Mitigation of Salt Stress in Tomato (Solanum lycopersicum L.) Through Sulphur, Calcium, and Nitric Oxide: Impacts on Ionic Balance, Nitrogen-Sulphur Metabolism, and Oxidative Stress

Abstract

Background: In this study, hydroponic experiments were conducted to examine the roles of sulphur (S), calcium (Ca), and nitric oxide (NO) in alleviating salt stress (20 mM NaCl) in tomato (Solanum lycopersicum L.) seedlings. Methods: Analyses included Na⁺/K⁺ contents, inorganic nitrogen (nitrate, nitrite, ammonium), nitrogen- and ammonium-assimilating enzymes (NR, NiR, GS, GOGAT), sulphur-assimilating enzymes (ATPS, OASTL), protein content, ROS (O₂^∙−, H₂O₂), and in vivo NO visualization were conducted. Results: We observed that salt stress increased Na⁺, reduced K⁺, disrupted nitrogen and sulphur metabolism, elevated ROS, and decreased NO, causing oxidative stress and reduced enzymatic activity. Supplementation with potassium sulphate (40 µM), calcium chloride (30 µM), and sodium nitroprusside (SNP; 40 µM) mitigated these effects, enhancing enzymatic activities, restoring Na⁺/K⁺ balance, improving protein content, and lowering ROS. The protective role of NO was confirmed using inhibitors L-NAME (500 µM) and cPTIO (100 µM), which reversed SNP’s benefits and aggravated stress damage. Conclusion: Overall, S, Ca, and NO were found to synergistically improve salt stress tolerance by modulating ion homeostasis, nitrogen and sulphur metabolism, and oxidative balance, offering nutrient- and signal-based strategies to enhance tomato resilience under salinity.

1. Introduction

Salt stress is a major abiotic factor that negatively impacts crop productivity worldwide, especially in arid and semi-arid regions [1]. Salt stress causes ionic imbalance, osmotic stress, and oxidative damage, ultimately disrupting various physiological and biochemical processes in plants. Tomato (Solanum lycopersicum L.), a vital vegetable crop globally, is highly vulnerable to saline conditions, which hinder its growth, development, and yield [2]. One of the most critical effects of salt stress is the excessive buildup of sodium (Na⁺) ions, which interfere with potassium (K⁺) homeostasis and disrupt essential cellular functions [3]. Additionally, salt stress hampers nitrogen (N) and sulphur (S) metabolism, crucial for plant growth and stress tolerance. Enzymes involved in nitrogen assimilation, such as nitrate reductase (NR) and nitrite reductase (NiR), which catalyze the reduction in nitrate (NO₃⁻) and nitrite (NO₂⁻) to ammonium (NH₄⁺), are significantly affected, disrupting nitrogen assimilation pathways [4]. Moreover, key sulphur assimilation enzymes like ATP sulfurylase (ATPS), responsible for activating inorganic sulphate (SO₄²⁻) to adenosine 5′-phosphosulfate (APS), and O-acetyl serine (thiol) lyase (OASTL), which catalyzes the final step in cysteine biosynthesis, are also impacted, disrupting sulphur assimilation pathways and ultimately reducing plant productivity [5].

Calcium (Ca), sulphur (S), and nitric oxide (NO) are essential signalling molecules crucial for modulating plant responses to salt stress. Ca controls the transport of Na⁺ and K⁺ across membranes, helping to maintain ionic balance and reduce toxicity [6,7]. sulphur supports stress tolerance by aiding in the biosynthesis of vital sulphur-containing compounds, such as glutathione, which acts as a strong antioxidant [8,9]. NO, a versatile signalling molecule, mediates stress responses by boosting antioxidant defences, scavenging reactive oxygen species (ROS), and maintaining redox homeostasis [10,11]. The interaction among Ca, S, and NO significantly affects key biochemical and physiological parameters under salt stress. Ca and NO work together to regulate Na⁺ and K⁺ homeostasis, thus preserving cellular ion balance during stress and also enhance the activities of nitrogen-assimilating enzymes, including NR and NiR, which improve the conversion of NO₃⁻ to NO₂⁻ and NH₄⁺ [12]. Additionally, sulphur and nitric oxide influence ammonium-assimilating enzymes like GS and GOGAT, facilitating the incorporation of ammonium into amino acids [13]. The roles of sulphur-assimilating enzymes such as ATPS and OASTL are amplified by calcium and NO, leading to increased production of sulphur metabolites that are crucial for stress tolerance [14]. Additionally, accelerated production of ROS under salt stress disrupts cellular redox balance, leading to oxidative injury and impaired physiological functions. Superoxide radicals and hydrogen peroxide, if not efficiently detoxified, can cause membrane lipid peroxidation and protein oxidation, ultimately hampering plant growth and development. NO acts both directly, by reacting with ROS to neutralize their effects, and indirectly, by upregulating the expression and activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). In conjunction with sulphur-containing compounds like glutathione (GSH), NO contributes to the regeneration of the cellular redox pool, thereby enhancing the plant’s capacity to maintain homeostasis under saline conditions [15].

Despite the extensive research on individual and dual roles of Ca, S, and NO, their combined regulatory mechanisms under salt stress remain unclear, particularly in the context of specific parameters like Na⁺ and K⁺ contents, nitrogen and sulphur metabolism, enzyme activities, protein content, and ROS dynamics. This gap in understanding is especially relevant in studies involving inhibitors such as 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and N-Nitro-L-arginine methyl ester (L-NAME), which are used to dissect the roles of endogenous and exogenous nitric oxide in stress responses. This study aims to elucidate the combined roles of Ca, S, and NO in regulating physiological and biochemical parameters under salt stress, with a focus on enhancing the salt tolerance of tomato and other solanaceous crops. By investigating these interactions, this research seeks to contribute to a deeper understanding of plant stress biology and inform strategies for sustainable agricultural practices in saline environments.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Seeds of Solanum lycopersicum L. var. NS 585 (tomato) were obtained from a certified supplier in Allahabad district. Tomato, a dicotyledonous vegetable, belongs to the family Solanaceae within the order Solanales. For surface sterilization, the seeds were treated with 2% (v/v) sodium hypochlorite for 15 min, thoroughly rinsed with double-distilled water (DDW), soaked in distilled water for 1–2 h, and placed in darkness for germination. After two days, uniformly germinated seeds were sown in sterilized sand (particle size: 0.2–1.6 mm) in plastic trays and kept under dark conditions at 25 ± 2 °C for two days until emergence. Seedlings were subsequently transferred to a controlled growth chamber (CDR model GRW-300 DGe, Athens, Greece) maintained at 25 ± 2 °C, 65–70% relative humidity, 200 μmol photons m⁻² s⁻¹ PAR, and a 16:8 h light–dark cycle. Irrigation was carried out on alternate days using half-strength Hoagland solution [16,17]. The experiment took place from May to June 2024 at Lovely Professional University, Punjab, India (located at approximately 31.2619° N, 75.5841° E, or 31°15′43″ N, 75°35′05″ E). After 21 days, once the secondary leaves had emerged, the seedlings were carefully uprooted, thoroughly washed to remove any adhering sand, and acclimatized in 50% Hoagland solution for 24 h. Thereafter, three healthy and uniform seedlings were transferred into each plastic bottle containing 15 mL of Hoagland solution, either alone or supplemented with the following treatments: NaCl (20 mM), Ca (30 µM from CaCl₂), S (40 µM from K₂SO₄), NO (40 µM from SNP; Sigma-Aldrich, St. Louis, MO, USA, Cat. No. 228710), L-NAME (100 µM; Sigma-Aldrich, Cat. No. N5751), and cPTIO (200 µM; Sigma-Aldrich, Cat. No. 217386). For salt treatment, seedlings were exposed to 20 mM NaCl with an electrical conductivity of 3.28 S/m. For exogenous NO treatment, seedlings were treated with 40 μM NO donor. The concentration of NaCl was selected based on its significant impact on the growth (fresh weight) of tomato. The concentration of SNP was chosen based on its effectiveness in alleviating NaCl toxicity, as determined through dose–response curves and previous studies [18]. The conductivity was readjusted every two days to maintain the NaCl dose until the 7th day of treatment. In line with the previous research findings by [19], we have employed cPTIO inhibitor with a concentration of 100 μM and L-NAME with a concentration of 500 μM to investigate the distinct roles of endogenous and exogenous NO in stress responses. The combinations, which likely draw on existing literature or preliminary studies, are designed to decipher the individual and combined effects of these elements on the physiological and biochemical responses in the test seedlings [20]. All chemicals were prepared in 50% Hoagland solution, and the following combinations were applied in the hydroponic system: (1) control (without NaCl/Ca/S/SNP/cPTIO/L-NAME), (2) NaCl, (3) NaCl + Ca, (4) NaCl + S, (5) NaCl + Ca + NO, (6) NaCl + S + NO, (7) NaCl + Ca + S + NO, (8) NaCl + Ca + L-NAME, (9) NaCl + S + L-NAME, (10) NaCl + Ca + cPTIO, (11) NaCl + S + cPTIO, (12) NaCl + Ca + S + L-NAME, (13) NaCl + Ca + S + cPTIO, and (14) NaCl + Ca + S + L-NAME + NO. These combinations were applied to both test seedlings to comprehensively assess the roles of Ca, S, and NO under stress conditions. Under aseptic conditions, an air bubbler was used to aerate the seedlings with sterile air, and the nutrient medium was changed every two days until the 7th day of treatment. Throughout this period, the pH of the Hoagland solution was regularly monitored with a pH metre and adjusted as needed with dilute acid or base to maintain optimal levels, ensuring consistent nutrient availability and optimal plant responses. After 7 days of treatment, seedlings from each pot were harvested, and various physiological and biochemical parameters related to growth regulation were analyzed.

2.2. Calculation of Na⁺ and K⁺ Content

Leaf samples were oven-dried at 80 °C for 48 h and 100 mg portion of the dried, ground sample was placed in a digestion vessel with 10 mL concentrated HNO₃ and left overnight at room temperature [21]. The next day, the sample was heated until NO₂ fumes dissipated, cooled, and then treated with 3 mL of 70% HClO₄. The mixture was reheated until a small volume remained, after which the digest was transferred to a flask, diluted with distilled water, and filtered through medium-grade filters into plastic bottles. Sodium (Na⁺) and potassium (K⁺) concentrations were determined using an Atomic Absorption Spectrophotometer (AAS, Model: [insert model, e.g., PerkinElmer Analyst 400, Waltham, MA, USA] equipped with hollow cathode lamps and operated with an air–acetylene flame under manufacturer-optimized parameters. Calibration was performed with certified Na⁺/K⁺ standard solutions (Merck, Darmstadt, Germany) in the range of 0.1–20 mg L⁻¹, and calibration curves consistently showed regression coefficients (R²) greater than 0.999.

2.3. Estimation of Inorganic Nitrogen Content (Nitrate, Nitrite, Ammonia)

Nitrate (NO₃⁻) content was estimated following the method of [22], which is based on the nitration of salicylic acid under acidic conditions. Nitrite (NO₂⁻) content was determined [23], while ammonium (NH₄⁺) content was measured using the method of [22]. For all assays, concentrations were calculated using calibration curves prepared with KNO₃, NaNO₂, and NH₄Cl as standards, respectively. Absorbance was recorded at the specific λmax of each assay (nitrate at 410 nm, nitrite at 540 nm and ammonium at 640 nm) using 1.0 cm path-length quartz or plastic cuvettes, or path-length–corrected microplates where applicable. Calibration curves were generated over defined concentration ranges with 6–8 calibration points, and quantification was performed using linear regression (R² ≥ 0.995) against reagent blanks.

2.4. Estimation of Nitrogen Assimilating Enzymes (Nitrate Reductase, Nitrite Reductase) Activities

For nitrate determination, plant material was homogenized using 100 mM potassium phosphate buffer (pH 7.4) containing 7.5 mM cysteine, 1 mM EDTA, 7.5 mM MgCl₂ and 1.5% (w/v) casein in a mortar and pestle. The homogenate was then centrifuged at 30,000× g for 15 min at 4 °C. A 0.1 mL extract was incubated with 0.5 mL 0.1 M potassium phosphate buffer (pH 7.4), 0.1 mL 0.1 M KNO₃ and 0.1 mL 0.15 mM NADH at 30 °C for 30 min, followed by termination of the reaction with 0.2 mL 1 M zinc acetate. The mixture was centrifuged at 3000× g for 10 min, and nitrite determination was performed by adding 1 mL 5.8 mM sulphanilamide (SA) in 1.5 N HCl and 1 mL 0.8 mM N-naphthyl ethylenediamine-dichloride to the centrifuged extract, incubating it for 20 min at room temperature. Absorbance was measured at 540 nm and nitrite concentration was calculated using a standard curve prepared with NaNO₂ [23,24].

For nitrite determination, enzyme extracts were prepared similarly to the nitrate reductase assay. A 0.1 mL extract was incubated with 0.4 mL 0.1 M potassium phosphate buffer (pH 7.4), 0.1 mL 15 mM sodium nitrite, 0.2 mL 5 mM methyl viologen and 0.2 mL 86.15 mM sodium dithionite in 190 mM NaHCO₃ at 30 °C for 30 min. The reaction was terminated by intense vortexing, and nitrite ions were assayed as in the nitrate reductase assay [25].

2.5. Estimation of Ammonium Assimilating Enzymes (Glutamine Synthetase and Glutamate Synthase) Activities

The protocols for Glutamine Synthetase (GS; EC 6.3.1.2) and Glutamate Synthase (NADH-GOGAT; EC 1.4.1.14) were as follows. For the GS assay [26], 1 g of leaf tissue was homogenized with sand for 75 s in 4 mL of 50 mM Tris-HCl (pH 7.8) containing 15% (v/v) glycerol, 14 mM 2-mercaptoethanol, 1 mM EDTA, and 0.1% (v/v) Triton X-100. The homogenate was squeezed through two layers of Miracloth and then centrifuged at 3000× g for 10 min at 0 °C. The standard reaction mixture was prepared with 50 mM L-glutamate, 10 mM ATP, 30 mM MgSO₄, 20 mM NH₂OH, and 100 mM Tris-HCl (pH 8.0). After adding 50 μL of the extract to start the reaction, it was incubated at 27 °C for 15 min, followed by termination with 2 mL of 2.5% (w/v) FeCl₂ and 5% (w/v) trichloroacetic acid in 1.5 M HCl. After centrifugation, the GS content was determined by measuring the absorbance at 540 nm. For the NADH-GOGAT assay [27], plant material was homogenized in 0.2 M sodium phosphate (pH 7.5), 50 mM KCl, 2 mM EDTA, 0.5% (w/v) Triton X-100, 0.1% (v/v) 2-mercaptoethanol and clean sand in a ratio of 1:4 (w/v) at 0–4 °C. The mixture was filtered through cheesecloth and centrifuged for 20 min at 20,000× g at 0 °C. Solid ammonium sulphate was added to the supernatant and incubated for 20 min, with precipitates from 30% and 55% saturation being re-dissolved in one-tenth of the original volume of 25 mM sodium phosphate (pH 7.5) containing 50 mM KCl, 1 mM EDTA, and 0.1% (v/v) 2-mercaptoethanol. The sample was then passed through a Sephadex G-75 column equilibrated with the same buffer. The assay mixture contains 0.5 mL of enzyme preparation, 0.4 mL of 20 mM L-glutamine, 0.1 mL of 100 mM KCl, 1 mM EDTA, 0.4 mL of 5 mM 2-oxoglutarate, 0.6 mL of 1 mM NADH, and 25 mM sodium phosphate (pH 7.5). The reaction was initiated by adding L-glutamine, and the decrease in absorbance at 340 nm was monitored for 5 min. The amount of NADH used was determined using a standard curve of NADH.

2.6. Estimation of Sulphur Assimilating Enzymes

The protocols for measuring ATP sulfurylase (EC 2.7.7.4) and O-acetylserine(thiol) lyase (OASTL) enzyme activities were as follows. For ATP sulfurylase activity [28], 100 mg fresh leaf and root tissue from control and treated seedlings was homogenized in 20 mM Tris-HCl (pH 8.0) containing 10 mM EDTA, 2 mM DTT and 0.01 g mL⁻¹ polyvinylpyrrolidone. The homogenate was then centrifuged at 20,000× g for 10 min at 4 °C. Two sets of reaction mixtures were prepared in test tubes: the first set contained 7 mM MgCl₂, 5 mM Na₂MoO₄, 2 mM Na₂ATP, 0.032 U mL⁻¹ sulphate-free pyrophosphatase, 80 mM Tris-HCl and 0.1 mL tissue extract; the second set contained all ingredients except 5 mM Na₂MoO₄. The reaction was initiated by adding the tissue extract and incubating at 37 °C for 15 min. Phosphate was then determined spectrophotometrically, and the ATP sulfurylase-mediated molybdate-dependent formation of pyrophosphate was calculated from the difference between the two reaction mixtures. For O-acetylserine(thiol) lyase (OASTL) activity [29], 100 mg leaf and root tissue was homogenized in 20 mM Tris-HCl (pH 8.0), and the homogenate was centrifuged at 13,000× g for 10 min at 4 °C. The reaction mixture consisted of 50 mM O-acetyl serine, 5 mM Na₂S, 33.4 mM DTT, 100 mM Tris-HCl (pH 7.5) and 50 µL tissue extract. The reaction mixture was incubated at 37 °C for 30 min, then the reaction was stopped by adding 1 mL acid ninhydrin reagent (0.8% w/v ninhydrin in a 1:4 concentrated HCl–acetic acid mixture). The absorbance of the reaction mixture was read at 560 nm, and the amount of cysteine was calculated using a standard curve.

2.7. Estimation of Protein Content

The method which was used for protein quantification, wherein the protein reagent was prepared by dissolving 0.01% Coomassie Brilliant Blue G-250 (100 mg) in 50 mL of 95% ethanol, adding 100 mL of 85% (w/v) phosphoric acid, and diluting the mixture to 1 L [29]. Fresh plant tissues (100–300 mg) were ground in liquid nitrogen using a precooled mortar and pestle, homogenized in cold 0.05 M Tris buffer (100–300 mg tissue per mL buffer) with 0.05 g of PVPP to minimize oxidation, and centrifuged at 14,000–19,000× g for 20 min at 4 °C; the clear supernatant obtained was either used immediately or stored at –20 °C. For the assay, 10–100 µg of protein extract was adjusted to 0.1 mL with buffer, mixed with 5 mL of protein reagent, vortexed, and absorbance was measured at 595 nm against a reagent blank (0.1 mL buffer + 5 mL reagent). Protein concentration was calculated using the Bradford assay with BSA standards (0–1000 µg/mL), and calibration curves consistently yielded R² > 0.998. All assays were performed under saturating substrate concentrations (well above Km), at a constant temperature, and volumes, with reaction times optimized to remain within the linear phase. Quality control included subtraction of procedural blanks (without enzyme), and reproducibility was confirmed with relative standard deviations below 5%.

2.8. Histochemical Detections of Superoxide and H₂O₂

Leaves were stained with 0.1% nitro blue tetrazolium (NBT) for superoxide anion (O₂^∙−) or 1% 3,3-diaminobenzidine (DAB) for hydrogen peroxide (H₂O₂). Leaves were incubated in the respective staining solutions for 8 h, with NBT staining performed in the dark and DAB staining in light, followed by decolorization in boiling ethanol [30]. Both low- and high-magnification representative images with scale bars were captured, and staining intensity was quantified using ImageJ (version 1.53t) by measuring area fraction and mean intensity. To minimize bias, blind scoring was employed, while incubation times were optimized and shortened to reduce background staining; where longer incubations were required, they were justified based on preliminary optimization. These refinements improved the accuracy, reproducibility, and specificity of ROS histochemical detection.

2.9. In Vivo Visualization of Nitric Oxide in Roots by Fluorescence

For detecting endogenous nitric oxide (NO) in roots, incubate root tips in Tris-HCl buffer (pH 6.5) containing 10 µM DAF-FM DA, dissolved in dimethyl sulfoxide (DMSO), for 30 min in the dark. Following incubation, the root tips were rinsed with distilled water and visualized using a fluorescence microscope with excitation at 495 nm and emission at 515 nm [31]. For detailed imaging, DAF-FM DA fluorescence was further examined using a [Zeiss LSM 710 confocal microscope, Carl Zeiss Microscopy, GmbH, Jena, Germany] equipped with a [40×/63×] oil immersion objective, with excitation at 488 nm and emission collected between 505 and 530 nm. Imaging parameters, including exposure time, gain, and laser intensity, were kept constant across all treatments to allow direct comparison. Fluorescence intensity was quantified with [ImageJ] by selecting uniform regions of interest (ROIs) and analyzing 8–10 random fields per replicate. Negative controls included dye-only (without tissue) and dye + cPTIO (NO scavenger) to verify signal specificity, while dye + sodium nitroprusside (NO donor) served as a positive control.

2.10. Estimation of Nitric Oxide Content

For measuring nitric oxide (NO) content in roots, prepare a reaction mixture containing 1 mL of supernatant, 1 mL of sulphanilamide, and 1 mL of N-(1-naphthyl) ethylenediamine dihydrochloride (NEDD) [32]. The mixture was incubated for 30 min, after which the absorbance was measured at 540 nm. The amount of NO was then determined by comparing the absorbance to a standard curve constructed using sodium nitrite (NaNO₂).

2.11. In Vivo Visualization of ROS (Reactive Oxygen Species) in Leaf and Roots

For visualizing reactive oxygen species (ROS), such as superoxide radicals (O₂^∙−) and hydrogen peroxide (H₂O₂), involves incubating leaves and roots in either nitroblue tetrazolium (NBT) prepared in potassium phosphate buffer (pH 6.4) containing 10 mM sodium azide or 1% 3,3′-diaminobenzidine (DAB) solution [33]. The samples were incubated in light for NBT (for O₂^∙−) and in the dark for DAB (for H₂O₂) until blue and brown spots, respectively, became visible. Afterward, the leaves and root tips were bleached by immersing them in boiling ethanol, which clears the tissue and allows for clear visualization of the blue and brown spots, which were then photographed.

2.12. Statistical Analysis

This study utilized data comprising the means and standard errors from three replicates (n = 3) to assess result accuracy. To determine the significance between the control and treatment group means, a one-way analysis of variance (ANOVA) was performed using SPSS-16 software. Subsequently, Duncan’s multiple range test (DMRT) was applied at a significance level of p ≤ 0.05. Before conducting the DMRT analysis, a multivariate test was employed to confirm the assumptions of variance homogeneity.

3. Results and Discussion

3.1. Estimation Na⁺ and K⁺ Content in Tomato

In tomato, Na⁺ content increased by 55.95%, while K⁺ content decreased by 39.87% under NaCl stress compared to control plants, demonstrating a significant disruption in ionic balance. Supplementation with calcium (NaCl + Ca), sulphur (NaCl + S), and their combination with nitric oxide (NaCl + Ca + S + NO) effectively alleviated these effects. The NaCl + Ca treatment reduced Na⁺ content by 21.26% and increased K⁺ content by 34.29%, while NaCl + S treatment reduced Na⁺ content by 18.02% and increased K⁺ content by 33.93%. The NaCl + Ca + S + NO combination was the most effective, reducing Na⁺ content by 32.20% and increasing K⁺ content by 98.52%, underscoring its potential to restore ionic balance compared to NaCl (

Table 1. Impact of exogenous supplementation of calcium (Ca), sulphur (S), nitric oxide (NO), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), and N-Nitro-L-arginine methylester (L-NAME) on the sodium to potassium ratio in the shoots of tomato seedlings exposed to NaCl stress.

S.No	Treatments	Tomato		Units (Both Parameters)
		Na+ Content	K+ Content
1	Control	4.45 ± 0.08 k	50.68 ± 0.06 b	mg g−1 DW
2	NaCl	6.49 ± 0.06 a	30.47 ± 0.09 i	mg g−1 DW
3	NaCl + Ca	5.11 ± 0.06 h	40.92 ± 0.04 e	mg g−1 DW
4	NaCl + S	5.32 ± 0.05 g	40.81 ± 0.06 e	mg g−1 DW
5	NaCl + Ca + NO	4.73 ± 0.07 j	50.48 ± 0.05 bc	mg g−1 DW
6	NaCl + S + NO	4.83 ± 0.06 i	50.28 ± 0.06 cd	mg g−1 DW
7	NaCl + Ca + S + NO	4.40 ± 0.06 k	60.49 ± 0.41 a	mg g−1 DW
8	NaCl + Ca + L- NAME	6.09 ± 0.03 c	30.83 ± 0.05 h	mg g−1 DW
9	NaCl + S + L- NAME	6.25 ± 0.05 b	30.73 ± 0.06 h	mg g−1 DW
10	NaCl + Ca + cPTIO	5.69 ± 0.02 e	40.48 ± 0.05 f	mg g−1 DW
11	NaCl + S + cPTIO	5.77 ± 0.04 e	40.22 ± 0.04 g	mg g−1 DW
12	NaCl + Ca + S + L-NAME	5.92 ± 0.05 d	40.36 ± 0.49 g	mg g−1 DW
13	NaCl + Ca + S + cPTIO	5.48 ± 0.04 f	40.67 ± 0.04 ef	mg g−1 DW
14	NaCl + Ca + S + L-NAME + NO	4.91 ± 0.05 i	50.10 ± 0.07 d	mg g−1 DW

Different superscript letters within a column indicate significant differences at p < 0.05.

). Conversely, L-NAME and cPTIO treatments exacerbated ionic imbalance under NaCl stress. L-NAME increased Na⁺ content by 40.44% and decreased K⁺ content by 39.36%, while cPTIO caused a 33.03% increase in Na⁺ content and a 20.63% decrease in K⁺ content compared to control.

In tomato plants, salt stress led to a significant disruption in ionic balance, characterized by increased Na⁺ accumulation and reduced K⁺ content. This imbalance reflects the deleterious effects of salinity on ion transport systems, leading to impaired growth and physiological performance. Supplementation with Ca and S, individually and in combination with NO, alleviated these effects by reducing Na⁺ uptake and enhancing K⁺ retention. The individual treatments (NaCl + Ca and NaCl + S) enhanced ionic balance by stabilizing membrane integrity and activating ion transporters, which are essential for maintaining Na⁺ exclusion and K⁺ uptake, as previously demonstrated in cotton and rice [34,35].

The combined treatment (NaCl + Ca + S + NO) demonstrated the most pronounced effects in restoring ionic homeostasis in tomato, emphasizing the synergistic roles of Ca, S, and NO. Nitric oxide likely stimulated plasma membrane H⁺-ATPase and H⁺/K⁺ antiporter activities, thereby promoting Na⁺ exclusion and K⁺ retention under salt stress in wheat [36]. This mechanism aligns with previous findings that NO modulates ionic transport by regulating transporter activity and signalling pathways in salt-stressed plants.

Conversely, treatments with L-NAME and cPTIO exacerbated ionic imbalances in tomato plants. These results highlight the indispensable role of endogenous and exogenous NO in ionic regulation. Inhibition of NO production by L-NAME and scavenging by cPTIO disrupted Na⁺ exclusion and K⁺ retention, highlighting NO’s regulatory role in ionic transport under salt stress in pea and barley [37,38].

3.2. Estimation of Inorganic Nitrogen Contents in Tomato

In tomato plants, NaCl treatment significantly decreased nitrate and nitrite levels by 34.14% and 32.85%, respectively, while increasing ammonium levels by 30.26% compared to control plants. Supplementation with calcium (NaCl + Ca), sulphur (NaCl + S), and their combination with nitric oxide (NaCl + Ca + S + NO) effectively mitigated these adverse effects. The NaCl + Ca treatment enhanced nitrate and nitrite levels by 40.74% and 34.04%, respectively, while reducing ammonium levels by 35.83%, compared to NaCl. Similarly, NaCl + S treatment increased nitrate and nitrite levels by 37.03% and 23.40%, respectively, but decreased ammonium levels by 30.18%. The combined NaCl + Ca + S + NO treatment demonstrated the most significant improvements, increasing nitrate and nitrite levels by 64.81% and 61.70%, respectively, while reducing ammonium levels by 47.16%, compared to NaCl (

Table 2. Impact of exogenous supplementation of calcium (Ca), sulphur (S), nitric oxide (NO), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), and N-Nitro-L-arginine methylester (L-NAME) on the inorganic nitrogen contents, including nitrate (NO₃⁻), nitrite (NO₂⁻), and ammonium (NH₄⁺), in tomato seedlings exposed to NaCl stress.

S.No	Treatments	Tomato			Units (All Parameters)
		Nitrate	Nitrite	Ammonium
1	Control	0.82 ± 0.004 b	0.84 ± 0.003 b	0.63 ± 0.005 h	(µmol g−1 FW)
2	NaCl	0.54 ± 0.011 j	0.54 ± 0.004 n	0.90 ± 0.010 a	(µmol g−1 FW)
3	NaCl + Ca	0.76 ± 0.007 de	0.74 ± 0.005 f	0.73 ± 0.006 f	(µmol g−1 FW)
4	NaCl + S	0.74 ± 0.004 ef	0.72 ± 0.005 g	0.73 ± 0.062 f	(µmol g−1 FW)
5	NaCl + Ca + NO	0.80 ± 0.011 bc	0.82 ± 0.003 c	0.68 ± 0.008 g	(µmol g−1 FW)
6	NaCl + S + NO	0.78 ± 0.004 a	0.79 ± 0.005 d	0.71 ± 0.005 f	(µmol g−1 FW)
7	NaCl + Ca + S + NO	0.89 ± 0.014 a	0.86 ± 0.003 a	0.58 ± 0.006 i	(µmol g−1 FW)
8	NaCl + Ca + L- NAME	0.64 ± 0.003 hi	0.62 ± 0.009 l	0.83 ± 0.006 bc	(µmol g−1 FW)
9	NaCl + S + L- NAME	0.62 ± 0.004 i	0.56 ± 0.005 m	0.86 ± 0.005 b	(µmol g−1 FW)
10	NaCl + Ca + cPTIO	0.71 ± 0.005 fg	0.69 ± 0.004 i	0.78 ± 0.005 de	(µmol g−1 FW)
11	NaCl + S + cPTIO	0.75 ± 0.052 g	0.68 ± 0.004 j	0.81 ± 0.008 cd	(µmol g−1 FW)
12	NaCl + Ca + S + L-NAME	0.67 ± 0.005 h	0.66 ± 0.005 k	0.82 ± 0.004 c	(µmol g−1 FW)
13	NaCl + Ca + S + cPTIO	0.72 ± 0.005 ef	0.70 ± 0.004 h	0.77 ± 0.005 e	(µmol g−1 FW)
14	NaCl + Ca + S + L-NAME + NO	0.77 ± 0.006 de	0.75 ± 0.005 e	0.72 ± 0.006 f	(µmol g−1 FW)

Different superscript letters within a column indicate significant differences at p < 0.05.

).

In contrast, the application of L-NAME and cPTIO adversely impacted nitrogen metabolism. L-NAME treatment lowered nitrate and nitrite levels by 24.03% and 30%, respectively, while increasing ammonium levels by 22.36%. Similarly, cPTIO treatment reduced nitrate and nitrite levels by 8.53% and 22.85%, respectively, but raised ammonium levels by 15.78% compared to control plants.

Salt stress significantly impairs nitrogen metabolism in tomato plants, as evidenced by reduced nitrate and nitrite levels and elevated ammonium levels. These changes suggest disruptions in the nitrogen assimilation pathway, likely due to inhibited activity of key enzymes such as nitrate reductase (NR) and nitrite reductase (NiR), which are crucial for the conversion of nitrate to nitrite and nitrite to ammonium. This disruption agrees with previous findings showing that salt stress impairs nutrient assimilation by affecting enzymatic activity and ion homeostasis in tomato and brinjal [39]. The observed increase in ammonium levels may reflect an imbalance in nitrogen metabolism or a stress-induced shift towards ammonium accumulation as a survival mechanism.

Supplementation with Ca and S, either individually or in combination with NO, mitigated these adverse effects and restored nitrogen metabolism. Ca supplementation improved nitrate and nitrite levels, potentially by stabilizing membrane integrity and enhancing enzymatic activities related to nitrogen uptake and assimilation. Similarly, S supplementation supported nitrogen metabolism by facilitating the synthesis of sulphur-containing amino acids and compounds critical for stress resilience. The combined NaCl + Ca + S + NO treatment produced the greatest improvements, indicating a synergistic interaction among these elements. Notably, NO appeared to play a central role in activating NR and NiR and in enhancing nitrogen-use efficiency under salt stress in wheat [40].

Conversely, the application of L-NAME and cPTIO, which inhibit NO synthesis or activity, exacerbated the suppression of nitrogen metabolism. These treatments led to a further decline in nitrate and nitrite levels while increasing ammonium accumulation, reflecting the critical role of NO in regulating nitrogen assimilation enzymes such as NR and glutamine synthetase. These results underscore the essential role of NO in sustaining nitrogen metabolism under salt stress in maize [41].

3.3. Enzymes Involved in Nitrogen Assimilation in Tomato

In this study, salt stress induced by NaCl alone led to significant reductions in NR and NiR activities, with decreases of 35.63% and 31.09%, respectively, in tomato, compared to control plants. Supplementation with calcium (NaCl + Ca), sulphur (NaCl + S), and their combination with sodium nitroprusside (NaCl + Ca + S + NO) effectively alleviated these negative effects. The NaCl + Ca treatment increased NR and NiR activities by 30.35% and 31.40%, respectively, while NaCl + S treatment improved these activities by 25% and 23.47%, respectively. The most substantial enhancements were observed with the NaCl + Ca + S + NO treatment, which resulted in increases of 69.64% and 49.68% in NR and NiR activities, respectively, compared to NaCl-stressed plants (Figure 1 and Figure 2). Conversely, the application of L-NAME and cPTIO further suppressed enzyme activities. L-NAME treatment caused reductions of 34.48% and 29.62% in NR and NiR activities, respectively, while cPTIO treatment led to decreases of 27.58% and 21.63% as compared to control.

In tomato plants, salt stress significantly suppressed the activities of nitrogen-assimilating enzymes NR and NiR. This inhibition reflects a disruption in nitrate and nitrite reduction processes, which are vital for nitrogen assimilation and overall plant growth. The reduction in enzyme activities under salt stress is likely due to oxidative damage and ionic imbalance, which impair enzymatic function, consistent with earlier reports in wheat [42].

Supplementation with Ca and S individually alleviated the inhibitory effects of salt stress on NR and NiR activities in tomatoes. This improvement is likely due to the stabilization of enzyme structures, enhanced ionic balance, and reduced oxidative stress. The combined treatment of NaCl + Ca + S + NO exhibited the most pronounced effects, restoring NR and NiR activities to near-optimal levels. These results are consistent with previous reports demonstrating the synergistic role of Ca, S, and NO in enhancing nitrogen assimilation by alleviating oxidative stress and stimulating nitrogen metabolism pathways in wheat and cabbage [43,44]. The role of NO as a signalling molecule is especially significant, as it regulates enzyme activity by mitigating ROS and enhancing nitrogen reduction processes in broccoli and soybean [45,46].

In contrast, the inhibition of NO biosynthesis or activity using L-NAME and cPTIO further exacerbated the suppression of NR and NiR activities under salt stress. These treatments interfered with the protective role of NO in nitrogen metabolism, highlighting its essential function in preserving enzyme activity under stress conditions in soybean [47].

3.4. Ammonium Assimilation Enzymes in Tomato

In tomato, the activities of ammonium-assimilating enzymes GS and GOGAT were significantly reduced under salt stress induced by NaCl, with decreases of 26.92% and 27.40%, respectively, compared to control plants. Supplementation with calcium (NaCl + Ca) improved GS and GOGAT activities by 27.51% and 28.18%, respectively, while sulphur supplementation (NaCl + S) enhanced these activities by 18.99% and 23.28%, respectively, compared to NaCl-stressed plants. The combined treatment of calcium, sulphur, and nitric oxide (NaCl + Ca + S + NO) yielded the most substantial recovery, with GS and GOGAT activities increasing by 41.62% and 42.64%, respectively, as compared to NaCl (Figure 3 and Figure 4). However, the application of L-NAME and cPTIO negatively affected these enzymes. L-NAME caused reductions of 25.08% and 22% in GS and GOGAT activities, respectively, while cPTIO treatment resulted in decreases of 17.13% and 17.43%, as compared to control exacerbating the effects of salt stress on ammonium assimilation.

Moreover, salt stress significantly suppressed the activities of ammonium-assimilating enzymes GS and GOGAT in tomato. This reduction disrupts the assimilation of ammonium into amino acids, a key step in nitrogen metabolism critical for plant growth and stress adaptation. The suppression of GS and GOGAT activities under salt stress is likely due to oxidative damage, ionic disturbances, and impairments in nitrogen metabolism pathways in rice [48].

Supplementation with Ca and S partially alleviated the adverse effects of salt stress on GS and GOGAT activities in tomatoes. However, the combination of Ca, S, and NO (NaCl + Ca + S + NO) proved to be the most effective, restoring enzyme activities to near-normal levels. This enhancement may be ascribed to the combined effects of these treatments in promoting ionic balance, minimizing ROS-mediated damage, and preserving enzyme stability in potato [13]. The role of nitric oxide in regulating GS and GOGAT is especially important, as it influences enzyme activity via post-translational modifications and alleviates oxidative stress in sage [49].

Conversely, treatments with L-NAME and cPTIO, which inhibit NO biosynthesis or activity, exacerbated the suppression of GS and GOGAT activities under salt stress. This highlights the crucial role of NO in controlling ammonium assimilation and sustaining nitrogen-use efficiency during stress in wheat [50].

3.5. Enzymes Involved in Sulphur Assimilation in Tomato

In tomato plants, salt stress induced by NaCl significantly reduced the activities of sulphur-assimilating enzymes ATPS and OASTL by 31.71% and 3.77%, respectively, compared to control plants. However, supplementation with calcium (NaCl + Ca) improved the activities of these enzymes by 37.91% and 37.14%, respectively. Sulphur supplementation (NaCl + S) resulted in even greater enhancements, with ATPS and OASTL activities increasing by 67.30% and 73.97%, respectively. The combined treatment (NaCl + Ca + S + NO) demonstrated the most pronounced improvements, increasing ATPS and OASTL activities by 71.82% and 125.57%, respectively, compared to NaCl-stressed plants (Figure 5 and Figure 6). Conversely, the application of L-NAME and cPTIO exacerbated the negative effects of salt stress, with L-NAME reducing ATP sulfurylase and OASTL activities by 16.74% and 22.89%, respectively, and cPTIO causing decreases of 13.65% and 22.89% as compared to control.

NaCl stress significantly suppressed the activities of ATPS and OASTL, reflecting the disruption of sulphur assimilation pathways due to ionic and osmotic stress in tomato. This reduction in enzyme activities led to impaired synthesis of sulphur-based antioxidants like glutathione, compromising the plant’s ability to mitigate oxidative damage and maintain stress tolerance. The reduced activity levels emphasize the susceptibility of sulphur metabolism to saline stress in rice [51].

Supplementation with calcium (NaCl + Ca), sulphur (NaCl + S), and their combination with nitric oxide (NaCl + Ca + S + NO) effectively restored and enhanced ATP sulfurylase and OASTL activities in tomato plants. The combined treatment (NaCl + Ca + S + NO) resulted in the most significant increases in enzyme activities, highlighting the synergistic effects of these molecules. Calcium supplementation stabilized enzyme structures and supported sulphur metabolism by reducing ionic toxicity and enhancing antioxidant defence [44]. Sulphur supplementation provided precursors for thiol and glutathione biosynthesis, further bolstering stress resilience. Nitric oxide stimulated the activity of sulphur-assimilating enzymes, leading to improved nutrient absorption and better stress tolerance in Arabidopsis [52].

In contrast, the inhibition of nitric oxide biosynthesis using L-NAME or its scavenging by cPTIO led to a further decline in ATP sulfurylase and OASTL activities in tomato plants. These findings highlight the essential role of endogenous NO in preserving the activity of sulphur-assimilating enzymes during stress in garlic and tomato [53,54].

3.6. Protein Content Estimation in Tomato

In tomato, salt stress induced by NaCl significantly reduced protein content by 32.86%, as compared to control, reflecting the detrimental effects of salt stress on protein biosynthesis and stability. However, supplementation with calcium (NaCl + Ca) improved protein content by 26.62%, while sulphur supplementation (NaCl + S) increased it by 20.55%, compared to NaCl-stressed plants. The combined treatment (NaCl + Ca + S + NO) had the most pronounced effect, enhancing protein content by 63.71% as compared to NaCl (Figure 7). Conversely, treatments with L-NAME and cPTIO, which inhibit nitric oxide-mediated signalling, exacerbated the effects of salt stress, causing reductions in protein content of 29.92% and 28.82%, respectively, compared to control plants. These findings highlight the protective role of nitric oxide and the synergistic effect of calcium and sulphur in promoting protein synthesis under salt stress.

In tomato plants, salt stress caused by NaCl led to a significant reduction in protein content, primarily due to disrupted nitrogen metabolism, impaired enzyme activities, and elevated proteolytic activity under salinity. Supplementation with Ca, S, and NO effectively alleviated these adverse effects, restoring protein biosynthesis and stability. Calcium preserved membrane stability and triggered stress-responsive signalling pathways, whereas sulphur supported the production of amino acids such as cysteine and methionine, which are vital for protein synthesis in brinjal [55]. The combined NaCl + Ca + S + NO treatment provided the most notable improvement in protein content, suggesting a synergistic interaction among these molecules. NO likely played a pivotal role in mediating the crosstalk between Ca and S, enhancing nitrogen assimilation, stabilizing protein structures, and mitigating oxidative damage through ROS regulation. These results are consistent with [56], which showed that NO enhances nitrogen use efficiency and promotes protein biosynthesis in pepper.

Conversely, treatments with L-NAME and cPTIO exacerbated protein degradation in tomato plants. This highlights the indispensable role of NO in regulating stress responses and maintaining protein biosynthesis under salt stress. The decline in protein content under these inhibitory treatment’s highlights NO’s role in safeguarding proteins from oxidative stress and preserving metabolic balance in wheat and pepper [57,58].

3.7. Visualization of ROS in Tomato: Histochemical Detection of Superoxide and Hydrogen Peroxide

In tomato plants, NaCl-induced salt stress resulted in pronounced oxidative stress in leaf tissues, as demonstrated by histochemical detection of reactive oxygen species (ROS). The accumulation of superoxide radicals (O₂·⁻) and hydrogen peroxide (H₂O₂) was visualized through blue formazan deposits and deep brown polymerisation products, respectively. These markers were especially evident along the leaf veins and margins, regions typically vulnerable to stress-induced damage. This excessive ROS build up correlated with visible symptoms such as chlorosis and necrosis, indicating impaired leaf health and metabolic dysfunction. These observations are consistent with earlier studies, such as those by [59,60], which associated ionic imbalance and osmotic stress under salinity with ROS generation, resulting in oxidative damage to lipids, proteins, and nucleic acids in wheat and pepper.

The application of calcium (NaCl + Ca) mitigated the intensity of both blue and brown staining in leaves, suggesting improved ROS detoxification. Further reduction in ROS levels was observed with sulphur supplementation (NaCl + S). Sulphur promotes GSH production, a key antioxidant that scavenges ROS, thereby bolstering the antioxidative defence system in Nostoc [61]. The most effective mitigation of oxidative stress was seen under the combined treatment of NaCl + Ca + S + NO, which led to minimal to no visible staining, reflecting restored redox balance and enhanced leaf greenness and structural integrity (Figure 8). Nitric oxide is crucial in this process, as it neutralizes free radicals, regulates antioxidant enzyme activities, and reduces ROS production at its origin in rice [62].

In contrast, treatments with L-NAME and cPTIO significantly intensified ROS accumulation, as evidenced by widespread blue and brown staining. This indicates that NO suppression disrupts key antioxidative pathways, exacerbating oxidative stress. These results are consistent with the findings of [63,64], who reported that inhibition of NO under stress conditions exacerbates ROS-mediated damage and cellular dysfunction in rice and mustard.

Of ROS in the roots. In vivo staining revealed intense blue and brown coloration in the root elongation zones, especially the root cap and meristematic regions (areas critical for cell division, elongation, and nutrient absorption). The elevated accumulation of ROS disrupted redox homeostasis and likely impaired root development as well as physiological processes in rice, bean, and tomato [63,64,65].

Under calcium treatment (NaCl + Ca), a notable reduction in ROS intensity was observed in root tissues. The reduced blue and brown staining, especially at the root tips, suggests enhanced stress tolerance, likely resulting from calcium’s role in safeguarding membranes and activating antioxidant enzymes in brinjal [66]. Sulphur supplementation (NaCl + S) provided further protection by boosting the root’s antioxidant capacity, likely through elevated synthesis of GSH and thiol compounds, which facilitate effective ROS detoxification in brinjal [67]. The uniform reduction in staining across the root system supports sulphur’s importance in redox buffering during stress. The NaCl + Ca + S + NO treatment provided the greatest relief from oxidative stress in root tissues (Figure 9). Roots treated with this combination exhibited faint or negligible ROS staining, confirming the synergistic effects of Ca, S, and NO. The role of NO in activating redox signalling, inducing antioxidant gene expression, and strengthening cellular stress responses has been well-documented in brinjal and pigeon pea [68,69]. On the other hand, treatments with L-NAME and cPTIO significantly exacerbated oxidative stress in the roots. The observed intense staining in these groups highlights the critical role of NO in ROS regulation. Likewise, inhibition of NO production or scavenging of its bioactive form disrupted redox homeostasis and intensified oxidative damage in maize [43].

3.8. In Vivo Visualization of Nitric Oxide in Roots by Fluorescence in Tomato

In tomato roots, the in vivo visualization of NO dynamics under salt stress and various treatments revealed significant variations in green fluorescence intensity. In NaCl-stressed plants, fluorescence intensity was substantially reduced, with weak green signals localized in the root tips and elongation zones, correlating with impaired growth and stress resilience as compared to control. However, supplementation with calcium (NaCl + Ca) enhanced green fluorescence moderately, particularly in the root cap and meristematic zones. S treatment (NaCl + S) further amplified this signal, with strong fluorescence extending into the elongation zone. The combined treatment (NaCl + Ca + S + NO) resulted in the brightest and most uniform fluorescence throughout the root structure, indicating elevated NO production and its pivotal role in mitigating salt stress as compared to salt stress plants (Figure 10). In contrast, L-NAME-treated tomato roots exhibited almost negligible fluorescence, while cPTIO treatment caused faint, scattered green signals, reflecting inhibited NO synthesis and scavenging effects as compared to control

In tomato roots, in vivo green fluorescence visualization under s NaCl stress revealed a marked reduction in NO levels, particularly in the tips and elongation zones. The decreased fluorescence intensity indicates lower levels of endogenous NO, signalling physiological disruptions caused by impaired NO-mediated stress mitigation pathways, such as antioxidant defence, ion balance, and signalling in tomato [70]. The weak fluorescence was accompanied by inhibited root growth and compromised stress resilience, emphasizing the detrimental effects of salt stress on NO dynamics.

Supplementation with Ca, S, and their combination with exogenous NO effectively restored NO levels, as evidenced by enhanced fluorescence intensity in root tissues. Moderate fluorescence under NaCl + Ca treatment indicates calcium’s role in stabilizing cellular membranes and facilitating NO synthesis. sulphur further increased fluorescence in NaCl + S-treated plants, supporting its role in providing precursors like cysteine and glutathione, which are essential for NO production and antioxidant defence in mustard and Arabidopsis [71]. The NaCl + Ca + S + NO treatment exhibited the brightest and most uniform fluorescence, highlighting the combined effects of Ca, S, and NO in enhancing nutrient absorption, reducing oxidative stress, and maintaining cellular homeostasis in Arabidopsis [72]. These findings validate the critical interplay between these elements in enhancing stress tolerance in tomato roots.

Conversely, treatments with L-NAME and cPTIO drastically reduced green fluorescence intensity, reflecting impaired NO synthesis and neutralization of bioavailable NO. The negligible fluorescence observed in L-NAME-treated roots confirmed the inhibition of NO biosynthesis, whereas the faint signals under cPTIO treatment indicated the scavenging of free NO in mustard [73]. Despite these reductions, tomato roots showed relatively better resilience compared to brinjal, highlighting species-specific differences in NO dynamics under stress.

3.9. Elucidation of Crosstalk Mechanism with Nitric Oxide

Salt stress triggers a complex regulatory network involving NO, Ca, and S, which collectively modulate key physiological and biochemical pathways to enhance plant resilience. NO interacts with Ca and S to regulate pigment stability, ion homeostasis, nitrogen and sulphur assimilation, oxidative stress responses, and antioxidant enzyme activities. Photosynthetic pigments such as chlorophyll and carotenoids are susceptible to degradation under salt stress due to oxidative damage. However, NO stabilizes chlorophyll biosynthesis by interacting with Ca, enhancing photosynthetic efficiency, while S contributes to the synthesis of thiol-containing antioxidants like glutathione, protecting chloroplasts from oxidative damage (Figure 11). Additionally, NO regulates ion transporters that maintain Na⁺ and K⁺ homeostasis, preventing ion toxicity and sustaining proper pigment synthesis and retention.

Nitrogen metabolism is another critical process influenced by NO, Ca, and S under salt stress. NO enhances nitrogen uptake and assimilation by modulating the activities of nitrogen-assimilating enzymes such as nitrate reductase (NR) and nitrite reductase (NiR), which convert inorganic nitrogen into bioavailable forms. Ca further stabilizes these enzymes, ensuring efficient nitrogen metabolism, while S provides essential cofactors necessary for enzymatic activity. Furthermore, NO interacts with ammonium-assimilating enzymes like glutamine synthetase (GS) and glutamate synthase (GOGAT), promoting amino acid synthesis and nitrogen assimilation, which are essential for cellular repair and growth during stress conditions. Sulphur assimilation is also enhanced through NO signalling, as NO upregulates the activity of sulphur-assimilating enzymes, ensuring an adequate supply of cysteine and glutathione. The coordinated interaction of NO, Ca, and S in nitrogen and sulphur metabolism enhances stress adaptation by improving metabolic efficiency and cellular homeostasis.

Salt stress leads to excessive production of ROS such as superoxide radicals and hydrogen peroxide, causing oxidative damage to cellular structures. NO plays a dual role in ROS regulation by acting as both a scavenger and a signalling molecule that modulates the expression of antioxidant enzymes. Ca enhances ROS-scavenging pathways, while S provides thiol groups necessary for antioxidant synthesis. The activities of key antioxidant enzymes such as SOD, CAT, GR and APX are regulated by NO to ensure efficient detoxification of ROS. In vivo visualization of NO in roots using fluorescence techniques, as well as ROS detection in leaves and roots, provides crucial insights into stress signalling dynamics. The crosstalk between NO, Ca, and S fine-tunes ROS accumulation, ensuring that oxidative stress remains within a controlled threshold, preventing cellular damage. This finely tuned regulatory network optimizes plant defence mechanisms, enabling plants to maintain metabolic stability, cellular integrity, and overall growth under salt-induced stress conditions.

4. Acknowledging Limitations

While the present study provides novel insights into the roles of calcium, sulphur, and nitric oxide in modulating salt stress responses in tomato and brinjal under hydroponic conditions, some limitations need to be considered. The experiments were conducted with a limited sample size under controlled laboratory settings, which may not fully represent the complex responses occurring in field-grown plants. Moreover, the study focused on physiological and biochemical indicators without assessing protein abundance or functional activity of transporters directly involved in ion regulation and stress tolerance. To address these gaps, future investigations should include quantification of transporter protein levels, ion-flux measurements, and membrane potential assays to unravel the mechanistic basis of ionic homeostasis. Field trials across different cultivars and environmental conditions are also necessary to validate the laboratory findings and to establish the practical relevance of Ca, S, and NO cross-talk in improving salt stress resilience in solanaceous crops

5. Conclusions

This study provides strong evidence that sulphur, calcium, and nitric oxide are closely associated with the alleviation of salt stress in tomato seedlings. Salt stress induced by NaCl disrupted ionic homeostasis, as reflected by elevated Na⁺ and reduced K⁺ levels, and impaired nitrogen and sulphur metabolism, leading to lower protein content and increased ROS (O₂^∙− and H₂O₂) accumulation. Moreover, endogenous NO levels declined under salinity stress. Treatments with K₂SO₄, CaCl₂, and SNP were correlated with the restoration of ionic balance, reduced ROS accumulation, and improved nitrogen and sulphur metabolism. These effects were accompanied by enhanced activities of key nitrogen-assimilating enzymes (NR, NiR, GS, GOGAT) and sulphur-assimilating enzymes (ATPS, OASTL), suggesting a supportive role of S, Ca, and NO in maintaining metabolic functions under stress. The reversal of these beneficial responses by NO inhibitors and scavengers (L-NAME and cPTIO) further indicates an association between NO signalling and improved stress tolerance. While the findings underscore the potential synergistic role of S, Ca, and NO in counteracting salt stress, the mechanistic links remain inferential, as direct protein-level or functional evidence was not assessed. Future studies incorporating transporter protein abundance, ion-flux assays, electrophysiology, and field validation are needed to substantiate these proposed mechanisms. Overall, this study highlights promising interactions among S, Ca, and NO in promoting salt stress resilience and provides a foundation for developing nutrient and signalling-based strategies to enhance tomato tolerance under salinity.