1 Introduction

Climate changes have generated critical issues of unfavorable conditions or stresses that negatively influence crop production and food security (El-Bially et al. 2018; Saudy and El-Metwally 2019; Abd–Elrahman et al. 2022; Sattar et al. 2023). Crop growth and yield are dramatically influenced by ecological pressures which involve salinity, inappropriate temperatures, water scarcity, heavy metal toxicity, and nutrient deficiency (El-Yazeid and Abou-Aly 2011; Saudy 2014; El-Bially et al. 2022a; El-Metwally et al. 2022a; Saudy et al. 2023a; Shaaban et al. 2023a; Shahin et al. 2023). Further, biological pressures have also pernicious influences on crop productivity and quality (Saudy 2015Saudy and Mubarak 2015; Saudy et al. 2020a, 2021b; Abou El-Enin et al. 2023). The ecological pressures which known as abiotic stresses cause disturbance in plant metabolism, suppressing crop potential (Abd El-Mageed et al. 2022; Hadid et al. 2023; Ramadan et al. 2023a; Saudy et al. 2023b). A major stress in this respect is soil salinization which poses a significant risk to crop productivity and world food security particularly with climate change intensifies (Mukhopadhyay et al. 2021; Shalaby et al. 2023). It may be naturally occurred through global warming and water evaporation, leading to accumulate huge amounts of salts in the soil surface (Corwin 2021). Moreover, there are many anthropogenic reasons including unsustainable agricultural practices and industrial wastes can increase the issues of salt affected soils worldwide (Litalien and Zeeb 2020).

Exposing plants to salinity stress can seriously affect water acquisition, osmotic equilibrium and ionic homeostasis (El Nahhas et al. 2021; Abdelaal et al. 2022; Darwish et al. 2023; Mansha et al. 2023). These effects occur due to reducing cell membrane stability and the toxic effect of the considerable amounts of the accumulated Na+ ions on the functioning of membrane transporter proteins (Alsamadany et al. 2022). Dysfunction of photosynthetic machinery and cellular oxidative burst are two other major biochemical markers of high salinity stress (Farag et al. 2022; Shehata and Ibrahim 2023; Vineeth et al. 2023). These responses have been found to be due to the excessive release of reactive oxygen species (ROS), such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide (H2O2) through hindering electron transport systems in chloroplast (Sun et al. 2016; Din et al. 2020; Gashash et al. 2022) and mitochondria (Analin et al. 2020). Methylglyoxal (MG) is also cytotoxic molecule in its high concentrations under saline conditions (Yadav et al. 2005). It is derived from glycolysis and can be involved in parallel with ROS in lipid peroxidation, photosynthesis disruption, and increasing the rates of DNA damage and cell death under adverse conditions (Wang et al. 2019; El-Metwally and Saudy 2021a; El-Mogy et al. 2022; Doklega et al. 2023). Moreover, salinity plays a primary role in controlling the photosynthetically fixed carbon by reducing the stomatal conductance and altering photosynthetic pigments (Youssef et al. 2021; Dourado et al. 2022). Hence, tolerance of plants to high saline conditions has been found to be due to maintain the performance of growth, photosynthetic apparatus, upregulation antioxidant defense systems, ion homeostasis and enhancing plant water relations (Mansour et al. 2021; Youssef et al. 2021).

Sorghum (Sorghum bicolor (L.) Moench; family; poaceae) is the most important cereal crop in the world after wheat, rice, maize, and barley (Jardim et al. 2020). It has been characterized as a C4 plant and tolerant to moderate saline environments (Huang 2018; Mansour et al. 2021). However, sorghum may encounter with the risk of high soil salinization in the arid and semi-arid regions, where temperatures and evaporation rates are high leading to accumulate toxic amounts of Na+ in the rhizosphere (Youssef et al. 2021). It has been found that sorghum can produce high biomass under salinity level up to 6.8 dS m− 1, but this production was declined to 50% when salinity level reached 10 dS m− 1 (Hossain et al. 2018). As well, both germination and seedling growth are the most susceptible stages to saline conditions which may determine the final plant performance of sorghum crop (Rajabi Dehnavi et al. 2020; Youssef et al. 2021). Therefore, it is robustly important to find a successful approach to improve seedling tolerance and survival under high salinity conditions. Plants have evolved various strategies to cope a variety of stressed environments while maintaining their survival and potential to grow and reproduce (Noureldin et al. 2013; Saudy et al. 2018, 2022; El-Metwally and Saudy 2021b). Plant growth responses to stresses are intricately modulated via multiple phytohormones which have vital works to orchestrate the various plant life processes and adaptability to stresses (Saudy et al. 2020b; El-Metwally et al. 2022b; Saudy et al. 2021d; Rizk et al. 2023).

Melatonin (N-acetyl-5-methoxytryptamine; MT) has been found to have an essential role in plant survival under biotic and abiotic stresses (Zhao et al. 2021a; El-Beltagi et al. 2023). As a powerful antioxidant, it can serve with high efficiency to scavenge ROS under various harsh environmental conditions i.e. drought (Huang et al. 2019; El-Metwally et al. 2021; Ramadan et al. 2023b; Shaaban et al. 2023b), heat stress (Buttar et al. 2020), cold (Qari et al. 2022), heavy metals (Hoque et al. 2021) and salinity (Li et al. 2019; Ali et al. 2021). Additionally, MT has been known as a phytohormone with a wide spectrum of functions and relationships with other phytohormones (Khan et al. 2022; Ali et al. 2024). Melatonin can stimulate the lateral root formation and regulate the root architecture by modulating auxin responses (Liang et al. 2017). Furthermore,, MT demonstrated an obvious ability to stimulate seed germination (Xiao et al. 2019), delay leaf senescence (Zhao et al. 2021b), alter flowering, fruit set and fruit ripening (Arnao and Hernández-Ruiz 2020) and enhance photosynthesis (Jahan et al. 2021). Under salinity stress, melatonin has been found to have the ability to regulate the antioxidant system, proline and carbohydrates metabolism (Siddiqui et al. 2019).

Very little research has been focused on the role of melatonin in mitigating the harmful effects of different salinity levels on sorghum plants at early sensitive stage of vegetative growth. As well, the optimal concentration of melatonin in this regard still needs to be determined based on stress level. The present study hypothesised that the appropriate MT concentration for alleviating salinity injuries could change as the salt stress level changed Therefore, the potential effects of different MT concentrations (0, 50, 100 and 200 µM) on alleviating the impacts of salinity stress at moderate (75 µM NaCl) and severe (150 µM NaCl) level were examined. Our results included different mechanisms were orchestrated together to confer salinity tolerance to sorghum plants i.e., regulating the vegetative growth, photosynthetic pigments and osmolytes, achieving cell membrane stability and ion homeostasis, suppressing the oxidative and carbonyl stress biomarkers and enhancing the antioxidant systems. These response mechanisms will help researchers to better understand the potential complex roles of melatonin in sorghum tolerance to saline conditions.

2 Materials and methods

2.1 Plant Materials and Treatments

Sorghum grains (Sorghum bicolor L. Moench; Cv. Dorado; purchased from Agricultural Research Center, Giza, Egypt) sterilized with 0.1% sodium hypochlorite for 5 min and washed 5 times with distilled water then sown in black plastic pots filled with 8 kg pre-washed sand. Each pot (20 cm diameter × 25 cm height) contained ten seedlings and was watered every two days with 250 ml of ½ strength Hoagland’s solution. After three weeks (full seedlings emergence), to explore the effects of moderate and severe salinity stress, the nutrient solution was modified by adding 75 and 150 µM NaCl, representing 6.85 and 13.70 dS m− 1. Over a period of 2 weeks after complete emergence, the pots received the salt solution at 2-day intervals. Three concentraions of melatonin, MT (50, 100 and 200 µM), in addition to a control treatment (0, MT, distilled water) were applied.The melatonin stock solution was prepared by dissolving melatonin in ethanol (100 mg of melatonin in 2 mL of ethanol absolute). After that different concentrations were prepared by dilution. Seedlings were foliar sprayed four times at 24, 27, 30 and 33 days after sowing with different MT concentrations. Everytime, each pot received 200 ml of MT solution. Tween-20, 0.05% (Sigma-Aldrich) were used as a surfactant with all MT (MT; Bio Basic, Markham, Canada) treatments. The experiment was terminated by collecting plant samples at 35 days after sowing for different physiological and biochemical investigations.

2.2 Assessements

2.2.1 Determination of Roots and Shoots Biomass and Leaf Pigments

At the end of the experiment, seedlings were collected to assess the fresh weights of roots and shoots per plant. Further, the concentration of chlorophyll a, chlorophyll b and carotenoids were determined spectrophotometry (Alan 1994). Pigment extraction in 80% acetone was performed using a fresh weight (0.5 g) of completely grown young leaves. The pigment extract was measured at 663, 644, and 452.5 nm for chlorophyll a, chlorophyll b, and carotenoids respectively, versus a blank of pure 80% acetone.

2.2.2 Leaf Relative Water Content (RWC) and Osmotic Molecules Determination

Ten fresh leaf discs were accurately weighed (FW) and maintained in distilled water for 1 h to gain turgidity (TW). The leaf discs were oven-dried for 24 h at 80 ℃ to determine their dry weight (DW). The RWC was calculated according to Hayat et al. (2007) using formula 1:

$$ \text{R}\text{W}\text{C} \left(\text{\%}\right)=\frac{\text{F}\text{W}-\text{D}\text{W}}{\text{T}\text{W}-\text{D}\text{W} } \times 100\dots \left(1\right)$$
(1)

where FW, DW and TW represent fresh, dry and turgid weights of leaf discs respectively. The percentage of relative water content (RWC) was measured according to Smart and Bingham (1974).

The ninhydrin reagent was used to estimate proline as described by Bates et al. (1973). For estimating total soluble sugars (TSS), the phenol-sulfuric acid technique was used (Chow and Landhäusser 2004). Free amino acids (FAA) were measured as glycine using the ninhydrin reagent (Hamilton et al. 1943).

2.2.3 Quantification of Hydrogen Peroxide (H2O2), Malondialdehyde (MDA), Methylglyoxylate (MG) and Cell Membrane Stability Index

For H2O2 determination, 0.3 g of leaf sample was homogenized in 3 mL of Trichloroacetic acid (TCA) 0.1% (w/v), followed by centrifugation at 10,000 x g for 10 min at 4 °C (Velikova et al. 2000). The supernatant (500 µL) was then combined with equivalent volume of 0.1 M potassium phosphate buffer (pH 7.8) and 1 mL of 1 M potassium iodine (KI). The combination was utilised to calculate the H2O2 by reading its absorbance at 390 nm. The level of lipid peroxidation was determined by measuring the amount of MDA at 535 nm and corrected for non-specific turbidity at 600 nm using thiobarbituric acid (TBA) reaction as performed by Heath and Packer (1968).

MG was measured in accordance with Hossain et al. (2009), with some adjustments. Fresh leaves (0.5 g) were homogenized in 3 mL of 0.5 M perchloric acid and incubated on ice for 15 min. The mixture was centrifuged at 4 °C for 10 min at 10,000 rpm. The supernatant was discolored by adding charcoal, then centrifuged at 10,000 rpm for 10 min. This supernatant was neutralized for 15 min at room temperature with a saturated potassium carbonate solution before being centrifuged at 10,000 rpm for 10 min. The reaction mixture contained 750 µL of 7.2 µM 1, 2-diaminobenzene, 250 µL of 5 M perchloric acid, and 2 mL of the supernatant was incubated at room temperature for 25 min then the absorbance was recorded at 335 nm. The stability of membrane was assessed as carried out by Premachandra et al. (1990) using the readings of EC meter for leaf discs before (EC1) and after (EC2) placing in autoclave at 120 °C for 20 min. The membrane stability index (MSI) of cell was estimated using the formula 2:

$$ \text{M}\text{S}\text{I}=1-\frac{\text{E}\text{C}1}{\text{E}\text{C}2} \text{x} 100\dots \left(2\right)$$
(2)

2.2.4 Nutrients Estimation

Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure the amounts of sodium (Na+), potasium (K+) and calcium (Ca+ 2) ions according to Jones Jr (2001). Further, K+/Na+ ratio was computed.

2.2.5 Antioxidant Enzymes Assay

For extraction of antioxidant enzymes, 0.2 g of fresh leaves were homogenized in 4 mL of 0.1 M ice-cold sodium phosphate buffer (pH 7.0) containing 1% (w/v) PVP and 0.1 µM EDTA, then centrifuged at 10,000 xg for 20 min at 4 C. The supernatant was used to measure peroxidase (POX, EC 1.11.1.7), catalase (CAT, EC EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (APX, EC 1.11.1.11) activities. Total soluble protein in the supernatant was also determined as described by Bradford (1976) to calculate the specific activity of different enzymes. POX activity was determined according to Reddy et al. (1985) at 470 nm. CAT activity was assayed following Cakmak et al. (1993) by reading the absorbance at 240 nm. The capability of SOD to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) at 560 nm was determined according to Beyer and Fridovich (1987). APX was determined at 290 nm (Nakano and Asada 1981),

2.2.6 Molecular Docking

The three-dimensional model structure of the receptors used in this molecular docking study were retrieved from UniProt and the Protein Data Bank (PDB) (PDB: https://doi.org/10.2210/pdb1EEA/ pdb). The following receptors were selected: (1) Ascorbate peroxidase (APX) (pdb ID: 8djs) with a binding pocket centered at coordinates (center_x = − 29.586, center_y = 28.561, center_z = 63.093); (2) Catalase (UniProt ID: C5Z2J6) with a binding pocket centered at coordinates (center_x = -1.311, center_y = − 3.311, center_z= -1.688); (3) Glutathione peroxidase (UniProt ID: Q6JAG4) with a binding pocket centered at coordinates (center_x = - -1.946, center_y = -13.748, center_z = -2.224); (4) superoxide dismutase (Uniprot ID: A0A194YHS4) with a binding pocket centered at coordinates (center_x = -8.466, center_y = -14.551, center_z = 13.116).Each with sized: 20 × 20 × 20The active sites on these receptors were identified using the Deepsite/PlayMolecule software (https://www.playmolecule.com/deepsite). The Deepsite results were retrieved from the PlayMolecule platform. All ligands’ SMILES and SDF (structure data files) were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The selected ligands were prepared for docking by performing energy minimization using Avogadro 1.2.0 with the MMFF94 force field. This step ensured that the ligands were in an energetically favourable conformation for docking. Melatonin was retrieved from pubchem with ID 896. The receptor proteins were prepared for docking using AutoDock Tools 1.5.6. This involved the addition of hydrogen atoms, removal of water molecules, and assignment of charges to the protein structure. The receptor’s grid box was centered on the active site coordinates mentioned earlier. Molecular docking was performed using AutoDock Vina. The prepared ligands were docked into the active sites of the receptors, and the software calculated the binding affinities and predicted binding poses. Multiple docking runs were conducted to explore different binding conformations. The docking results were analyzed to identify potential binding modes and interactions between ligands and receptors. The binding affinities and RMSD (Root Mean Square Deviation) values were examined to assess the quality of the docking poses.

2.3 Statistical Analysis and Data Visualization

The correlation analysis was performed according to hierarchical clustering and two-dimensional heatmap plotting was constructed using R-studio and Orange3 module. Statistical analysis was conducted using the one-way analysis of variance (ANOVA) test followed by SAS software. Tukey’s multiple range test at 5% was carried out to determine the significance of means using triple replication.

3 Results

3.1 Effect of Melatonin on Plant Biomass under Salt Stress

Plants subjected to moderate (75 µM) and high concentrations (150 µM) of NaCl stress showed significant (p > 0.05) reductions in the root and shoot fresh weight (FW), which was partially improved by the application of exogenous melatonin (MT) compared to the control (Fig. 1). Root and shoot FW was observed to be improved under each slinity level with supplying MT at the concentrations of 50, 100 and 200 µM without significant differences (p < 0.05) between them. However, under severe salinity (150 µM), MT did not differe than the control treatment for root FW.

Fig. 1
figure 1

Influence of melatonin (MT) concentration on roots and shoots fresh weight of sorghum seedling under salinity levels. MT0, MT50, MT100 and MT200: application of MT concentration at 0, 50, 100 and 200 µM, respectively; S1, S2 and S3: salinity level at 0, 75 nad 150 NaCl µM, respectively. The means in each column followed by the same lower-case-letter refer to that there is no significant difference according Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data were expressed as the mean ± standard deviation of 3 replications

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3.2 Effect of Melatonin on Pigments Content under Salt Stress

NaCl stress (both moderate and high levels) brought about significant (p > 0.05) reductions in chlorophyll a, chlorophyll b, total chlorophyll and carotenoids (Fig. 2). Generaly, melatonin supply improved chlorophylls and carotenoids pigment compared to the control plants either in the presence or absence of salinity stress. Different MT levels showed differential effects on chlorophylls and carotenoids content at moderate and higher levels of salinity stress. Herein, MT100 was more effective in increasing chlorophyll a, and carotenoids at moderate concentration of NaCl stress, surpassing MT0, MT50 and MT200 treatments by 40.3 and 40.7%, 18.4 and 19.7% and 10.1 and 19.1%, respectively. Also, MT100 along MT200 exhibited greater values of chlorophyll b and total chlorophyll under modertae slinity and carotenoids under high salinity than MT0 and MT50.

Fig. 2
figure 2

Influence of melatonin (MT) concentration on chlorophyll a, chlorophyll b, total chlorophyll and carotenoids of sorghum seedling under salinity levels. MT0, MT50, MT100 and MT200: application of MT concentration at 0, 50, 100 and 200 µM, respectively; S1, S2 and S3: salinity level at 0, 75 nad 150 NaCl µM, respectively. The means in each column followed by the same lower-case-letter refer to that there is no significant difference according Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data were expressed as the mean ± standard deviation of 3 replications

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3.3 Effect of Melatonin on RWC and Osmolyte Accumulation under Salt Stress

The effects of NaCl stress and melatonin supplementation on the modulation of relative water content, proline, sugar and free amino acids were significant (p > 0.05) as dipected in Fig. 3. Salinity stress markedly reduced RWC at 75 µM and 150 µM NaCl stress, where the later resulted in a serious deterioration in RWC content. Conversely, exogenous MT treatments resulted in an obvious improvement in RWC either in the presence of 75 µM or 150 µM NaCl stress. With exception on unstressed conditions (no salinity), MT100 and MT200 under 75 µM NaCl and MT100 under 150 µM NaCl wer the efficient practices for maintining RWC showig increases of 3.3, 2.4 and 3.4%, compared to the counterpart control treatment (MT0), respectiveky. Concenrning the osmolytes, the accumulation of proline, total soluble sugars and free amino acids showed significant (p > 0.05) increases in the presence of NaCl stress (Fig. 3). Overall, the highet increase in proline was observed with MT100 or MT200 under 75 µM NaCl. While MT100 and MT50 showed the maximal total soluble and free amino acids, respectively, under150 µM NaCl.

Fig. 3
figure 3

Influence of melatonin (MT) concentration on relative water content (RWC), total soulable sugars (TSS), proline and free amino acids (FAA) of sorghum seedling under salinity levels. FW: fresh weight; MT0, MT50, MT100 and MT200: application of MT concentration at 0, 50, 100 and 200 µM, respectively; S1, S2 and S3: salinity level at 0, 75 nad 150 NaCl µM, respectively. The means in each column followed by the same lower-case-letter refer to that there is no significant difference according Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data were expressed as the mean ± standard deviation of 3 replications

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3.4 Effect of Melatonin on H2O2, MDA, MG Accumulation and MSI under Salt Stress

Plants grown under NaCl stress exhibited a sharp increase in H2O2 content accompanied by elevation in MDA and MG content (Fig. 4). Thus, under each of moderate or severe salinity, no MT supply (MT0) treatment gave the highest H2O2, MDA and MG accumulation. Application of exogenous MT resulted in substaincial (p > 0.05) reductions in H2O2, MDA and MG accumulation. Both the intermediate and high concentrations of MT (100 and 200 µM) were effective in reducing the effect of NaCl stress of H2O2, MDA and MG. Accordingly, compared to the counterpart control treatment (MT0), reductions in H2O2, MDA and MG were 17.8 and 20.7%; 47.6 and 43.4% and 27.5 and 28.3% due to application of MT100 and MT200, respectively, under 75 µM NaCl stress. As well, the corresponding reductions under 150 µM NaCl stress were 22.0 and 23.9%; 29.1 and 23.8%; and 26.0 and 40.4%, respectively.

Membrane stability index (MSI %) was seen to be reduced in the presence of NaCl stress. However, an obvious and significant (p > 0.05) improvement was observed implementing MT treatments (Fig. 4). The impact of MT levels was more evident under severe salt stress, since MT100 along MT50 possessed the best improvement in MSI %. While, the difference between MT50, MT100 and MT200 in MSI % under moderate salt stress was not significant (p < 0.05).

Fig. 4
figure 4

Influence of melatonin (MT) concentration on hydrogen peroxide (H2O2), malondialdehyde (MDA), methyglycoxal (MG) and membrane stablity index (MSI) of sorghum seedling under salinity levels. FW: fresh weight; MT0, MT50, MT100 and MT200: application of MT concentration at 0, 50, 100 and 200 µM, respectively; S1, S2 and S3: salinity level at 0, 75 nad 150 NaCl µM, respectively. The means in each column followed by the same lower-case-letter refer to that there is no significant difference according Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data were expressed as the mean ± standard deviation of 3 replications

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3.5 Melatonin Regulates Ion Homeostasis under Salt Stress

The effect of NaCl stress on ion levels was analyzed by measuring Na+, K+, Ca2+ content and calculating Na+/K+ ratio in sorghum roots and shoots (Fig. 5). Na+ showed 1.8 and 3.4-fold increase in roots and 3.5 and 6.2-fold increase in shoots under moderate and severe salinity level compared to the unstressed plants. While, K+ levels showed decreases of 28.0 and 41.3% in roots and 19.5 and 34.3% in shoots of plants subjected to 75 and 150 µM NaCl stress, respectively. Accordingly, Na+/K+ ratio was observed to exhibit sharp increase up 2.2 and 5.9% in roots as well as 4.4 and 9.5% in shoots of plants subjected to 75 and 150 µM NaCl stress, respectively. Further, NaCl stress at 75 and 150 µM NaCl resulted in reduction in Ca2+ content by 27.4 and 40.8% in roots and 33.9 and 50.9% in shoots, respectively. Exogenous MT significantly reduced the accumulation of Na in both root and shoot systems under moderate and higher salinity levels. In this context, these responses were in parallel with enhancing the content of K+ in both organs (root and shoot) and consequently reducing the ratio of Na+/K+. The treatments of MT at 100 and 200 µM were more potent in this respect. Moreover, MT treatments specifically at 100 and 200 µM exhibited a significant (p > 0.05) increase in Ca+ 2 content in the root and shoot systems of the saline stressed plants whether with 75 or 150 µM NaCl.

Fig. 5
figure 5

Influence of melatonin (MT) concentration on sodium (Na+), potassium (K+), Na+/K+ ratio and calcium (Ca+ 2) of sorghum seedling under salinity levels. DW: dry weight; MT0, MT50, MT100 and MT200: application of MT concentration at 0, 50, 100 and 200 µM, respectively; S1, S2 and S3: salinity level at 0, 75 nad 150 NaCl µM, respectively. The means in each column followed by the same lower-case-letter refer to that there is no significant difference according Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data were expressed as the mean ± standard deviation of 3 replications

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3.6 Melatonin Regulates the Activities of Antioxidant Enzymes under Salt Stress

Plants subjected to salinity stress (75 µM and 150 µM) displayed dramatic increase in the activity of antioxidant enzymes (POX, CAT, SOD and APX) compared to the unstressed plants (Fig. 6). The treatment of MT at 100 µM led to the highest significant (p > 0.05) increase in the activity of POX compared to the untreated plants under both moderate and higher salinity levels. Under moderate salinity stress (75 µM NaCl), application of MT100 and MT200 showed the highest significant (p > 0.05) increase in the activity APX and CAT, respectively. Meanwhile, maximum activity of SOD was observed under the higher salinity level (150 µM NaCl) with supplying MT100.

Fig. 6
figure 6

Influence of melatonin (MT) concentration on the activity of peroxidase (POX, µmol mg− 1 protein min− 1), catalse (CAT, µmol mg− 1 protein min− 1), superoxide dismutase (SOD, Unit mg− 1 protein) and ascorbate peroxidase (APX, µmol mg− 1 protein min− 1) of sorghum seedling under salinity levels. DW: dry weight; MT0, MT50, MT100 and MT200: application of MT concentration at 0, 50, 100 and 200 µM, respectively; S1, S2 and S3: salinity level at 0, 75 nad 150 NaCl µM, respectively. The means in each column followed by the same lower-case-letter refer to that there is no significant difference according Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data were expressed as the mean ± standard deviation of 3 replications

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3.7 Molecular Docking

As illustrated in Fig. 7a, b, the test samples had very good interactions with the target enzymes, as shown by binding energies and binding mode. Melatonin has top-ranked confirmations with target antioxidant enzymes, according to obtained docking results. The molecular docking results reveal that there were several favourable binding affinities between MT and antioxidant enzymes APX and CAT (Fig. 7a) and POX and SOD (Fig. 7b). MT with APX and CAT shows the strongest binding affinities with ΔG values of − 8.7 and − 8.3 kcal/mol respectively. MT forms multiple hydrogen bonds with key amino acid residues for glycine (A: 162), tryptophane (A: 41), leucine (A: 165), tyrosine (A: 235) in the active site of ascorbate peroxidase. The alkyl interactions with leucine (A: 37), arginine (A: 38) and cysteine (A: 168) also contribute to its high affinity.

Fig. 7
figure 7

Docking view of melatonin on the binding sites of antioxidant enzymes; (A) Ascorbate peroxidase; (B) Catalase. Left are the 2D interaction diagrams, and right are the complex structures in 3D. Docking view of melatonin on the binding sites of antioxidant enzymes; (C) Peroxidase; (D) Superoxide dismutase. Left are the 2D interaction diagrams, and right are the complex structures in 3D

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4 Discussion

Salinity stress has been considered a major threat for crop production due to its destructive impacts on plant growth and development in many regions worldwide. In this study, roots and shoots fresh weights were significantly suppressed by exposing to moderate or sevre saline levels (75 and 150 µM NaCl). According to its level, salinity stress can affect plant cell cycle regulation, leading to reduce cell division rates and cell numbers in leaves, roots, or the shoot meristem (Qi and Zhang 2020). Melatonin can act as a plant growth regulator with a similar action of indolyl-3-acetic acid (Arnao and Hernández-Ruiz 2014). It has different signalling pathways and crosstalk reactions with other phytohormones under normal and stressful conditions (Khan et al. 2022). Under saline conditions, many previous studies have revealed that exogenous MT can stimulate plant growth and development in different plant species i.e., maize (Chen et al. 2018), cucumber (Zhang et al. 2020), tomato (Ali et al. 2021), snap bean (El-Beltagi et al. 2023) and sorghum (Nie et al. 2023). In the present study, applied-MT at different concentrations (50, 100 and 200 µM) significantly improved shoot fresh weight under unstressed and various saline conditions (75 and 150 µM NaCl). Meanwhile, root fresh weight was enhanced by MT-treatments under unstressed and moderate saline conditions (75 µM NaCl), but this effect was less clear under higher salinity level (150 µM NaCl). This effect might be attribute to that high salinity level can affect the rate and direction of carbon flow between different plant tissues (Dong and Beckles 2019).

Subjecting sorghum plants to salinity stress sharply reduced the leaf content of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids compared to the unstressed conditions. Salinity stress can provoke the photosynthetic apparatus to release excessive amounts of ROS, accelerating degradation rate of leaf pigments, inducing leaf senescence, and eventually causing leaf cell death (Liang et al. 2015; Youssef et al. 2021; El-Bially et al. 2023). However, applied-MT led to an obvious and significant increase in chlorophylls (a & b) and carotenoids of sorghum leaf. Several earlier reports have revealed that under saline conditions, applied-MT plays an important role in improving the photosynthetic efficiency and maintaining the leaf pigments (Wang et al. 2016; Chen et al. 2018; Altaf et al. 2020; Jiang et al. 2021). These influences may be due to the ability of MT to regulate the transcription of leaf-pigments related genes, protecting of photosynthetic proteins, promoting antioxidant systems and activating the xanthophyll cycle (Yang et al. 2022).

Relative water content (RWC) appears to be an important parameter for measuring the ability of plants to be survived under saline conditions. In this study, MT-treated plants exhibited a significant improvement in RWC compared to those of unstressed conditions. The accumulation of osmoprotectants i.e., proline, soluble sugars and free amino acids are common adaptive responses in several plants species under saline conditions (Ali et al. 2021; Youssef et al. 2021; Farag et al. 2022). As well, these organic molecules can be orchestrated together to regulate plant water uptake and preventing the dehydration of tissues through readjustment the cellular osmotic potential under adverse conditions (Batista-Silva et al. 2019; Dong and Beckles 2019; Ramadan et al. 2022). Earlier works have ascertained the role of MT in improving different osmolytes concentration under salinity stress (Siddiqui et al. 2019; Ali et al. 2021; Nie et al. 2023). In the present work, increase of proline, soluble sugars and the oscillation in free amino acids in MT-treated plants implies that MT may be involved in the biosynthesis and metabolism of osmolytes under saline conditions.

NaCl stress (moderate and high level) led to a marked elevation in H2O2 content, lipid peroxidation (MDA), MG content, while reduced MSI %. Application of exogenous MT was effective in reducing the levels of H2O2, MDA and MG content. According to Zhang et al. (2021b) MT is effective in instigating the gene expression of antioxidative enzymes including ascorbate-peroxidase, hence exogenous melatonin reduced H2O2 levels and restored cell membrane integrity in NaCl-stressed sugar beets. Findings of the present work revealed that exogenous MT possibly appears beneficial in normalizing the redox imbalance by reducing H2O2 content and maintain cell membrane stability (low MDA content and increased MSI %). Various reports have provided instances of the effect of MT in reducing H2O2 and MDA content under salt stress (Li et al. 2012; Wang et al. 2016; Jiang et al. 2021; El-Beltagi et al. 2023).

Alharbi et al. (2021) have reported the modulatory role of MT in regulation of glyoxalase system. In the present study, NaCl stress significantly increased MG content which was however, partially reduced by MT treatment. Thus, modulation of glyoxalase system in the presence of MT is evident to exert regulatory effects on MG content under NaCl stress. Glyoxalase system is known to be an important regulator of ROS and MG content during salinity and other abiotic stress in plants (Hasanuzzaman et al. 2014; Nahar et al. 2016; El-Yazied et al. 2022). Previous investigations also reported the effect of MT in improvement the membrane stability index (MSI%) under abiotic stress (Ali et al. 2021; Chen et al. 2021). In the present work, MT treatments reduced lipid peroxidation under salinity stress which was accompanied by a concomitant increase in MSI%. Therrfore, reduced H2O2 and MG content in the presence of exogenous MT was observed to be correlated with improving membrane stability and oxidative tolerance in plants subjected to NaCl stress.

As for ion homeostasis, it has been reported that the unfavorable circumistances cause dengerous impacts of nutrients uptake and utilization affecting the related plant metabolism (Saudy et al. 2020c, 2021c; Salem et al. 2022; Ali et al. 2023). NaCl stress significantly increased Na+ content in both roots and shoots which was accompanied by a reduction in K+ and Ca2+ content. Contrariwise, exogenous MT (at all concentrations) reduced the accumulation of Na+ in both roots and shoots which was accompanied by subsequent increase in K+ and Ca2+ content. Thus, observations recorded in the present work imply that exogenous MT effectively reduced Na+/K+ ratio during NaCl stress. Apart from its role as a potent antioxidant, MT has been ascertained to be an important biostimulant which alleviates NaCl stress-induced ionic imbalance in plants. Herein, maize seedlings treated with NaCl stress have shown a reduction in Na+/K+ ratio in the presence of MT treatment (Yan et al. 2020). Furthermore, seedlings of Malus sp. have also exhibited an obvious increase in K+ content and higher K+/Na+ ratio in the presence of MT treatment (Li et al. 2012). Interestingly, MT-induced ion homeostasis during NaCl stress indicates increased activity of SOS2, AKT and NHX (Zhan et al. 2019). Although MT-induced regulation of ion homeostasis during NaCl stress requires further investigations, it is well evident that MT functions as a positive regulator of Na+/H+ antiporter and K+ influx system in cell and tonoplast. Zhang et al. (2021a) reported that melatonin treatment improved Na+ efflux and K+ influx systems in NaCl-stressed sugar beet plants. Thus, Na+ compartmentalization, and its efflux is accompanied by increase in K+ content during MT supplementation under NaCl stress. We also recorded a marked increase in Ca2+ which indicates that MT was effective in restoring ion and electropotential gradient across the plasma membrane of plants subjected to NaCl stress. An interesting report by Liu et al. (2020) has provided evidence of melatonin-induced instigation of Ca2+ signalling during NaCl stress. The authors reported MT-induced increase in calcineurin B-like/calcineurin B-like-interacting protein kinase and calcium-dependent protein kinase expression in salinity stressed rice plants. Moreover, endogenous and exogenous melatonin has been advocated to be important for improving Ca+ content and Ca2+-associated signalling pathways during salinity tolerance in cotton plants (Zhang et al. 2021b) Thus, briefly, our present work substantiates the fact that exogenous MT in variable concentrations are effective in reducing Na+/K+ ratio and improving Ca2+ content under moderate and high levels of NaCl stress.

Salinity stress can disturb the physiological/biochemical processes in plants by stimulating the excessive generation of free radicals as superoxide radical and hydrogen peroxide which can directly attack membrane lipids and led to the loss in MSI (Miller et al. 2010; Youssef et al. 2021). In order to prevent the damage triggered by oxidative stress, plants can evolve enzymatic antioxidant scavenging mechanism for eliminating the adverse effect of ROS (Mansour et al. 2021; Makhlouf et al. 2022; Saudy and El-Metwally 2023). In the current study, the activities of some antioxidant enzymes as SOD, CAT, POX and APX were significantly increased under salinity stress. The higher activities of these enzymes afford the tolerance mechanism in plants by scavenging ROS induced by salinity stress (Chen et al. 2018; El-Beltagi et al. 2023; Nie et al. 2023). As well, the results in this investigation showed that the exogenous application of MT induced further increase in the activities of antioxidant enzymes (SOD, CAT, POX and APX) under salinity stress, leading to attenuate the accumulation of ROS levels (El-Bially et al. 2022b; (El-Metwally et al. 2022c). These effects may confirm the protecting effect of MT as powerful antioxidant under saline conditions (Zhang et al. 2020; Ali et al. 2021).

Furthermore, in this study, it was observed that MT had high affinity with different studied antioxidant enzymes (SOD, CAT, POX and APX) with docking score of -8.3. The treatments of MT showed three polar interactions with the active residues of the target CAT enzyme. It was observed that Arg A:62, Arg A: 344 and Gly A:173 established hydrogen bond, also additional pi-sigma, alkyl, and pi-alkyl interactions with residues Ala A: 123, Typ A: 348, Val A: 130 and Arg A: 102. Melatonin form van der waals force, alkyl and pi-alkyl contacts with POX but have lower affinity (-5.1 kcal/mol) than APX and CAT enzymes. In summary, MT with target antioxidant enzymes confer better binding potential which can be explained by their ability to form multiple hydrogen bonds and pi-pi stacked conformations with amino acids in the active site. Structural modifications of antioxidant enzymes to integrate more hydrogen bonding functional groups could further improve their affinity and activator potential. The ligand melatonin shows the strongest predicted binding affinity with SOD, having a ΔG of -6.8 kcal/mol. It forms a hydrogen bond interaction with the key active site residue Arg A: 339 and Ser A:292. It forms alkyl and pi-alkyl interactions with residues Val A:72, Lue A: 258 and leu A: 287. The combination of hydrogen bonds and hydrophobic interactions with multiple active site residues leads to its positive and expected binding. To link the results of the molecular docking study with the estimation of target antioxidant enzymes activity, based on previous studies, MT has a stimulant effect on these enzymes. This feature has been related to the influence of MT, which reduced lipid peroxidation and improved ROS metabolism (Sati et al. 2023). In another study, Jiao et al. (2022) found the enhanced antioxidant properties in MT -treated kiwifruit by increasing SOD activity. As well, Onik et al. (2021) observed a rising trend in CAT, SOD, and POX activity in apples treated with MT followed by a corresponding decrease in MDA level.

5 Conclusions

Despite sorghum plant is a relatively moderate tolerant to salinity stress, the results of this study confirmed its high sensitivity to a wide range of saline conditions at early growth stages. Applied-melatonin enhanced different morphological, physiological and biochemical mechanisms which orchestrated together to confer the stressed seedlings the required tolerance against salt stress. These mechanisms included strengthening photosynthesis by elevating the biosynthesis of photosynthetic pigments, stabilizing cell membranes, enhancing osmotic balance, and nutrients homeostasis. Moreover, melatonin had the ability to enhance the antioxidant capacity by altering the activities of antioxidant enzymes. The molecular docking modeling studies showed top-ranked confirmations between melatonin and the target antioxidant enzymes. The highest powerful affinity has been showed between melatonin treatments and ascorbate peroxidase compared to the other enzymes.