By 2050, the global population is expected to exceed 9.6 billion, necessitating a 70% increase in food production (FAO, 2017). This demand poses a significant challenge for agriculture, especially in regions already constrained by environmental stressors. Soil salinity has emerged as a critical abiotic stress, particularly in arid and semi-arid regions, where it severely hampers crop productivity and land sustainability (Deolu-Ajayi et al., 2024; Hassan et al., 2025). Salinization affects over 20% of irrigated land globally and continues to expand, primarily due to unsustainable irrigation and poor drainage practices (Selma, 2025). Each year, hundreds of thousands of hectares become agriculturally unproductive due to salt accumulation (FAO, 2017). Salinity imposes osmotic and ionic stress, disrupting water uptake and leading to toxic levels of sodium (Na⁺) and chloride (Cl^-), nutrient imbalance, and cellular injury. Plants respond to this stress in two stages: an early osmotic phase causing reduced cell expansion and stomatal closure, followed by a chronic ionic phase that leads to ion toxicity, senescence, and metabolic suppression (Selma, 2025). Photosynthesis, which contributes up to 90% of plant biomass, is highly sensitive to salinity. Reduced stomatal conductance limits CO₂ assimilation, while excessive ROS production damages chloroplasts and enzymes (Tomaškinová et al., 2025). To counteract these effects, plants activate antioxidant systems and osmolyte synthesis pathways, including proline metabolism, to restore redox balance and maintain physiological functions (Nurhidayati et al., 2024). Recent studies emphasize the importance of integrated strategies such as bio-stimulants, salt-tolerant genotypes, and molecular breeding to enhance crop resilience under saline conditions (Hassan et al., 2025). These insights are vital for developing sustainable agricultural practices in the face of escalating soil salinity and global food insecurity.

Brassinosteroids (BRs), a group of steroidal plant hormones and essential regulators of key physiological activities, including cell division and elongation, vascular tissue differentiation, and floral development (Vriet et al., 2012). The discovery and characterization of BR-deficient and BR-insensitive mutants in recent years have significantly advanced our understanding of BR signaling pathways and their contribution to plant growth and the development of improved agronomic traits (Nolan et al., 2020). BRs are known to promote both cell division and expansion, and they function by interacting with other hormone signaling networks at the transcriptional level to coordinate plant growth responses (Gudesblat and Russinova, 2011; Khan et al., 2023). In metabolically active tissues, BRs exhibit concentration and sensitivity-dependent effects, regulating growth and development while modulating antioxidant defenses, especially under stress-induced overproduction of ROS. BRs have also been reported to enhance plant tolerance to several stresses, including salinity, drought, extreme temperatures, and heavy metal toxicity (Bajguz and Hayat, 2009; Khan et al., 2024). Despite these advances, the interaction between BRs and other phytohormones under high salinity conditions, particularly about their combined effects on proline metabolism and antioxidant systems, remains poorly understood. On the other hand, melatonin (ML), a recently identified plant bio-regulator, has attracted considerable attention due to its diverse physiological roles, such as delaying leaf senescence, promoting root and shoot development, enhancing nutrient uptake, and improving heat stress tolerance (Ahammed et al., 2018). The role of ML in plant stress physiology has been investigated using both synthetic ML applications and transgenic approaches that elevate endogenous ML levels (Erland et al., 2018). Exogenous ML treatment has been demonstrated to protect plants from diverse abiotic stresses by mitigating damage caused by ROS (Karumannil et al., 2023; Yusuf et al., 2024). Specifically, ML-mediated stress tolerance has been linked to enhanced synthesis of antioxidants, activation of stress-responsive enzymes, regulation of polyamine metabolism, and efficient ROS scavenging (Ni et al., 2018). However, the potential crosstalk between BRs and ML in mediating plant tolerance to high salinity, particularly through the modulation of proline metabolism and antioxidant systems, has not yet been elucidated. Exploring this interaction may uncover novel strategies for enhancing stress resilience in crops under saline environments. Proline serves as a key osmoprotectant and antioxidant, playing a central role in mitigating oxidative damage by scavenging ROS, thereby enhancing plant resilience to abiotic stresses and reducing associated agricultural losses. Despite its importance, the regulatory mechanisms underlying proline metabolism in response to external stimuli remain incompletely understood.

This study aims to investigate the modulation of proline metabolism under high salinity stress following exogenous co-application of ML and/or EBL. Specifically, the research evaluates the capacity of ML to enhance salt stress tolerance in the presence of EBL, with a particular focus on photosynthetic performance, proline accumulation, and antioxidant defense responses.

Plants frequently face environmental stresses such as drought, salinity, and temperature extremes, which elicit diverse responses at morphological, biochemical, and molecular levels. In the present study, exposure of tomato plants to 300 mM NaCl led to a significant increase in oxidative stress markers, including lipid peroxidation, electrolyte leakage, and H₂O₂ accumulation, indicators of severe cellular damage. These responses reflect impaired redox homeostasis and membrane destabilization under salinity stress. However, foliar application of ML, EBL, or their combination effectively mitigated these stress-induced elevations. This suppression of oxidative markers underscores their protective roles in enhancing cellular resilience. Notably, elevated levels of TBARS, a proxy for LPO, are a well-established hallmark of membrane deterioration under oxidative stress (Cavalcanti et al., 2007). Under salinity, increased ROS levels further compromise membrane integrity and may trigger programmed cell death through ionic imbalance and calcium-mediated signaling pathways (Demidchik et al., 2014). Salinity-induced disruption of electron transport chains in chloroplasts and mitochondria exacerbates ROS production, contributing to the accumulation of H₂O₂ in leaf tissues (Chawla et al., 2013). Our findings align with this mechanism, as salt-exposed plants exhibited heightened H₂O₂ levels ( Figure 3C ), which were significantly reduced by ML and EBL treatments. ML is known for its broad-spectrum antioxidative action across various stress contexts, including drought, cold, heat, and salinity, primarily through enhancement of the antioxidant defense system (Hasan et al., 2019; Kaya et al., 2020; Ahammed et al., 2020; Yusuf et al., 2024). In our study, ML application significantly lowered MDA, electrolyte leakage, and H₂O₂ levels in salt-stressed tomato, corroborating earlier findings in maize and other crops (Ahammed et al., 2020). Similarly, EBL application alleviated oxidative damage by reducing membrane injury markers, consistent with studies in wheat (Dong et al., 2017; Khan et al., 2024) and perennial ryegrass (Wu et al., 2017). BRs, including EBL, have been shown to enhance membrane stability through the upregulation of membrane-associated protective proteins (Li et al., 2012; Gao et al., 2015). These effects, as observed in our study, emphasize the role of BRs in stabilizing cellular structures and limiting ROS-mediated damage under saline conditions.

ROS are continuously generated in plant cells as byproducts of metabolic processes. Under optimal conditions, these are tightly regulated by a robust antioxidative system. However, under abiotic stresses such as high salinity, ROS production increases dramatically, overwhelming the cellular antioxidant machinery and leading to oxidative damage. This includes membrane lipid peroxidation, elevated H₂O₂ accumulation, and increased electrolyte leakage ( Figures 3A–C ), which impair cellular function and integrity. To combat oxidative stress, plants initiate a multifaceted defense response involving both enzymatic (CAT, POX, SOD, and GR) and non-enzymatic antioxidants. In our study, tomato plants subjected to 300 mM NaCl showed a pronounced increase in the activity of CAT, POX, SOD and GR. This response was significantly amplified by the application of ML, EBL, and most prominently by their combined treatment ( Figures 5A–D ). The upregulation of antioxidant enzymes under salinity is well-established. Early studies (Xue et al., 2008; Wu et al., 2011; Yusuf et al., 2024) demonstrated similar increases in enzymatic activity under NaCl stress. More recently, Sahu et al. (2025) reported that combined phytohormone treatments, including ML and BRs, enhance antioxidant capacity and confer improved salt tolerance in rice and wheat. Similarly, Li et al. (2025) found that exogenous ML application significantly boosted ROS-scavenging enzymes in salinity-stressed maize. ML is known to enhance antioxidative defense by upregulating genes encoding antioxidant enzymes through transcriptional activation, potentially mediated by ROS-responsive signaling pathways (Cui et al., 2017; Ahammed et al., 2020). Additionally, studies by Ali et al. (2024) highlight ML’s regulation of NAC and WRKY transcription factors under oxidative stress, further supporting its protective role. Moreover, EBL acts through a distinct but complementary mechanism and modulates stress-responsive transcription factors, including BIN2 and BES1/BZR1, and activates downstream genes such as DREB, WRKY, bZIP, and MYB/MYC (Bartwal and Arora, 2020). This transcriptional network contributes to redox balance and improved stress tolerance. EBL has also been shown to influence the det2 gene, enhancing the synthesis of protective enzymes under stress (Cao et al., 2005). Our data align with these findings, as EBL application significantly reduced markers of oxidative stress, including MDA, H₂O₂, and electrolyte leakage under salinity ( Figures 3A–C ). These observations affirm that strengthening antioxidant metabolism through ML and EBL applications is an effective strategy to mitigate salinity-induced oxidative damage. This approach holds promise for improving stress resilience in crop species, as evidenced by our results and corroborated by other recent studies (Fariduddin et al., 2014; Hasan et al., 2019; Sahu et al., 2025).

Proline serves a dual function in plant stress responses, acting as both an osmoprotectant and a redox buffer with significant antioxidant potential (Kavi Kishor and Sreenivasulu, 2014). Its metabolism is tightly regulated through key enzymes such as P5CS, P5CR, ProDH, and δ-OAT. During salt stress, the accumulation of proline helps maintain cellular osmotic balance and protects macromolecules from ROS-induced damage. Our findings show that salt-stressed tomato plants treated with a combination of ML and EBL exhibited significantly higher proline content and enhanced activity of P5CS, ProDH, and δ-OAT enzymes compared to untreated controls ( Figures 4A–D ). This suggests a coordinated upregulation of proline metabolism under the influence of ML and EBL. Similar results have been reported by Antoniou et al. (2017), who found that ML treatment increased both the transcription and activity of P5CS and P5CR in Arabidopsis. Vendruscolo et al. (2007) also demonstrated that P5CS overexpression enhances proline levels, reduces lipid peroxidation, and improves antioxidant defense. Likewise, Roosens et al. (2002) and Liu et al. (2014) reported that δ-OAT and P5CR play critical roles in stress-induced biomass enhancement and photosynthetic efficiency. EBL are known to stimulate proline accumulation under salinity by modulating the expression of biosynthetic genes (Özdemir et al., 2004; Zeng et al., 2010). Recently, Sahu et al. (2025) noted that exogenous EBL application in rice enhanced proline biosynthesis through transcriptional activation of stress-responsive pathways, while Xia et al. (2025) demonstrated that combined ML and BR treatment synergistically boosted proline metabolism and ROS scavenging capacity in maize.

Photosynthesis is among the first cellular processes disrupted by salinity stress due to its sensitivity to redox imbalance (Biswal et al., 2011; Mathur et al., 2014). In our study, salinity severely impaired photosynthetic parameters, including chlorophyll content, net photosynthesis, stomatal conductance, and RuBisCO activity. However, application of ML and EBL, individually or in combination, significantly restored these traits ( Figures 2A–E ), alongside improving leaf relative water content ( Figure 1D ). The strong correlation observed between net photosynthetic rate and Rubisco activity ( Figure 6A ) further supports our finding that EBL and ML, either individually or in combination, significantly enhance photosynthetic efficiency. This recovery may stem from the stabilization of photosystem components and enhancement of Calvin cycle enzyme activity. Chloroplast dysfunction under salt stress, manifesting as thylakoid swelling and plastoglobuli accumulation, has been reported to impede light-harvesting efficiency (Meng et al., 2016). Salt-induced ABA signaling also limits water availability, reducing leaf relative water content and biomass (Davies et al., 2005; Zhang et al., 2006). ML has been shown to counter these effects by promoting RuBisCO activity, maintaining chlorophyll content, and enhancing the expression of photosynthesis-related genes (Zhao et al., 2015; Liang et al., 2015; Shi et al., 2015). In our data, ML-treated plants showed improved CO₂ assimilation and sugar content even under stress ( Figures 2B–D , 3D ), underscoring its regulatory influence on carbon metabolism. EBL also improved photosynthetic efficiency, likely through enhanced carboxylation capacity and upregulation of Calvin cycle enzymes, as previously reported by Yu et al. (2004) and confirmed in our results ( Figures 2A, C ). Zhao et al. (2017) similarly observed increased RuBisCO activity following EBL application under both stress and non-stress conditions. Furthermore, BRs have been linked to increased PSII efficiency and stabilization of chloroplast structure (Fariduddin et al., 2014; Nazir et al., 2021).

Salinity stress also triggered oxidative damage in our study, evidenced by increased lipid peroxidation, H₂O₂ accumulation, and electrolyte leakage ( Figures 3A–C ), ultimately reducing plant growth and productivity (Gupta and Huang, 2014). This was reflected in the reduced root and shoot lengths, lower biomass, and diminished leaf area ( Figures 1A–C ). A strong correlation between SPAD chlorophyll values and leaf area ( Figure 6B ) further supports the close link between photosynthetic efficiency and morphological development. The auxin-like activity of ML derived from the shared tryptophan precursor with IAA may underlie its role in nutrient uptake and growth promotion (Arnao and Hernández-Ruiz, 2007; Zhang et al., 2017). EBL, a potent growth regulator, operates through transcription factors like BZR1 and BES1/BZR2 to modulate growth-related genes even under stress (Gudesblat and Russinova, 2011). Our findings confirm that ML and EBL not only alleviate stress but also promote growth recovery, in line with previous reports (Fariduddin et al., 2014).

This study provides compelling evidence that ML and EBL, either alone or in combination, effectively mitigate salinity-induced oxidative stress in tomato plants. Their application enhances antioxidant capacity, activates proline metabolism, and reduces key stress markers such as lipid peroxidation, hydrogen peroxide levels, and electrolyte leakage. Importantly, the combined treatment of ML and EBL demonstrated superior effectiveness, preserving photosynthetic pigment content and enhancing RuBisCo activity under both stress and non-stress conditions. These findings suggest that the synergistic use of ML and EBL offers a promising, sustainable, and environmentally friendly strategy to boost salt tolerance and overall plant performance. This approach holds strong potential for practical application in saline agriculture, contributing to improved crop productivity and resilience in salt-affected regions.

As a future direction, it would be valuable to conduct transcriptomic or proteomic analyses to elucidate the precise molecular pathways and gene networks regulated by ML and BRs during salt stress. Additionally, time-course experiments across different developmental stages and field-level trials under varying salinity gradients could validate the efficacy and practical applicability of ML and EBL co-application in diverse tomato cultivars or other crops.