced protein levels compared to the control group. Conversely, higher melatonin doses (800\u0026ndash;1000 \u0026micro;M) resulted in moderately increased osmotic regulator content relative to the control, although these differences were less obvious.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Melatonin impacts on plants under salt-alkali stress conditions\u003c/h2\u003e \u003cp\u003eAmong numerous abiotic stresses, salt-alkali is one of the most common environmental factors that restrict plant growth and development. Under salt-alkali stress, the concentration of Na\u0026thinsp;+\u0026thinsp;ions in plants increases, resulting in an imbalance between Na\u0026thinsp;+\u0026thinsp;and K+, which harms the plants\u0026rsquo; functionality (Khan et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Melatonin, a pleiotropic signaling molecule, has garnered significant attention in plant physiology for its dual capacity to modulate growth dynamics and enhance resilience to abiotic stress (Zhang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study systematically evaluates the role of melatonin in mitigating salt-alkali stress in poplar (\u003cem\u003ePopulus\u003c/em\u003e spp.), focusing on its regulatory effects on oxidative enzyme activity and soluble metabolite profiles. To delineate melatonin\u0026rsquo;s stress-alleviating potential, we employed a dual-phase experimental design: (1) initial screening of salt-alkali stress intensity using various concentrations (0, 40, 80, 120, 160 mmol\u0026middot;L⁻\u0026sup1;), and (2) subsequent application of melatonin at varying doses (0, 200, 400, 600, 800, 1000 \u0026micro;mol\u0026middot;L⁻\u0026sup1;) under pre- and post-treatment schedules. The initial stress response identified 120 mmol\u0026middot;L⁻\u0026sup1; as the threshold salt-alkali concentration inducing significant physiological disruption, which was then adopted as the baseline stress condition, aligning with previous studies (Song et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This concentration was subsequently applied to assess melatonin\u0026rsquo;s efficacy in alleviating stress when administered either before or after exposure to stress. The dual experimental approach comparing stress-only conditions with melatonin-augmented interventions provides a comprehensive understanding of how melatonin influences stress adaptation in poplar.\u003c/p\u003e \u003cp\u003eSalt-alkali stress primarily damages plants by disrupting cell membrane integrity and inducing ROS accumulation, which plants counteract via antioxidant enzyme systems such as SOD, CAT, and POD. In this study, melatonin pretreatment enhanced salt-alkali tolerance in poplar seedlings by controlling antioxidant enzyme activity. In particular, under stress conditions, SOD and CAT activities increased, with further amplification following melatonin application. However, enzyme responses demonstrated concentration-dependent biphasic behavior: SOD and CAT activities peaked at 200 \u0026micro;M melatonin but declined at higher concentrations (\u0026ge;\u0026thinsp;400 \u0026micro;M), whereas POD activity initially decreased at 200 \u0026micro;M before rebounding at elevated doses, highlighting its multifaceted role in stress signaling. These findings align with previous studies demonstrating that exogenous melatonin at 100 \u0026micro;M boosts SOD and CAT activities in stressed poplar and apple plants, mitigating oxidative damage by scavenging ROS and stabilizing membranes (Song et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xian et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Notably, while post-treatment melatonin slightly increased CAT and POD activities, SOD activity remained unchanged at lower doses, and all enzymes declined at higher concentrations, emphasizing the critical role of dosage optimization. Similarly, managing melatonin as a pretreatment notably improves the salinity tolerance of poplar seedlings. This enhancement is achieved through efficiently scavenging of ROS and improvement of cellular membrane stability, thereby effectively reducing oxidative damage caused by salt (Song et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Interestingly, a recent study on the transcriptome and metabolome shown of poplar seedlings has demonstrated that melatonin affects genes and metabolites associated with stress tolerance, with findings indicating that lower concentrations (100 \u0026micro;M) are supported by recent multi-omics studies (Duan et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Furthermore, prior research has shown that melatonin treatment significantly enhances the salt and drought tolerance of rice plants. This effect is realized by strengthening antioxidant defense mechanisms and upregulating stress-responsive genes, including \u003cem\u003eOsSOS\u003c/em\u003e, \u003cem\u003eOsNHX\u003c/em\u003e, \u003cem\u003eOsHSF\u003c/em\u003e, and \u003cem\u003eOsDREB\u003c/em\u003e, in rice (Khan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Notably, we strongly suggest prioritizing the identification of regulatory genes underlying stress response pathways in poplar plants subjected to abiotic stressors. Similarly, our results align with earlier research indicating that low levels of exogenous melatonin can significantly enhance antioxidant enzyme activity in \u003cem\u003eTriticum aestivum\u003c/em\u003e and \u003cem\u003ePennisetum glaucum\u003c/em\u003e when exposed to salt or drought stress (Awan et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These findings collectively highlight melatonin\u0026rsquo;s potential as a priming agent to enhance stress tolerance, though its concentration-specific effects require further mechanistic investigation.\u003c/p\u003e \u003cp\u003eIn another, melatonin pre-treatment application induced distinct morphological responses in poplar seedlings exposed to salt-alkali stress. Plants treated with 400 \u0026micro;mol\u0026middot;L⁻\u0026sup1; melatonin showed significant increases in leaf number and expanded leaf area compared to untreated controls, demonstrating consistent stress-mitigating effects across this concentration range. Interestingly, pretreatment with 200 \u0026micro;mol\u0026middot;L⁻\u0026sup1; melatonin resulted in a decrease in leaf number compared to controls, despite causing an increase in leaf area. This indicates a dose-dependent variation in physiological responses. Additionally, most recent studies have shown that low concentrations of pretreatment melatonin (100 \u0026micro;mol\u0026middot;L⁻\u0026sup1;) can promote plant growth and help alleviate the negative impacts of saline-alkali stress on tomato plants (Dou et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, an early study on cotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e) demonstrated that the combined stress of salt and drought results in a significant reduction in plant growth and chlorophyll content (Ibrahim et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In contrast, the post-treatment application of melatonin reversed these benefits; under stress conditions, leaf number significantly increased compared to controls exposed only to stress. The most notable alleviation was observed at 400 \u0026micro;mol\u0026middot;L⁻\u0026sup1; across different growth stages, while higher concentrations, such as 1000 \u0026micro;mol\u0026middot;L⁻\u0026sup1;, exhibited reduced effectiveness. Notably, melatonin pretreatment did not statistically alter ground diameter, plant height, or overall leaf area, though subtle similarities between pre- and post-treatment conditions were observed. Seedlings subjected to saline-alkaline stress exhibited increased plant height, stem diameter, leaf number, and leaf area, implying that elevated melatonin concentrations may be required post-stress to fully mitigate damage, as stress conditions likely increase physiological demands. This differential response highlights the nuanced role of melatonin dosage and timing in modulating morphological adaptations, with optimal stress alleviation achieved at intermediate concentrations (400 \u0026micro;mol\u0026middot;L⁻\u0026sup1;), while excessive doses may disrupt beneficial effects. The observed trends suggest that melatonin\u0026rsquo;s efficacy in enhancing salt-alkali tolerance depends on balancing its regulatory influence on growth parameters with its capacity to counteract stress-induced oxidative and ionic imbalances.\u003c/p\u003e \u003cp\u003eThis study revealed the dose-dependent effects of melatonin on osmoregulation in poplar seedlings under salt-alkali stress. Lower melatonin concentrations (200 \u0026micro;mol\u0026middot;L⁻\u0026sup1;) suppressed the accumulation of soluble sugars and proteins during pretreatment, while higher concentrations (\u0026ge;\u0026thinsp;400 \u0026micro;mol\u0026middot;L⁻\u0026sup1;) significantly enhanced their production, underscoring melatonin\u0026rsquo;s dual role as both a signaling molecule and a stress modulator in osmoregulation. Notably, the 200 \u0026micro;mol\u0026middot;L⁻\u0026sup1; pretreatment emerged as the optimal dose for improving stress resilience despite its inhibitory effect on osmolyte levels. A similar pattern was observed after salt-alkali stress induction: seedlings treated with 200 \u0026micro;M or 400 \u0026micro;M melatonin exhibited consistently lower soluble sugar levels and slightly reduced protein content compared to untreated controls. This aligns with previous findings by Song et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which demonstrated that low concentrations of exogenous melatonin can significantly alter osmoregulation activity in plants.\u003c/p\u003e \u003cp\u003eSaline-alkaline stress triggers initial root-derived stress signals that disrupt aboveground plant growth by degrading photosynthetic pigments, accelerating leaf senescence, and reducing photosynthetic capacity. Exogenous melatonin application mitigates these effects by slowing chlorophyll decline and preserving membrane integrity in poplar plants (Song et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, we confirmed that melatonin pretreatment significantly reduced MDA levels, a marker of oxidative membrane damage, with the lowest MDA observed at 200 \u0026micro;mol\u0026middot;L⁻\u0026sup1;. A recent study has demonstrated that pretreatment with melatonin reduces MDA content in rice plants under salt stress (Ubaidillah et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Conversely, post-stress melatonin supplementation showed concentration-dependent efficacy: MDA levels decreased progressively with higher doses, with the 1000 \u0026micro;M treatment group showing the most significant alleviation of oxidative stress. In parallel, pretreatment with melatonin did not statistically alter chlorophyll levels; however, post-treatment application under stress revealed a dose-dependent recovery. At 200 \u0026micro;M, chlorophyll concentrations dropped to minimal levels, yet higher doses (400\u0026ndash;1000 \u0026micro;M) induced progressive recovery, particularly during the T2 phase (14 days), where chlorophyll rebounded significantly. This suggests that post-stress conditions required higher melatonin concentrations to counteract severe oxidative damage as stress intensifies physiological demands. Notably, melatonin\u0026rsquo;s ability to stabilize membranes and scavenge free radicals aligns with previous findings (Dou et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), where lower concentrations (100 \u0026micro;mol\u0026middot;L⁻\u0026sup1;) alleviated stress by reducing cellular damage. Our results highlight melatonin\u0026rsquo;s dual role in osmoregulation and antioxidant defense, demonstrating optimal efficacy at 400\u0026ndash;1000 \u0026micro;M post-stress. This concentration enhances the synthesis of photosynthetic pigments and enhances resilience by counteracting chlorophyll degradation and oxidative damage. These findings emphasize the significance of dosage and timing in melatonin\u0026rsquo;s protective function, indicating its potential as a targeted intervention for enhancing plant tolerance under saline-alkaline stress.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, our findings demonstrate that exogenous melatonin application is a promising strategy for enhancing salt-alkali stress tolerance in poplar seedlings. More importantly, it primarily contributes to improving plant growth, chlorophyll synthesis, antioxidant enzyme activity (SOD, CAT), and osmoregulatory capacity, collectively improving ROS scavenging capacity and significantly restoring the growth and development of salt-alkali-stressed poplar seedlings by enhancing physiological and morphological resilience. While pre-melatonin application serves as a cost-effective strategy to improve stress resistance, post-treatment supplementation has also proven effective in alleviating the effects of stress, further enhancing tolerance, and reducing agricultural losses. These dual-phase benefits highlight melatonin\u0026rsquo;s versatility as a protective agent, employing both preventive and reparative mechanisms to counteract stress-induced damage, as illustrated in the proposed model (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor Contributions: Conceptualization, Z.N. and X.Z.; methodology, Z.N. and X.Z.; validation, Z.N. and X.Z.; formal analysis, J.N, X.Z., W.H., T.M., X.H., Z.L., X.L., X.L., J.S.; data curation, J.N, and X.Z.; writing original draft preparation, J.N, X.H., X.L., and J.S.; writing review and editing, J.N, W.H., T.M., Z.L., X.L., J.S.; visualization, X.Z.; project administration, X.Z.; funding acquisition X.Z. \u0026nbsp;All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding: We are grateful to the researchers who are contributing to this field. This paper was funded by the Scientific Research Project of the Education Department of Jilin Province (JJKH20250597KJ).\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate: Not applicable.\u003c/p\u003e\n\u003cp\u003eInformed Consent Statement: Not applicable.\u003c/p\u003e\n\u003cp\u003eData Availability Statement: Research data are available upon request from the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConflicts of Interest: The authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAwan SA, Khan I, Rizwan M, Brestic M, Wang X, Zhang X, Huang L (2024) Exogenous melatonin regulates the expression pattern of antioxidant-responsive genes, antioxidant enzyme activities, and physio-chemical traits in pearl millet under drought stress. 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Plant Sci 287:110184. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plantsci.2019.110184\u003c/span\u003e\u003cspan address=\"10.1016/j.plantsci.2019.110184\" 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":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Melatonin, Salt-alkali stress, oxidative enzymes, soluble metabolites, Poplar plant","lastPublishedDoi":"10.21203/rs.3.rs-8293236/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8293236/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMelatonin plays a crucial role in modulating plant stress responses; however, its potential for mitigating salt-alkali stress remains incompletely understood. The present study evaluates the efficacy of exogenous melatonin in alleviating moderate salt-alkali stress (120 \u0026micro;M) in poplar (\u003cem\u003ePopulus spp\u003c/em\u003e.) seedlings, investigating both pre- and post-stress treatments (0\u0026ndash;1000 mmol\u0026middot;L⁻\u0026sup1;). Physiological and morphological parameters including chlorophyll content, antioxidant enzyme activities, and osmolyte accumulation were analyzed to assess stress responses. Under salt-alkali stress, seedlings exhibited elevated stress markers and osmolyte levels, indicating activated stress responses. More importantly, melatonin at a concentration of 200 mmol\u0026middot;L⁻\u0026sup1; was identified as the most effective in mitigating stress effects, significantly enhancing antioxidant enzyme activities such as superoxide dismutase (SOD) and catalase (CAT), while restoring chlorophyll content and reducing oxidative damage markers like malondialdehyde (MDA). Additionally, it contributed to the regulation of osmotic regulators in the leaves, indicating improved cellular stability under stress conditions. Notably, post-stress application of melatonin required slightly higher concentrations to achieve comparable levels of recovery compared to pre-treatment, underscoring the critical influence of application timing on its efficacy. These findings highlight the valuable insights into the strategic use of melatonin for stress mitigation and provide a foundation for molecular breeding efforts aimed at developing salt-alkali-tolerant poplar varieties.\u003c/p\u003e","manuscriptTitle":"Impact of Melatonin on Oxidative Enzymes and Soluble Metabolites in Salt-Alkali Stressed Poplar (Populus spp.): A Comparative Study of Pretreatment and Post-Treatment Effects","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 08:54:55","doi":"10.21203/rs.3.rs-8293236/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ad694b6-2782-4165-a7fb-e1d87ddb11b8","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-20T14:41:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 08:54:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8293236","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8293236","identity":"rs-8293236","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}