Tomatoes (Solanum lycopersicum L.) are an important industrial crop that is susceptible to a variety of environmental stresses. As one of the most important environmental stresses, water or soil salinity is increasingly a serious menace to crop production (Ghorbani et al., 2021; Hao et al., 2021). Unless effective management measures are implemented, roughly 50% of agricultural lands are anticipated to suffer from salt stress by 2050 (Wang et al., 2022). Excess sodium (Na⁺) in irrigation water or soil inhibits photosynthesis, absorption of essential nutrients, and protein synthesis, resulting in serious disruptions in plant vital metabolism, development, and yield (Shabala and Pottosin, 2014). Salt-stressed plants maintain potassium (K⁺) and Na⁺ homeostasis through a variety of mechanisms. Sodium/hydrogen antiporters (NHXs) in the tonoplast regulate the homeostasis of cellular cations, modulate stomatal function, and maintain cellular pH through sequestering Na and adsorbing K into vacuoles (Bassil and Blumwald, 2014). The NHX family includes six members (NHX1-6) in maize and rice and eight members (NHX1-8) in Arabidopsis (Khan et al., 2018). NHX1–4 isoforms were specified in tomato that are implicated in the accumulation of K in vacuoles, with the NHX1 and NHX2 transporters being the most significant (Gálvez et al., 2012). Furthermore, NaCl toxicity can lead to the overproduction of reactive oxygen species (ROS) and damage to various cellular organelles. These destructive effects induced by salinity are reduced by the cellular antioxidant defense system, such as antioxidant enzymes (e.g., peroxidases, catalase, and glutathione reductase), by diminishing the accumulation of free radicals (Li et al., 2017; Ghorbani et al., 2018b).

Melatonin is a plant-synthesized indoleamine that has been discovered to have crucial functions in root and shoot development, seed germination, and circadian growth rhythms (Murch and Erland, 2021). Melatonin also improved plant adaptation to biotic and abiotic stresses such as low temperature (Qari et al., 2022), high temperature (Byeon and Back, 2014), cadmium toxicity (Kaya et al., 2019), and salinity (Ghasemi-Omran et al., 2021). It has been shown that melatonin treatment can maintain ionic homeostasis in apple and maize plants during salinity stress (Li et al., 2012; Jiang et al., 2016). In addition, melatonin has been indicated to be involved in the adjustment of phytohormones and signaling molecules such as gibberellin, hydrogen peroxide (H₂O₂), nitric oxide (NO), and abscisic acid in NaCl-exposed plants (Zhang et al., 2021; Liu et al., 2015; Liang et al., 2015). NO has a vital function in plant stress responses as a redox signaling molecule. Melatonin has been demonstrated to modulate plant adaptation to different stresses through interaction with NO metabolism (Fancy et al., 2017; Yan et al., 2020; Feng et al., 2021). Liu et al. (2015) showed that melatonin reduced the levels of H₂O₂, free toxic radicals, and malondialdehyde (MDA) and improved the activities of catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) during sodic alkaline toxicity via interaction with NO. Yan et al. (2020) suggested that the application of melatonin by inducing nitrate reductase (NR)-synthesized NO up-regulated the H+-pump activity of the vacuole membrane and plasma membrane and, consequently, maintained K⁺/Na⁺ balance in rice under NaCl stress.

Apart from the outstanding defensive effects of NO and melatonin on the induction of plant adaptation under environmental stress, there is no accurate information on the regulatory role of melatonin treatment on NO metabolism in NaCl-stressed tomatoes and/or the function of melatonin and NO interaction in the molecular mechanisms of K/Na hemostasis in tomato plants. Accordingly, we examined the regulation of NO metabolism by melatonin as well as the cross-talk between NO and melatonin in the expression of NHXs transporters and the adjustment of K⁺/Na⁺ balance in tomato plants during NaCl toxicity. In addition, the effects of melatonin and NO interaction on chlorophyll and proline metabolism, the antioxidant machinery, and ionic homeostasis in tomato leaves were investigated under salinity conditions. The findings of this study may provide a new physiological basis for further elucidating the regulatory mechanisms of NaCl toxicity tolerance in tomato plants induced by the melatonin-NO interaction.

The results showed that NaCl treatment lessened the height and biomass of tomato seedlings, which is in line with earlier declared results on tomato (Ghorbani et al., 2018a), rice (Yan et al., 2020), and cotton (Zhang et al., 2021). NaCl stress has been shown to disrupt various metabolic pathways in plants by interfering with water status and nutrient uptake (Abdelaziza et al., 2019; Ghorbani et al., 2019). Interestingly, melatonin-induced mitigation of NaCl stress was evident through endogenous NO content, verified by the use of cPTIO (a NO scavenger), indicating an inhibition of melatonin-induced impacts in the presence of NaCl toxicity. The beneficial impacts of the exogenous application of melatonin on improving the growth and biomass of various plants have already been documented (Li et al., 2017; Chen et al., 2018; Alharbi et al., 2021). Our results confirm evidence of an interaction between melatonin and NO signaling in diminishing NaCl toxicity in tomato seedlings.

The application of melatonin induced a positive impact on the metabolism of photosynthetic pigments during NaCl toxicity, as evidenced by an increase in ALA accumulation associated with a subsequent upregulation in ALAD activity. The ALAD enzyme is required for the formation of pyrrole compounds by combining two ALA molecules, which is a crucial precursor for chlorophyll production (Killiny et al., 2018). It has been shown that high levels of ALA under stressful conditions can have positive impacts on biomass, the antioxidant defense system, and osmotic regulation under environmental stress (Wu et al., 2019). Moreover, melatonin diminished the chlorophyll degradation process in NaCl-stressed plants by downregulating Chlase activity, which may have protective effects on the performance of the photosynthetic apparatus during NaCl stress. Decreased expression of the Chlase gene by melatonin has been earlier documented by Weeda et al. (2014). The positive effect of melatonin on chlorophyll metabolism in NaCl-stressed plants can be caused by reducing oxidative stress and improving the activity of enzymes involved in chlorophyll metabolism (Liu et al., 2020), as well as improving ionic homeostasis and providing more Mg and Fe elements (Yan et al., 2020). However, cPTIO reversed the beneficial impacts of melatonin on chlorophyll metabolism, indicating the regulatory role of melatonin-mediated NO in modulating chlorophyll metabolism during NaCl stress. Additional examinations are needed to specify the exact role of melatonin and its interaction with NO in the biosynthesis of intermediates and chlorophyll metabolism.

It has been shown that the accumulation of osmoregulatory compounds such as proline under environmental stresses maintains ROS scavenging, cellular osmolality, redox homeostasis, and the function of bio-macromoleculars in plants, and is a significant help in improving plant adaptation in stressful conditions (Ghorbani et al., 2018a). NaCl toxicity enhanced the leaf content of proline by decreasing ProDH activity and enhancing P5CS activity, which can be described by putative water stress caused by salinity, as confirmed by Jiang et al. (2021) and Ghorbani et al. (2018a). Guan et al. (2020) revealed that upregulating the expression and activity of enzymes involved in proline biosynthesis (P5CS) increases proline accumulation and, as a result, improves plant adaptation to NaCl toxicity. Melatonin, by modulating enzymes in proline metabolism, provoked a further enhancement in proline in tomato leaves under NaCl stress, which can play a key function in enhancing plant adaptation. Similarly, Jiang et al. (2021) showed that melatonin, by increasing the proline content, protected the photosynthetic apparatus and improved the biomass of the cotton plant during salinity. As a result, employing cPTIO, melatonin-mediated proline metabolism was reversed, confirming the important regulatory function of melatonin-induced NO in the regulation of proline metabolism.

One of the serious damages induced by salinity stress is the excessive production of ROS (H₂O₂ and superoxide anion) in plants, which causes serious oxidative damage to important cell components, including biomembranes and the induction of EL. Plants have machinery to balance the endogenous level of toxic free radicals. The NaCl treatment increased the accumulation of toxic compounds such as superoxide anions and H₂O₂, resulting in increased MDA and EL, which indicate the induction of oxidative stress in tomato seedlings. NaCl toxicity-induced oxidative stress in tomato (Yin et al., 2019) and stevia (Ghasemi-Omran et al., 2021) plants has also been previously documented. Salinity stress disrupts the function of the photosynthetic apparatus and induces an imbalance between the production and consumption of electrons, causing the transmission of excess electrons to the oxygen molecule and the generation of ROS (AbdElgawad et al., 2016). However, melatonin effectively increased the activity of antioxidant enzymes, thereby reducing the level of ROS and protecting the biomembranes. The helpful impacts of melatonin on the antioxidant machinery and the decline of oxidative stress under salinity stress have already been confirmed by Li et al. (2017) and Jiang et al. (2021). Sun et al. (2021) indicated that melatonin upregulated the expression of antioxidant genes, such as SOD, CAT, APX, and GR, by enhancing the internal contents of NO and, as a result, reduced oxidative stress and enhanced plant biomass under salinity, which is in accordance with the findings of this research. Arora and Bhatla (2017) indicated that melatonin-mediated antioxidant defense in sunflower plants during salinity was dependent on NO. The addition of cPTIO prevented the beneficial impacts of melatonin on the antioxidant machinery, exhibiting the critical function of melatonin-caused internal NO in augmenting the defense system of NaCl-stressed tomatoes.

Key enzymes involved in N and NO metabolism (GOGAT), GS, NiR, and NR) play a crucial function in plant adaptation during environmental stresses (Chen et al., 2021). Melatonin significantly increased the leaf activity of enzymes in N assimilation and the leaf content of NO in NaCl-exposed seedlings, which can effectively improve the tolerance of tomato seedlings. Similar results have already been documented by Talaat (2021) and Ma et al. (2021). An increase in melatonin-induced N metabolism can improve N uptake and transfer between source and sink parts, thereby accelerating the transfer of nutrients between different plant organs under salinity stress (Talaat, 2021). Melatonin was shown to upregulate the expression of NiR, NR, GS, and GOGAT genes, which significantly enhanced the adaptation of the plant during stress (Liang et al., 2018). Due to the high sensitivity of GS and GOGAT enzymes to oxidative stress, increasing their activity under melatonin treatment can be due to the melatonin-caused alleviation of oxidative stress (Bose and Howlader, 2020; Zhao et al., 2021). However, cPTIO reduced the activity of enzymes in N assimilation in melatonin-treated plants, indicating the induction of melatonin-mediated N metabolism through the internal NO content under salinity.

Depolarization of the plasma membrane resulting from the influx of Na ions into the cell causes a continuous efflux of K from the cell under salinity stress, which results in a drop in the ratio of K/Na and, consequently, serious damage to vital cell processes (Shabala et al., 2006; Munns and Tester, 2008). NaCl treatment enhanced the accumulation of Na and declined the accumulation of K in the leaf and root of tomato seedlings, which was associated with a decrease in the K/Na ratio. Similar results of reducing the K/Na ratio in tomato (Ghorbani et al., 2019) and rice (Yan et al., 2020) have already been shown. Melatonin effectively reduced Na uptake and transport to the leaves, which maintained K homeostasis and increased the K/Na ratio under NaCl stress. Yan et al. (2020) in rice and Jiang et al. (2016) in maize plants under salt stress reported similar effects of melatonin’s regulatory role in diminishing Na uptake and improving the ratio of K/Na. Melatonin also diminished the translocation of Na to the leaves under salinity, which could be due to decreased Na loading in the xylem (Yan et al., 2021). A report showed that melatonin maintained K homeostasis and improved plant tolerance under salinity by alleviating oxidative stress and regulating K transporters, including HAK5 (Liu et al., 2020). NaCl treatment also reduced the concentration of nutrient minerals (P, N, Mg, and Ca) in the leaves, which could be due to oxidative stress-induced damage to biomembranes, as well as disruption of membrane potential due to the entry of excessive Na levels into the cellular cytoplasm (Ghorbani et al., 2019; Yan et al., 2021). Melatonin significantly improved the concentration of mineral nutrients in NaCl-stressed seedlings, which could result in the reduction of toxic radicals and the stabilization of cell membranes (Liu et al., 2020). The helpful impacts of melatonin on maintaining ionic homeostasis under salinity have been earlier confirmed by Zahedi et al. (2021) and Alharbi et al. (2021). Thus, melatonin improves plant growth and biomass under salinity stress by maintaining ionic homeostasis and reducing Na uptake. Numerous studies have shown that NO treatment effectively reduces Na uptake, improves K/Na, and increases the uptake of mineral nutrients in plants under salinity stress (Zhang et al., 2006; Wang et al., 2009; Hasanuzzaman et al., 2021). The inductive impacts of melatonin on the maintenance of ionic balance and the reduction of Na uptake in cPTIO-treated plants were reversed, indicating that the impacts of melatonin improvement on the maintenance of ionic homeostasis are achieved through NR-mediated NO production.

Salinity stress increases the entry of Na into the cell cytoplasm, which, by damaging cytosolic enzymes, causes a serious disruption in the vital processes of the plant (Fukuda et al., 2011). Therefore, regulating the cytosolic concentrations of Na and K and maintaining the cytosolic ratio of K/Na under salinity can play a critical function in enhancing plant adaptation to salt stress. The three main mechanisms for preventing the cytosolic accumulation of Na include: inhibiting Na influx into the cytoplasm, inducing Na efflux, and sequestering Na in vacuoles (Padan et al., 2001). The NHX gene family, located on the tonoplast membrane, is implicated in the compartmentalization of Na into the vacuoles, which can play an important function in reducing the potential toxicity of Na during salinity (Padan et al., 2001; Fukuda et al., 2011). Increased expression of NHX1, NHX2, NHX3, and NHX4 genes has been shown to decline Na toxicity and, consequently, enhance plant growth under NaCl toxicity (Abdelaziza et al., 2019; Ghorbani et al., 2019). Tan et al. (2021) indicated that the heterologous expression of the melatonin-synthesizing gene was increased by enhancing the expression of NHXs and inducing an efflux of Na in the plants under NaCl toxicity, which was accompanied by increased growth and adaptation of the plant under salinity. The results showed that melatonin significantly increased the relative expression of NHX1, NHX2, NHX3, and NHX4 genes in the root and leaf of tomato seedlings during NaCl toxicity, which could play an important function in Na detoxification by sequestering in vacuoles. Increased expression of NHX genes in the root was stronger than in the leaf, which is consistent with a decrease in Na translocation to the leaves. However, when the melatonin-subjected plants were treated with cPTIO, the inducible effects of melatonin on the expression of the NHX gene were inhibited, indicating that melatonin-induced upregulation of the NHX gene arises through NR-mediated NO during NaCl stress.

Our findings provide crucial insights into the function of exogenous melatonin and melatonin-mediated internal NO signaling in handling NaCl adaptation in tomato. The results confirmed that exogenous application of melatonin by interaction with endogenous NO increased plant growth and biomass during NaCl stress, indicating that NO as a signaling molecule may be involved downstream of the melatonin-mediated defense response in NaCl-exposed tomato seedlings. To confirm the function of NO downstream of the melatonin-induced signaling pathway, cPTIO was used as a NO scavenger, which showed that cPTIO prevented the beneficial role of melatonin in chlorophyll and proline metabolism, the antioxidant defense system, ionic homeostasis, and modulation of NHX gene expression. Therefore, by adjusting N metabolism and enhancing the internal content of NO, melatonin improves chlorophyll and proline metabolism, strengthens the antioxidant defense system, preserves ion balance, and modulates the expression of transporter genes involved in K/Na homeostasis, thus improving the growth and tolerance of tomato seedlings under NaCl toxicity. In the future, the interaction of other enzymes or signaling molecules in increasing the tolerance of abiotic stresses induced by melatonin should be examined.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

Methodology and Conceptualization, AG; Investigation and Validation, AG and LP; Resources, KVS; Analysis, AG; Writing original, AG; Review and Editing, M-XC. All authors contributed to the article and approved the submitted version.