Melatonin Enhances Tomato Salt Tolerance by Improving Water Use Efficiency, Photosynthesis, and Redox Homeostasis

Abstract

Salinity stress is a primary abiotic constraint limiting global crop productivity, with progressive soil salinization inducing growth inhibition and physiological dysfunction in plants. Although melatonin (MT) has been extensively documented to enhance stress adaptation, the underlying mechanisms through which it mediates salt tolerance by integrating physiological processes remain unclear. This study investigated the effects of varying MT concentrations on photosynthetic performance, plant water relations, water-use efficiency, and stress-responsive physiological parameters in tomatoes, aiming to identify the key physiological pathways for MT-mediated salt stress mitigation. The results showed that salt stress significantly reduced the leaf relative water content and root hydraulic conductivity, suppressed the photosynthetic rate, and ultimately caused significant reductions in the aboveground and root biomass. MT spraying effectively improved leaf water status and root water uptake capacity, enhancing the photosynthetic rate and water-use efficiency, thereby providing material and energy support for plant growth. Furthermore, MT spraying increased the total antioxidant capacity in leaves and promoted the synthesis of phenolic and flavonoid compounds, thereby reducing oxidative damage. Simultaneously, it stimulated the accumulation of osmolytes to enhance cellular osmotic adjustment capacity and optimized ion uptake to maintain cellular ion homeostasis. Among the tested concentrations, 100 μM MT showed the most significant alleviative effects. This concentration comprehensively enhanced the salt tolerance and growth performance of tomato plants by synergistically optimizing water use, photosynthetic function, antioxidant defense, and ion balance. In conclusion, these findings provide experimental evidence for elucidating the physiological mechanisms underlying MT-mediated salt tolerance in tomatoes and offer theoretical references for the rational application of MT in crop production under saline conditions.

1. Introduction

Salt stress is a global agricultural challenge that affects approximately 20% of cultivated land and 33% of irrigated areas worldwide [1]. The annual expansion of saline soils at a rate of 2 million hectares continues to threaten a substantial proportion of arable land [2]. Soil salinity impairs plant growth and development through multifaceted disruptions of physiological processes, including photosynthesis, ion homeostasis, antioxidant defense systems, and osmotic regulation [3], ultimately leading to substantial yield losses [4].

Plants exhibit complex physiological adaptations under salt stress. Saline conditions significantly reduce chlorophyll content and PSII photochemical efficiency, thereby compromising photosynthetic apparatus integrity [5]. Elevated soil salinity restricts radial root expansion and diminishes root hydraulic conductivity through inhibited water transport [6]. Another primary threat stems from osmotic stress-induced ion toxicity, particularly due to excessive Cl⁻ and Na⁺ accumulation, which disrupts Ca²⁺/K⁺ balance and impairs ionic homeostasis [5]. Furthermore, salt stress triggers an excessive overaccumulation of reactive oxygen species (ROS), damaging cellular components including membrane lipids, proteins, and nucleic acids, ultimately inducing programmed cell death [7,8]. To counteract salinity, plants activate integrated defense systems comprising both enzymatic (SOD, CAT, POD, APX) and non-enzymatic (phenols, flavonoids, ascorbic acid) antioxidants [9,10]. These components show salinity-induced upregulation, and their activity levels correlate with plant tolerance capacity. Moreover, osmolyte accumulation (proline, soluble sugars) plays an essential role in mitigating salinity damage by maintaining ROS homeostasis in chloroplasts and mitochondria [5,11]. When salt stress exceeds the adaptive thresholds, defense mechanisms become insufficient, resulting in apoptotic cell death and severe growth inhibition [12]. Although breeding salt-tolerant cultivars remains a viable strategy, the polygenic nature and pleiotropic effects of salt tolerance traits complicate genetic selection [13]. Consequently, exogenous protectant application has emerged as a promising complementary approach for enhancing plant salt tolerance.

Exogenous MT has emerged as a potent antioxidant and signaling molecule with significant potential for alleviating salt stress [14,15]. MT directly scavenges ROS and harmful oxidizing agents while concurrently enhancing key antioxidant enzyme activity through its signaling functions [16]. Studies have shown that MT-treated plants exhibit reduced ROS accumulation, ionic leakage, and cellular damage, accompanied by increased plant height and biomass, compared to untreated plants [17]. MT application plays a pivotal role in maintaining ionic homeostasis under salinity by promoting K⁺ uptake and inhibiting Na⁺ accumulation, thereby reducing salt-induced cellular damage [18]. Moreover, MT stimulates osmolyte accumulation while maintaining critical cellular functions, including growth regulation and biosynthesis, under saline conditions [19,20]. The growth-promoting effects of MT under salt stress have been validated in multiple crops, including tomatoes [21], wheat [22], and soybean [23]. However, the dose-dependent effects of melatonin on plant growth are well documented; lower concentrations generally promote growth, whereas higher concentrations may exert inhibitory effects [24]. The physiological mechanisms underlying the effects of MT on plants require further investigation.

Tomatoes have become a major global crop owing to their desirable taste and nutritional value, with extensive cultivation in China [25]. However, the development of modern protected cultivation systems has led to excessive fertilizer application and improper irrigation practices, resulting in increased soil salinity [26]. Salt stress is a primary constraint on tomato growth and yield [25]. Therefore, there is an urgent need to develop new strategies to improve the salt tolerance of tomatoes and mitigate the negative effects of climate-related salt stress. Although previous studies have reported the role of MT in mitigating abiotic stress damage, research on the regulatory mechanisms by which exogenous MT enhances tomato salt tolerance through multiple pathways remains scarce. This study aimed to systematically investigate MT-regulated salt tolerance mechanisms in tomatoes through comprehensive analyses of photosynthetic performance, water relations, antioxidant defense, osmoregulatory capacity, and ionic homeostasis, ultimately evaluating tomato salt tolerance. The findings of this study offer novel insights into the physiological basis of exogenous MT application for enhancing salt tolerance in tomatoes.

2. Materials and Methods

2.1. Plant Growth Conditions

The experiment was conducted at the Nongcui Experimental Station of Anhui Agricultural University (117.26 E 31.86 N) from July to August 2024. The main local cultivar ‘Wanza 18’ was selected as the experimental material, with seeds originating from the Anhui Academy of Agricultural Sciences. ‘Wanza 18’ is an indeterminate, pink-fruited hybrid tomato cultivar specifically bred for protected cultivation in subtropical regions. Characterized by high yield potential, disease resistance, and low-light tolerance, this cultivar is an ideal model system for investigating abiotic stress responses in tomatoes. Melatonin reagent (CAS #73-31-4; BR, 99%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China. Uniform seeds were selected and sterilized with 15% (w/v) sodium hypochlorite for 5 min, thoroughly rinsed with sterile distilled water, placed in a constant-temperature incubator for germination, and sown in seedling trays on July 10th. The physicochemical properties of the test soil were as follows: total nitrogen, 0.49 g kg⁻¹; total phosphorus, 0.61 g kg⁻¹; total potassium (K₂O), 14.4 g kg⁻¹; alkaline dissolved nitrogen, 31.23 mg kg⁻¹; and effective phosphorus, 15.10 mg kg⁻¹. When the seedlings developed two true leaves, they were transplanted into plastic pots (18 cm diameter × 25 cm height). The test soil was prepared by mixing organic fertilizer, potting soil, and sand in a 1:2:1 ratio.

2.2. Experimental Design

At the transplantation stage, all treatments were irrigated with equal volumes of water to ensure uniformity and consistent seedling growth. To minimize environmental interference, all pots were transferred to an artificial growth chamber (RTOP-B, Zhejiang Topu Yunnong Technology Co., Ltd., Hangzhou, China) during the experiment. Plants were maintained in a controlled growth chamber under the following conditions: temperature 18–25 °C, relative humidity 60%, CO₂ concentration 400 µmol mol⁻¹, and a 14-h photoperiod with 350 µmol m⁻² s⁻¹ photosynthetic photon flux density. When the seedlings developed four true leaves, tomato plants exhibiting uniform growth were selected for melatonin spraying. Based on preliminary experiments and previous studies [27], three melatonin concentrations (M1: 50 µM, M2: 100 µM, M3: 150 µM) were established. Due to the photolability of melatonin, spraying was conducted 1 h after dark adaptation. Both the adaxial and abaxial leaf surfaces were uniformly sprayed with melatonin solution until droplet runoff was observed. Melatonin-sprayed seedlings received daily applications for five consecutive days, whereas non-primed control plants were sprayed with an equivalent volume of tap water.

Salt stress treatment was initiated after melatonin spraying. Based on previous studies [21,27], a 150 mM NaCl solution was applied to the soil to establish salt stress conditions. Five treatments were implemented, as follows: M1S (50 µM melatonin + salt stress), M2S (100 µM melatonin + salt stress), M3S (150 µM melatonin + salt stress), SS (0 µM melatonin + salt stress), and CK (control, 0 µM melatonin + no salt stress), with 12 replicates (pots) per treatment. The salt stress treatment employed a pulsed application-leaching regime to simulate field salinity dynamics, consisting of (1) 100 mL of 150 mM NaCl solution applied every 2 days (6 total applications), increasing soil electrical conductivity (EC) by approximately 1.3–1.5 dS m⁻¹ per application; and (2) 200 mL tap water flushing every 5 days to maintain target EC levels (4.5 ± 0.3 dS m⁻¹), preventing excessive salt accumulation. Control plants received equivalent volumes of tap water with an identical leaching frequency to maintain consistent soil moisture conditions.

2.3. Sampling and Measurement

2.3.1. Plant Sampling

Leaf and root samples were collected at the 4th, 8th, and 12th days after stress (D4, D8, and D12). Samples were immediately transported to the laboratory to determine leaf relative water content, root hydraulic conductivity, and membrane stability index. Na⁺, K⁺, Mg²⁺, and Ca²⁺ contents were quantified using dried leaf and root samples. The remaining samples were stored at −80 °C for subsequent determination of antioxidant enzyme activity, phenol and flavonoid content, osmotic regulators, reactive oxygen species levels, and malondialdehyde content. All the aforementioned physiological and biochemical parameters were analyzed using three biological replicates (n = 3), with each measurement performed in triplicate.

2.3.2. Determination of Growth Traits

Growth traits were measured in tomato plants at D12. Plant height was measured using a digital caliper, and root samples were collected. Root length was determined by scanning the root systems and analyzing them using WinRhizo software, Basic version (Winrhizo, Regent Ltd., Quebec, QC, Canada). Subsequently, both aboveground and root samples were oven-dried at 80 °C to a constant weight.

2.3.3. Determination of Gas Exchange and Plant Water Relations

Gas exchange parameters were measured at D4, D8, D12, and D16 using an LI-6400XT portable photosynthesis system (LI-COR Biosciences, Lincoln, NE, USA). Parameters, including net photosynthetic rate (P_n) and stomatal conductance (g_s), were measured under 400 µmol mol⁻¹ CO₂ concentration at a constant flow rate of 500 mL min⁻¹. Instantaneous water-use efficiency (WUE_i) was determined as the ratio of P_n to T_r. Leaf relative water content (LRWC) was determined using the following formula:

LRWC (%) = [(FW − DW)/(SW − DW)] × 100

(1)

where FW is leaf fresh weight, DW is leaf dry weight, and SW is saturated leaf weight.

Root hydraulic conductance (L_p) was determined using excised roots placed in a pressure chamber with the cut stem exposed to the atmosphere. Increasing pressure gradients were applied to measure the volumetric flow rates until the root water potential was reached. The final L_p values were calculated according to Kang and Zhang [28].

2.3.4. Determination of Antioxidant Enzyme Activity

Superoxide dismutase (SOD) activity was determined as described by Giannopolitis and Ries [29]. The reaction mixture (3 mL) consisted of 50 mmol⁻¹ potassium phosphate buffer (pH 7.0), 13 mM methionine, 75 μM nitroblue tetrazolium (NBT), 2 μM riboflavin, and 0.2 mL of enzyme extract. One unit of SOD activity was defined as the enzyme quantity that inhibited 50% of NBT reduction at 560 nm. Peroxidase (POD) activity was analyzed according to Cakmak and Marschner [30] by adding 16 mM guaiacol, 0.2 mL enzyme extract, and 10 mM hydrogen peroxide to 50 mM phosphate buffer (pH 7.0), and the absorbance was recorded at 470 nm. Catalase (CAT) activity was measured using the protocol described by Havir and Mchale [31], where 0.2 mL of enzyme extract was added to 50 mM phosphate buffer (pH 7.0) containing 12.5 mM H₂O₂, and the absorbance was monitored at 240 nm.

The total antioxidant capacity (TAC) was evaluated as described by Fatima [32]. Briefly, 100 μL of the extract was mixed with 900 μL of reagent (0.6 M sulfuric acid, 4 mM ammonium molybdate, and 28 mM sodium phosphate) and incubated at 95 °C for 90 min. Absorbance was measured at 695 nm after cooling, using ascorbic acid for standard curve calibration.

2.3.5. Determination of Phenols and Flavonoids Contents

The total phenol content was quantified using the method of Yu and Dahlgren [33]. The reaction mixture (2 mL) contained 18 µL of the extract, 70 µL of Folin–Ciocalteu reagent, 175 µL of 20% (w/v) sodium carbonate solution, and 1.75 mL of distilled water. After 30 min of incubation at 45 °C, the absorbance was measured at 750 nm. The total flavonoid content was determined according to Arvouet–Grand [34] by mixing 1 mL of the extract with 1 mL of 2% methanol-aluminum chloride solution, incubating in the dark for 20 min, and measuring the absorbance at 415 nm.

2.3.6. Determination of ROS, Malondialdehyde, and Membrane Stability Index

Hydrogen peroxide (H₂O₂) content was assessed following Velikova [35]. Leaf tissue was homogenized in 5 mL 0.1% (w/v) trichloroacetic acid (TCA) at 8000 rpm and 4 °C. The supernatant was mixed with 10 mM potassium iodide (KI), and the absorbance was recorded at 390 nm. The superoxide anion (O₂⁻) content was measured using a commercial reagent kit (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China). Malondialdehyde (MDA) content was analyzed via the thiobarbituric acid reaction [36], where 1 mL extract was mixed with 4 mL 20% trichloroacetic acid containing 0.5% thiobarbituric acid, heated at 95 °C for 30 min, cooled on ice, and absorbance measured at 532, 600, and 450 nm.

The membrane stability index (MSI) was calculated according to the method of Rady [37].

MSI (%) = [1 − (EC₁/EC₂)] × 100

(2)

where EC₁ is the conductivity of the leaf sample solution heated at 40 °C for 30 min, and EC₂ is the conductivity after heating at 100 °C for 20 min.

2.3.7. Determination of Proline and Soluble Sugar Contents

The proline content was determined using the method described by Bates [38]. The reaction mixture containing 1 mL extract, 2 mL ninhydrin reagent, and 2 mL glacial acetic acid was boiled for 1 h, cooled on ice, extracted with 3.5 mL toluene, and the absorbance was measured at 520 nm. Soluble sugar content was analyzed following Yemm and Willis [39] by mixing 100 μL of the extract with 3 mL of anthrone reagent, heating at 95 °C for 10 min, cooling to room temperature, incubating for 20 min, and measuring the absorbance at 625 nm.

2.3.8. Determination of Ion Contents

The Na⁺, K⁺, Ca²⁺, and Mg²⁺ contents were quantified from dried leaf and root samples. The samples were ground into powder using a centrifugal mill (ZM 200, Retsch GmbH, Haan, Germany) at 8000 rpm. Digestion was performed with 30% hydrogen peroxide and concentrated nitric acid, followed by ion content analysis using flame atomic absorption spectrometry according to Sousa [40].

2.4. Data Analysis

Data were presented as mean  ±  standard deviation (SD). Statistical analysis was performed using one-way ANOVA followed by Duncan’s multiple range test (p < 0.05) using SPSS 22.0 (IBM Corp., Armonk, NY, USA). All figures were generated using Origin 2021 software (OriginLab, Northampton, MA, USA). The relationships between physiological parameters were examined using Pearson correlation analysis. Principal component analysis (PCA) was performed using R software (version 4.1.0). Data normalization was conducted using within-group maximum normalization (observed value/maximum value within the group), transforming all values to a relative 0–1 scale to eliminate unit differences and facilitate heatmap visualization and intergroup comparisons. Tomato salt tolerance was quantitatively evaluated using the membership function method described by Sun [41].

3. Results

3.1. Aboveground and Root Growth of Tomato

Exogenous melatonin (MT) significantly alleviated salt stress-induced growth inhibition in tomato plants (Figure 1A). At D12, salt stress reduced the aboveground dry mass and plant height by 26% and 19%, respectively, compared to the control. MT spraying increased the aboveground dry mass by 5%, 17%, and 16% in M1S, M2S, and M3S plants, respectively, compared to CK. M2S and M3S treatments demonstrated more pronounced growth promotion than M1S, with plant height increases of 16% and 15%, respectively, compared to salt-stressed plants. Root growth parameters were severely impaired under salt stress, showing 35% and 38% reductions in root dry mass and length, respectively. MT spraying effectively alleviated these adverse effects, increasing root dry mass by 10%, 27%, and 18% in M1S, M2S, and M3S plants, respectively (Figure 1B).

3.2. Plant Water Relations and Gas Exchange Parameters

The leaf relative water content (LRWC) progressively declined under prolonged salt stress (Figure 2A). At D4, MT-primed plants maintained significantly higher LRWC than salt-stressed (SS) plants. At D8, M1S, M2S, and M3S plants exhibited 2%, 5%, and 6% higher LRWC than SS plants. The M2S and M3S plants retained relatively higher LRWC than the SS plants at D12. Salt stress reduced root hydraulic conductivity (L_p), impairing water uptake capacity. At D4, L_p in SS plants decreased by 17% compared to CK, whereas M2S and M3S plants showed only 5% reduction and 2% increase, respectively, and the positive effect of M2 treatment on L_p gradually diminished with prolonged stress exposure.

P_n progressively declined under salt stress (Figure 2B). At D8, M3S plants maintained P_n comparable to CK and were 26% higher than that of SS plants. Although M3S plants showed greater P_n reduction at D12, their values remained 39% higher than those of SS plants. At D16, P_n in M2S and M3S plants increased by 63% and 52% compared to SS plants, respectively. For g_s, M2S, and M3S plants exhibited 1.17- and 1.28-fold higher g_s than SS plants at D8 and 1.43- and 1.63-fold higher g_s at D12. At D16, g_s increased by 80% and 89% in M2S and M3S plants, respectively, compared with SS. MT application effectively mitigated the salt-induced reduction in T_r, particularly in the M2 and M3 treatments. At D8, D12, and D16, T_r in M2S and M3S treatments were significantly higher than in SS. M2S and M3S treatments maintained relatively high WUE_i at D8, D12, and D16, with M3S showing 8.6% and 24.9% higher WUE_i than SS at D12 and D16, respectively.

3.3. Phenols, Flavonoids, and Osmoregulatory Substances Contents

Salt stress significantly increased the total phenol content in leaves (Figure 3A). At D8, the total phenol content was substantially higher in SS plants than in CK, with M2S and M3S showing 6% and 23% increases compared to SS, respectively. At D12, M1S, M2S, and M3S plants maintained 16%, 42%, and 27% greater phenol content than SS plants, respectively. During early stress (D4), the total flavonoid content in M2S and M3S plants was significantly lower than that in SS plants (Figure 3B). At D8, M1S and M2S plants accumulated significantly higher flavonoid levels than SS plants. At D12, M2S and M3S plants maintained 35% and 27% higher total flavonoid content than SS plants. MT spraying progressively enhanced proline accumulation with stress duration (Figure 3C). At D8, the proline content in M3S plants increased by 35% compared to SS plants. At D12, M3S plants maintained the highest proline levels. Similarly, salt stress markedly increased the soluble sugar content (Figure 3D). At D4, the soluble sugar content of M3S plants was 24% higher than that of SS plants. M1S and M2S plants showed substantial increases in soluble sugar at D12, whereas the soluble sugar content in M3S plants declined sharply. The soluble sugar content in M1S, M2S, and M3S plants reached 2.7-, 3.2-, and 1.9-fold higher than that in SS plants, respectively.

3.4. Membrane Stability and Reactive Oxygen Species Contents

Salt stress significantly reduced the membrane stability index (MSI) (Figure 4A). At D8, M2 and M3 treatments effectively alleviated this decline, exhibiting 13% and 14% higher values than SS plants, respectively. At D12, the MSIs in M1S, M2S, and M3S plants remained 14%, 30%, and 26% higher than those in SS plants, respectively. MDA content increased markedly under salt stress (Figure 4B). At D4, MDA levels in SS plants rose to 2.06-fold of control values, whereas M1S, M2S, and M3S plants showed increases of 1.86-, 1.66-, and 1.74-fold, respectively. At D12, the MDA contents in M2S and M3S plants were 41% and 44% lower than those in SS plants, respectively. Salt stress induced the accumulation of O₂⁻ and H₂O₂ in leaves, with progressively stronger effects over time (Figure 4C,D). At D8, the O₂⁻ levels in SS plants were 140% higher than those in the controls, whereas M3S plants exhibited only a 74% increase. The regulatory effects of MT on H₂O₂ were evident at D8. SS plants showed a 74% increase in H₂O₂ content compared to the control, whereas M2S plants displayed a mere 30% increase. At D12, M2S plants retained 22% lower H₂O₂ content than M1S plants.

3.5. Antioxidant Enzyme Activity

Salt stress significantly enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) (Figure 5A). At D8, the SOD activity in M2S plants was 16% higher than that in SS plants. At D12, M3S plants exhibited substantially higher SOD activity than M1S, M2S, and SS plants. For POD, SS plants showed an 89% increase at D8 compared to CK (Figure 5B), whereas M2S and M3S plants displayed even greater increases of 127% and 115%, respectively. During the late stress stage (D12), M2S and M3S plants maintained 1.57- and 1.34-fold higher POD activity than SS plants at D12. CAT activity in M2S plants increased by 12% at D8 compared to SS plants (Figure 5C). At D12, CAT activity in M1S and M2S plants further increased to 1.96- and 2.12-fold higher levels than in SS plants, respectively. Salt stress also significantly elevated the total antioxidant capacity (TAC) of tomato plants (Figure 5D). The positive effects of MT spraying on TAC levels became more pronounced with prolonged stress exposure. At D12, TAC levels in M2S and M3S, plants were 33% and 29% higher than those in SS plants, respectively.

3.6. Ion Contents of Leaves and Roots

The leaf Na⁺ content in M1S, M2S, and M3S plants decreased by 22%, 19%, and 44% at D4, compared to SS plants, respectively (Figure 6A). Root Na⁺ content in M2S and M3S plants was 15% and 28% lower than that in SS plants at D4. At D12, M3S plants showed further reductions, with leaf and root Na⁺ contents 29% and 46% lower than those of SS plants, respectively. Salt stress suppressed K⁺ uptake in both leaves and roots (Figure 6B). At D8, M2S and M3S plants displayed 14% and 13% higher leaf K⁺ content and 41% and 30% higher root K⁺ content than SS plants, respectively. At D12, the root K⁺ content in M2S and M3S plants was 93% and 80% higher than that in SS plants, respectively. The K⁺/Na⁺ improved markedly with melatonin treatment (Figure 6C). At D4, leaf and root K⁺/Na⁺ levels in M3S plants were 1.73- and 1.76-fold higher than those in SS plants (Figure 6C). At D8, leaf K⁺/Na⁺ in M2S and M3S plants exceeded SS levels by 57% and 84%, respectively, whereas root K⁺/Na⁺ increased by 78% and 104%, respectively. At D12, the root K⁺/Na⁺ in M2S and M3S plants was significantly higher than that in SS plants.

Salt stress inhibited Mg²⁺ absorption in the leaves and roots (Figure 6D). At D4, M2S plants showed 14% higher leaf Mg²⁺ content than SS plants. At D8, leaf Mg²⁺ content in M2S and M3S plants was significantly elevated, with M1 and M3 showing stronger effects on root Mg²⁺ content than M2S. At D12, M2S plants achieved 39% higher leaf Mg²⁺ content, while M1S, M2S, and M3S plants showed 42%, 29%, and 51% increases in root Mg²⁺ content compared to SS plants, respectively. For Ca²⁺ content, M3S plants exhibited 6% and 10% higher leaf and root levels than SS plants at D4 (Figure 6E). At D8, M2S and M3S plants showed 13% and 17% increases in leaf Ca²⁺ content compared to SS plants, respectively. At D12, the leaf Ca²⁺ content in M1S, M2S, and M3S plants reached 1.30-, 1.39-, and 1.56-fold of SS levels, respectively, with similar enhancement patterns observed for root Ca²⁺ content in M2S and M3S plants.

3.7. Relationship Among Physiological Characteristics and Salt Tolerance Evaluation

3.7.1. Relationships Among Physiological Characteristics

Under salt stress conditions, leaf Na⁺ content was positively correlated with TAC, whereas leaf K⁺, Mg²⁺, and Ca²⁺ contents were significantly negatively correlated with TAC. Under MT spraying, the improved leaf K⁺, Mg²⁺, and Ca²⁺ contents contributed to a higher TAC. MSI decreased significantly with increasing leaf Na⁺ content, but demonstrated positive correlations with leaf K⁺, Mg²⁺, and Ca²⁺ contents. L_p showed a significantly negative correlation with root Na⁺ content and a significant positive correlation with root K⁺, Mg²⁺, and Ca²⁺ contents (Figure 7).

3.7.2. Comprehensive Evaluation of Salt Tolerance

Principal component analysis (PCA) revealed that salt-stressed (SS) plants clustered with physiological markers (PH, TAC, O₂⁻, FL, and Pro along the right abscissa, where Pro displayed strong positive correlations with FL, O₂⁻, and TAC (Figure 8A). Significant positive correlations between Mg²⁺ and Ca²⁺ contents were observed in leaves and roots, whereas all measured ions showed strong associations with MSI. Under MT spraying, the data points of the M2S and M3S treatments shifted rightward relative to the SS treatment. Furthermore, the data points of the M1S, M2S, and M3S treatments were closer to the upper side of the coordinate axis than those of the SS treatment, indicating that MT spraying effectively improved multiple physiological processes to enhance salt tolerance.

Heatmap analysis corroborated the PCA results (Figure 8B), demonstrating that MT spraying mitigated the decreases in P_n, water relations, and ion absorption (excluding Na⁺), while enhancing antioxidant enzyme activities and non-enzyme and osmoregulatory substance contents, particularly under M2 treatment. Eight key indicators of comprehensive salt tolerance were evaluated (Figure 8C), revealing a progressive improvement in M1S and M2S plants during stress, with M2S plants achieving the highest tolerance index at D12. Overall, M2 spraying provided superior salt tolerance in tomato plants, followed by M3 treatment.

4. Discussion

4.1. Exogenous Melatonin Improves Tomato Growth by Regulating Photosynthesis

Photosynthesis is a highly sensitive physiological process that is affected by salt stress. Salt stress substantially impairs plant photosynthetic capacity via stomatal closure and photosystem damage [5]. This study revealed that salt stress reduced g_s in tomato leaves, which may be attributed to decreased LRWC and L_p. Melatonin (MT) treatment, particularly at 100 µM concentration, markedly enhanced g_s and alleviated the stomatal limitation induced by salt stress. These findings are consistent with those reported by Ye [42], indicating that MT can improve plant gas exchange capacity by regulating stomatal aperture. Furthermore, the results demonstrated a progressive decline in the net photosynthetic rate (P_n) with prolonged salt stress exposure. Shi [17] proposed that melatonin exerts beneficial regulatory effects by upregulating the transcription of photosynthesis-related genes and protecting the photosynthetic apparatus. The current study confirmed that MT treatment significantly improved the P_n of tomato leaves subjected to salt stress. Notably, 100 and 150 μM melatonin outperformed 50 μM in sustaining photosynthetic rates. The improvement in P_n may be associated with the upregulation of genes encoding PSI-related proteins (PsaK and PsaG), the PsbO and PsbP subunits, and the oxygen-evolving complex (OEC) protein in the PSII reaction center [43].

This study found that salt stress significantly inhibited the growth parameters of both aboveground parts and roots of tomato plants (Figure 1). Growth suppression can be primarily attributed to salt-induced stomatal closure, which reduces P_n and ultimately leads to diminished biomass accumulation [44]. Previous studies have confirmed that MT is a regulator of plant growth and development [21,22]. For instance, low concentrations of MT can promote root elongation, whereas higher concentrations may inhibit growth, as observed in wild mustard [45]. Consistent with previous findings, we found that 100 μM MT exhibited superior regulatory effects on tomato growth compared to 50 μM and 150 μM treatments, which aligned with the findings of Bajwa [46] in Arabidopsis. We speculated that this dose-dependent response may be associated with hormonal regulation and the activation of salt-tolerance-related genes [47]. MT may alleviate salt-induced growth inhibition in tomatoes by regulating the expression of genes encoding K⁺ channels and Na⁺/H⁺ antiporters [48], thereby maintaining leaf ion homeostasis. Furthermore, the growth-promoting effects of MT have been well documented in other crops, including maize [49] and rice [50].

4.2. Exogenous Melatonin Enhanced Tomato Salt Tolerance by Improving Osmoregulation

Osmolyte accumulation plays a vital role in maintaining cellular homeostasis and counteracting salt stress toxicity [20]. Our study revealed significant proline accumulation in tomatoes under salt stress, with this trend becoming more pronounced during the later stages of stress. Under salt stress conditions, plants coordinately enhance proline accumulation by upregulating key biosynthetic genes (e.g., P5CS1 and P5CR) while suppressing catabolic genes (e.g., ProDH/PDH), thereby improving salt tolerance [51]. Notably, MT spraying further strengthened this accumulation effect. This is consistent with the findings of Zhang [52], who demonstrated that MT application significantly increased betaine and proline content in plants, while upregulating the expression of related synthesis genes (BADH, P5CS, etc.), consequently enhancing osmoregulatory capacity under saline conditions. These results collectively suggest that MT application elevates proline levels in plants, and the accumulation of proline can increase leaf osmotic potential and promote water absorption by plants, thereby providing favorable conditions for photosynthesis and ultimately improving growth performance [19,53]. Notably, the regulatory effects of MT on proline synthesis exhibited significant concentration-dependent variations; 150 μM demonstrated optimal efficacy in promoting proline accumulation throughout the stress period, whereas 50 μM had the weakest effect. These results confirmed that MT regulation of proline metabolism is concentration-dependent.

In the current study, salt stress significantly increased soluble sugar content in leaves, while MT treatment exhibited a distinct concentration-dependent effect; medium MT concentration (100 μM) further enhanced sugar accumulation, whereas both low (50 μM) and high (150 μM) concentrations reduced it. These findings are consistent with previous studies demonstrating that MT-induced accumulation of osmolytes reduces cellular osmotic potential, thereby enhancing plant osmoregulatory capacity and improving water status under stress conditions [54,55]. In this study, MT-sprayed plants exhibited significantly higher LRWC and L_p than those subjected to salt stress treatment, which directly confirmed this mechanism. The improved water status in salt-stressed plants may also be attributed to melatonin-mediated promotion of root growth and aquaporin activity, which enhances water absorption and transport [56]. Additionally, higher cellular water content can induce stomatal opening in leaves, facilitating the exchange of H₂O and CO₂ between leaves and the external environment and providing sufficient raw materials for photosynthesis. This also explains why the MT application under salt stress increased g_s and P_n in tomato plants.

4.3. Exogenous Melatonin Enhanced Tomato Salt Tolerance by Regulating the Antioxidant System

Under salt stress, excessive Na⁺ accumulation induces ROS overproduction, leading to lipid peroxidation and membrane disruption [7,8]. Our study showed significant increases in H₂O₂ and O₂⁻ levels due to persistent Na⁺-induced ion toxicity, which was closely related to the osmotic stress induced in the early stage of salt stress and the ionic toxicity caused by long-term Na⁺ accumulation [57]. Nevertheless, melatonin spraying at varying concentrations effectively reduced the levels of these oxidative markers, with 100 µM MT demonstrating significantly superior ROS-scavenging capacity compared to 50 μM and 150 μM treatments. Notably, the inhibitory effect of MT on ROS did not diminish with the prolongation of salt stress. After 8–12 days of stress, O₂⁻ and H₂O₂ levels remained substantially lower in MT-treated plants than in those subjected to salt stress treatment alone. This phenomenon has been observed in various crops. For instance, continuous MT application reduced ROS accumulation in tea plants under long-term arsenic stress [58], and after 15 days of salt stress, the malondialdehyde content and electrolyte leakage in the MT-treated group of alfalfa were significantly lower than those in the control group [59]. These results indicate that the ROS-scavenging capacity of MT in the short term is not affected by the extension of stress duration, which may be related to the sustained high levels of endogenous melatonin in plants after MT spraying [60]. Similarly, studies on cucumber have also found that salt stress led to an increase in H₂O₂ concentration, while MT spraying maintained a lower level of H₂O₂ throughout the experiment [61]. MT also reduces the O₂⁻ content in tomato seedlings under salt stress [62]. Our data further showed that melatonin spraying reduced MDA levels and improved the membrane stability index, which coincided with decreased ROS generation, suggesting its role in maintaining membrane integrity through oxidative damage alleviation [63]. In terms of membrane protection, both 100 and 150 µM melatonin outperformed 50 µM. This protective effect may be associated with the activation of the MAPK cascade by melatonin, a signaling pathway that not only promotes the expression of WRKY transcription factors [64] but also effectively mitigates membrane lipid peroxidation [65].

Plants have evolved complex antioxidant systems to counteract ROS accumulation during stress. Our results demonstrated that exogenous MT combined with salt stress synergistically enhanced SOD, POD, and CAT activities, indicating that MT spraying effectively upregulated antioxidant enzyme activities in tomato leaves, thereby scavenging excess ROS and protecting plants from oxidative damage. Notably, 100 μM MT treatment demonstrated significantly greater enhancement of antioxidant enzyme activity compared to 50 μM and 150 μM treatments. Zhang [66] also confirmed that MT treatment significantly upregulated the expression of POD, APX, SOD, and CAT genes in cucumbers under salt stress conditions. Similar results have been reported in naked oats [64] and rapeseeds [67]. The mechanism may involve MT enhancing antioxidant enzyme activity by upregulating the expression of antioxidant enzyme-related genes and reducing the degradation of biomacromolecules [66,68]. However, studies have shown that MT did not enhance the activities of CAT and POD under salt stress but instead reduced them [69]. These differences may be related to the MT concentration and salt stress intensity.

This study revealed that the antioxidant strategy of MT-sprayed plants was not fixed but was dynamically adjusted with prolonged stress duration. During the 0–8 days of salt stress, MT-sprayed plants primarily relied on the antioxidant enzyme system to scavenge ROS. This enzyme-dependent mechanism demonstrates both high efficiency and substrate specificity, with its activity positively correlated with plant salt tolerance [70]. The results further demonstrated that the activities of the antioxidant enzymes showed a trend of first increasing and then decreasing with the extension of stress duration. Interestingly, although MT cannot directly scavenge O₂⁻ and H₂O₂, it maintains cellular redox homeostasis by activating antioxidant enzymes. During the late stage of stress, MT-sprayed plants relied on small-molecule antioxidants to eliminate ROS. This is because toxic ROS, such as O₂⁻ and H₂O₂, are difficult to completely remove by the enzyme system, necessitating the synergistic effects of proline, flavonoids, and polyphenols [71]. Our data demonstrated that M2S plants exhibited multiplicative increases in phenol and flavonoid content compared to salt-stressed plants during later stress stages, although excessively high concentrations of MT had an inhibitory effect. Our results are consistent with the study on rubber trees [72], while uniquely revealing that the magnitude of antioxidant accumulation during late stress substantially exceeds that observed during the initial phase, indicating that MT-sprayed tomatoes primarily rely on antioxidant substances to maintain cellular homeostasis in the later stages of stress.

4.4. Exogenous Melatonin Enhanced Tomato Salt Tolerance by Regulating Ion Homeostasis

Salt stress-induced plasma membrane depolarization activates non-selective cation channels and K⁺ outward-rectifying channels, leading to excessive Na⁺ influx into cells and subsequent Na⁺ toxicity [57]. This study also observed similar phenomena, where salt stress significantly increased the Na⁺ content in tomatoes while reducing the K⁺ content. Accumulating evidence indicates that MT enhances salt tolerance by regulating key ion transporter genes to maintain cellular ion homeostasis. For instance, Li [73] found that MT alleviated plant damage in high-salt conditions by upregulating the expression of the MdNHX1 gene (Na⁺/H⁺ antiporter). In sugar beets, MT application under salt stress not only enhanced root plasma membrane H⁺-ATPase activity and H⁺ efflux capacity but also upregulated the relative expression of HKT1 and NHX1 in roots [74], further confirming the key role of MT in maintaining ion homeostasis under salt stress. The results demonstrated that MT spraying significantly enhanced K⁺ accumulation while reducing Na⁺ content in both leaves and roots of tomato plants, corroborating the findings of Zahedi [75] in tomatoes and confirming that MT can improve plant salt tolerance by regulating ion transport systems. Similarly, in salt-stressed cucumber roots, MT upregulated 77 differentially expressed genes, including important transcription factors such as MYB, WRKY, NAC, and ERF, whose expression is closely related to cucumber salt tolerance [76]. Moreover, we observed that salt stress-induced K⁺/Na⁺ imbalance compromised cell membrane integrity, whereas exogenous MT enhanced salt tolerance by upregulating this ratio. This may be attributed to MT promoting the expression of SOS1, NHX1, and/or AKT1, thereby maintaining K⁺/Na⁺ homeostasis [77]. Maintaining a relatively high K⁺/Na⁺ ratio helps alleviate ion toxicity, protect cell membrane integrity [78], and ensure normal photosynthesis [79].

At the initial stage of salt stress, the Ca²⁺ content in leaves and roots exhibited a transient increase. This phenomenon is often coordinated with reactive oxygen species (ROS) generation, both as a stress signaling mechanism and to regulate Na⁺ homeostasis through the activation of the membrane-bound Na⁺/H⁺ antiporter SOS1 [80,81]. With prolonged stress exposure, a progressive decline in Mg²⁺ and Ca²⁺ contents was observed in both leaves and roots. Nevertheless, MT effectively alleviated the inhibitory effect of Na⁺ on the absorption of Mg²⁺ and Ca²⁺ by suppressing Na⁺ accumulation and promoting K⁺ retention. Under salt stress conditions, MT stimulates Ca²⁺ release by upregulating the expression of genes involved in InsP3 and InsP6 synthesis [52]. Notably, leaf Na⁺ accumulation was positively correlated with TAC, which may represent an adaptive response of plants to oxidative stress. However, this Na⁺-induced enhancement of antioxidant capacity exhibited inherent limitations, as evidenced by a significant decline in the membrane stability index with increasing Na⁺ content, indicating that ROS scavenging alone cannot fully alleviate Na⁺-mediated membrane damage. MT treatment altered this relationship by simultaneously promoting the uptake and retention of K⁺, Mg²⁺, and Ca²⁺, thereby providing additional reinforcement to the antioxidant defense systems. Particularly noteworthy is the strong positive correlation between increased in K⁺, Mg²⁺, and Ca²⁺ content and membrane stability index, suggesting that MT protects membrane integrity through a dual mechanism; on one hand, by reducing Na⁺-induced membrane lipid peroxidation, and on the other hand, by enhancing the stabilizing effect of essential cations on membrane structure [82]. At the root level, the negative correlation between Na⁺ content and L_p revealed the inhibitory effect of salt stress on water transport in roots. In contrast, melatonin-sprayed plants exhibited highly significant positive correlations between K⁺, Mg²⁺, and Ca²⁺ content and L_p, confirming that melatonin maintained root water uptake efficiency by regulating ion homeostasis [83].

4.5. Comprehensive Evaluation of Tomato Drought Tolerance

To overcome the limitations of single-indicator evaluations, this study employed the membership function method to identify the optimal melatonin concentration for alleviating salt stress. The results showed that 50 and 100 μM MT-sprayed plants showed sustained increases in the comprehensive salt tolerance index, whereas 150 μM MT-sprayed plants exhibited an increasing and then decreasing trend. Prolonged stress exposure (12 days) resulted in a dramatic decline in the comprehensive salt tolerance index of non-primed plants, indicating that severe salt stress exceeded the tolerance threshold of non-primed plants, ultimately causing substantial growth suppression. Notably, the beneficial effects of 150 μM melatonin on tomato plants were markedly diminished at D12 compared to those at D8. Under prolonged salt stress, high concentrations of melatonin may reduce tomato salt tolerance by disrupting ion homeostasis and damaging the antioxidant defense system. Moreover, long-term treatment with high concentrations of MT inhibits the expression of key genes in the SOS signaling pathway, thereby affecting the regulation of Na⁺/K⁺ homeostasis [82]. These findings collectively indicate that melatonin’s protective effects exhibit a distinct ‘optimal concentration window’ in plants, and excessively high concentrations or prolonged treatment durations may lead to negative effects.

While this study delineated the physiological responses of tomato plants to exogenous melatonin under salt stress, the underlying mechanisms require further elucidation. Future investigations should focus on (1) transcriptomic sequencing and targeted gene quantification to decipher melatonin’s regulation of ion homeostasis and antioxidant-related genes; (2) multi-season field validation of the optimal 100 μmol/L concentration coupled with the development of cost-effective delivery systems (e.g., drip irrigation); and (3) concentration gradient refinement (75–125 μmol/L) and extension to tomato cultivars with varying salt tolerance and other vegetable crops. These approaches bridge the gap between controlled-environment findings and agricultural applications, offering practical solutions for saline-affected regions.

5. Conclusions

This study investigated the effects of exogenous melatonin on tomato plants subjected to salt stress (Figure 9). The results demonstrated that 100 μM melatonin (M2) exhibited the most pronounced alleviative effects, particularly during 8–12 days of treatment, showing superior performance over both 50 μM and 150 μM treatments in enhancing leaf relative water content, root hydraulic conductivity, net photosynthetic rate, and biomass accumulation, whereas higher concentrations displayed inhibitory effects. The regulatory efficacy of M2 treatment became increasingly prominent with prolonged stress duration, operating through multiple integrated physiological pathways, activating the antioxidant system and promoting phenolic and flavonoid accumulation to mitigate oxidative damage; elevating osmolyte content and optimizing ion homeostasis to enhance osmotic adjustment capacity; and improving root architecture to facilitate water uptake. A comprehensive evaluation of salt tolerance confirmed that 100 μM melatonin enabled tomato plants to achieve optimal salt tolerance. These findings elucidate the physiological mechanisms through which melatonin alleviates salt stress via a coordinated ‘water-photosynthesis-antioxidant-ion’ network, providing a scientific basis for the precise application of melatonin in tomato production under saline conditions.