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Exogenous melatonin increases salt tolerance in bitter melon by regulating ionic balance, antioxidant system and secondary metabolism-related genes

Abstract

Background

Melatonin is a multi-functional molecule widely employed in order to mitigate abiotic stress factors, in general and salt stress in particular. Even though previous reports revealed that melatonin could exhibit roles in promoting seed germination and protecting plants during various developmental stages of several plant species under salt stress, no reports are available with respect to the regulatory acts of melatonin on the physiological and biochemical status as well as the expression levels of defense- and secondary metabolism-related related transcripts in bitter melon subjected to the salt stress.

Results

Herewith the present study, we performed a comprehensive analysis of the physiological and ion balance, antioxidant system, as well as transcript analysis of defense-related genes (WRKY1, SOS1, PM H+-ATPase, SKOR, Mc5PTase7, and SOAR1) and secondary metabolism-related gene expression (MAP30, α-MMC, polypeptide-P, and PAL) in salt-stressed bitter melon (Momordica charantia L.) plants in response to melatonin treatment. In this regard, different levels of melatonin (0, 75 and 150 µM) were applied to mitigate salinity stress (0, 50 and 100 mM NaCl) in bitter melon. Accordingly, present findings revealed that 100 mM salinity stress decreased growth and photosynthesis parameters (SPAD, Fv/Fo, Y(II)), RWC, and some nutrient elements (K+, Ca2+, and P), while it increased Y(NO), Y(NPQ), proline, Na+, Cl, H2O2, MDA, antioxidant enzyme activity, and lead to the induction of the examined genes. However, prsiming with 150 µM melatonin increased SPAD, Fv/Fo, Y(II)), RWC, and K+, Ca2+, and P concentration while decreased Y(NO), Y(NPQ), Na+, Cl, H2O2, and MDA under salt stress. In addition, the antioxidant system and gene expression levels were increased by melatonin (150 µM).

Conclusions

Overall, it can be postulated that the application of melatonin (150 µM) has effective roles in alleviating the adverse impacts of salinity through critical modifications in plant metabolism.

Peer Review reports

Background

Salinity is of the major constraints affecting world agricultural production, appearing as one of the major challenges to be alleviated because of its retarding effects on growth, development and productivity of crops [1, 2]. Nearly 50% of irrigated land and 10% of soils in the world are under exposure to high levels of salinity [3]. Due to their sessile nature, plants cannot escape from the environmental cues and for that reason, they have to evolve an elaborate system as well as adaptive responses against salt stress. Corresponding to the high levels of salinity, the sodium and chloride ions accumulate in the soil, which in turn reduces the availability of essential nutrients (such as K+) and water in plants [4]. K+/Na+ homeostasis is one of the key mechanisms for salinity tolerance in plants and in this regard, regulation/compartmentalization of Na+ and K+ homeostasis in plants is critical for enhanced salt stress tolerance [5]. In cytosol, plasma membrane Na+/H+ antiporter (SOS1), SKOR K+ channel, and the PM H+-ATPase regulate Na+/K+ ion homeostasis under salinity stress. Furthermore, reactive oxygen species (ROS) signaling exhibits a crucial role linked to salinity tolerance [6]. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is the main source of apoplastic ROS production which leads to salinity tolerance in the various plant [7]. In addition, gene family of WRKY, a plant-specific transcriptions factor (TF) group, plays key functions in various response pathways. For example, WRKY1 is involved in plant tolerance against drought [8] and salinity [9]. SOAR1, a cytosolic-nuclear pentatricopeptide repeat protein, has a vital role in plant response to salinity and drought [10, 11]. Furthermore, phenylalanine ammonia-lyase (PAL) is a well-known precursor to increase production of major secondary metabolites which are, in general, crucial for plant adaptation against biotic and abiotic stress factors [12, 13].

Melatonin (MT) has been shown to have various potential physiological functions in plants under stressful and non-stressful conditions [14]. Among the known functions, MT has been revealed to be effective in alleviating the oxidative damage of stress factors, viz. heavy metal [14], high temperature [15], salt [16], drought [17] and cold stress [18]. In this context, Zhang and Zhang [19] and Reiter et al. [20] reported that MT is a mitochondria-targeted antioxidant that achieves this action directly (detoxification of RONS) or indirectly (by inducing antioxidant enzymatic activity and suppressing pro-oxidant enzymatic activity). It has also been reported that MT as a regulator of growth and/or biostimulator in plants [21, 22] and could be effective in triggering germination by biosynthesis regulation and catabolism GA4 and ABA in cucumbers [23], stimulating development of roots owing to the regulation of auxins synthesis, signaling and transport, as observed in tomatoes and Arabidopsis [24], increasing berry quality of grape [25] and improving the postharvest conservation of fresh fruits and vegetables [26]. Moreover, exogenous applications of MT increased the secondary metabolite contents in cabbage plants via up-regulating the expression of related biosynthetic genes [27].

Momordica charantia L., commonly known as bitter melon or bitter gourd is an important member of family cucurbitaceae and it widely grows in tropical and sub-tropical areas. The fruit and leaves of bitter melon are rich in phytochemicals including nutraceutical and nutritional components [28]. Bitter melon has a wide range of medical applications to treat cancer, hypertension, T2DM, bacterial and viral infections, obesity, and even AIDS [29]. Anti-HIV protein, MAP30, and α-momorcharin (α-MMC) are Type-I RIPs (Ribosome-Inactivating Proteins) having single enzyme chain, which was isolated from bitter melon and demonstrated to have efficacy against HIV infection and cancer. Polypeptide-P, another bioactive peptide isolated from bitter melon, showed hypoglycemic activity in diabetes [30].

The excellent functions of MT as anti-stressor have been widely reported for several crops. For instance, MT critically altered plant responses against stress through reducing the levels of H2O2, activating the ROS-metabolizing enzymes and inducing Na+ and K+ transporters. Those modifications assisted in alleviating the adverse effects of salinity [21, 31]. Interestingly, it has been surmised that melatonin produce minor metabolites, which then co-work in combating with the stress. For that reason, Back [32] hypothesized that melatonin and its metabolites are together in the case exogenous applications of melatonin, suggesting that marked responses of the plants cannot be exclusively attributed to the melatonin alone. For that reason, the studies linked to reveal the roles of MT are required. In this regard, we herein carried a comprehensive study in bitter melon subjected to salt stress. The regulatory roles of MT on the expression levels of defense- and secondary metabolism-related related transcripts in bitter melon subjected to the salt stress were, for the first time, investigated. The key objectives of the current work were: (i) to study the potential function of MT in ameliorating the negative effect of salinity, (ii) to decipher the expression profile of defense and secondary metabolism-related genes induced by MT under salt stress, and (iii) to examine the physiological, biochemical, and nutritional state of MT-primed plants under salt stress, mainly aiming to investigate molecular regulatory components involved in the response to salinity.

Materials and methods

Growth conditions and plant treatment

This experiment was conducted in a growth chamber of the Department of Plant Biotechnology, University of Tabriz. The experiment was performed as a factorial using a completely randomized design (CRD) with three biological replications and each replication consisted of two plants. The bitter melon (Palee F1) seeds were provided from Victoria Companies, India. Seeds were sterilized using sodium hypochlorite solution (1%) for 5 min and were then left for germination under darkness at 25 °C for 48 h for pre-germination. Following one-week germination, the seeds those were homogeneously and uniformly germinated were transferred to the trays for cultivation comprising coco-peat and watered with ½ Hoagland’s modified solution. The seeds containing trays were then placed in the growth chamber with 28/22°C (day/night) and relative humidity of 62–80% 360 µmol m− 2 s− 1 light intensity. At the time of third true leaf emergence, healthy and uniform seedlings were selected and separated into three groups: (i) Hoagland’s nutrient solution (HNS, as a control); (ii) HNS + 75 µM melatonin (MT); and (iii) HNS + 150 µM MT. The concentrations of melatonin were selected according to Yin et al. [33]. Plants were grown in melatonin-supplemented Hoagland solution for one week with 0, 75 and 150µM MT (MT added to Hoagland’s nutrient solution). MT was dissolved in ethanol were then diluated with MilliQ water. The MT purchased from Sigma–Aldrich, company, USA. Four days after MT supplementation, plants were exposed to different levels of salinity stress (0, 50 and 100 mM NaCl) for three day period. After treatment, leaves and roots samples were harvested for RNA extraction and study of gene expression, frozen in liquid nitrogen promptly after harvest, and stored at ˗80 °C until further analyses.

Growth parameters

Fresh and dry weights of shoot and root, as well as height of shoot and root, were measured at the end of three-day salt stress.

Chlorophyll-related parameters

The chlorophyll index in five fully-expanded leaves was recorded with a SPAD-502 chlorophyll meter (Minolta Co. Ltd., Japan). Chlorophyll fluorescence parameters (Fv/Fo, Y(II), Y(NO) and Y(NPQ)) of leaf samples were assessed with a chlorophyll fluorometer (Dual-PAM-100, Heinz Walz, Efeltrich, Germany) under dark adaption for 20 min.

Relative permeability, proline contents and relative water content

Relative permeability was determined according to the method of Nanjo et al. [34]. Proline content in leaf samples was determined with detailed method of Bates et al. [35]. Freshly sourced leaves were macerated in 3% sulphosalicylic acid solution and were then centrifuged for 10 min at 15,000 × g at 4 °C. One ml of prepared supernatant solution was placed in a tube and reacted with one ml acid ninhdrin and one ml of acetic acid. The resultant mixtures were heated for 60 min at 100ºC. The assay reaction was stopped by putting the reaction assay on ice. After that, toluene (2 mL) was used for the extraction of assay mixture. The two phases were separated by keeping the reaction assay at room temperature for the period of 30 min. Finally, supernatants absorbance was read at 520 nm on a spectrophotometer (UV-1800 Shimadzu, Japan) and toluene was served as blank. The relative water content (RWC) of leaf samples was measured following the protocol described by Sairam and Srivastava [36]. Initial fresh leaf samples were weighted using digital balance for fresh weight (FW). Then, turgid weight (TW) was recorded by placing the leaf samples in double-distilled water for 24 h. Finally, dry weights (DW) of samples were assessed after 24 h drying at 70 ºC. RWC was measured by the equation: RWC= (FW − DW)/(TW − DW)×100.

Malondialdehyde (MDA) and hydrogen peroxide (H2O2) content

The concentration of malondialdehyde (MDA) was determined with previous protocol [37]. The 0.3 g fresh leaf sample was ground in 20% trichloroacetic acid (TCA) and centrifuged for 15 min at 13,000×. Thereafter, TCA (20%, 4 mL) was incorporated into 1 ml of the solution of supernatant. The mixture was the boiled in water bath (95 °C) for 30 min. Afterward, the mixture was quaickly cooled in an ice bath and absorbance was noted at 600 nm and 532 nm. Finally, MDA was calculated by used 155 mm− 1 cm− 1 as a coefficient of molar absorption.

H2O2 was determined with protocol of Allen [38]. Briefly, 0.2 g sample of the leaves was macerated in an ice bath which contained 0.1% TCA (3 mL) and it was centrifuged for 15 min at 20,000×g. After that, 500 µL assay mixture was reacted with 10 mM concentrated phosphate buffer (500 µL, 7.0 pH) comprising 2 M KI. The resultant assay was kept under darkness for 60 min at room temperature for the incubation. Finally, H2O2 content was assayed at 390 nm on a spectrophotometer.

Antioxidant enzymes activities

In order to assay the activity of antioxidant enzymes, 0.5 g homogenized leaf sample was macerated in 0.05 M phosphate buffer (1% PVP, 1 M MEDTA, 7.8 pH), and subjected to centrifugation for 20 min at 12,000× g (4 °C). The collected supernatants were used for peroxidase (POD) [39] and superoxide dismutase (SOD) activities determination [40]. To determine POD activity, assay reaction comprising enzymes extract, 5 µL of 10% (w/v) H2O2, 100 mM phosphate buffer (pH 6.0), and 16 mM guaiacol. The absorbance was at 470 nm for 1 min as mmol produced tetraguaiacol per minute per mg soluble proteins (U mg− 1). As well as one unit of SOD activity was enzyme amount needed to cause 50% inhibition of NBT (nitro blue tetrazolium) at 560 nm.

Content of nutrient elements

For quantifying the content of major macro-elements, 100 mg of ground, oven-dried root tissue was digested in concentrated nitric acid (110 °C for 6 h). The concentration of potassium (K+) and sodium (Na+) in the digested extracts was quantified by flame emission spectrometry, while calcium (Ca2+) and phosphorus (P) were determined by atomic absorption spectrometry (AA-7000, Shimadzu). For chloride (Cl) determination, oven-dried root samples were extracted with deionized water at 100 °C for 2 h, after which Cl content was measured by ion chromatography (ICS 2000, Dionex, Sunnyvale, CA, USA).

RNA isolation and quantitative real-time PCR (RT-qPCR) assay

Total RNA extracted from the leaf and root samples (using CinnaGen kit, Iran) was used for cDNA synthesis (using Yekta Tajhiz Azma kit, Iran). The primers for the α-tubulin 1a internal control gene and studied genes (MAP30, α-MMC, polypeptide-P, SOS1, H+-ATPase, SKOR, SOAR1, Mc5PTase7, and WRKY1) are shown in Table S1. The RT-qPCR reaction mixtures (25 µL) contained 12.5 µL of master mix (AMPLIQON), 1 µL of primer (10 µM), 2 µL of cDNA, and 9.5 µL of nuclease-free water. The reaction parameters were used in all cycle sequencing reactions: initial denaturation at 95 °C for 30 s; denaturation at 95 °C for 5 s, annealing at 60 °C for 20 s, 30–40 cycles; 55° to 95 °C increased by 0.5 °C every 30 s, 81 cycles. Three replicates were calculated for each sample and gene the relative expression of the gene was calculated by the comparative Ct (2−ΔΔCt) method.

Statistical analysis

The data obtained were analyzed by Statistica-13 (Statsoft, Tulsa, USA). Factorial ANOVA, in which the concentration of MT solution and degree of salinity stress were used as categorical variables revealed a significant difference between treatments. When a significant difference was found, Duncan’s post-hoc analysis was used to find homogeneous groups (p < 0.05, significant difference).

Results

Effect of exogenous melatonin on growth parameters under salinity stress condition

To assess the effects of MT and salt stress in bitter melon, four-week-old bitter melon seedlings were subjected to MT pre-treatment, and were then treated with 50 and 100 mM NaCl stress for three days. In relation to the control, the higher levels of salinity critically decreased shoot height, root height, shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight up to 51.25%, 57.23%, 48.51%, 48.46%, 42.18%, and 42.01% respectively (p < 0.05) (Table 1). As expected, 150 µM concentration of MT substantially increased shoot height (15.04% and 30.46%), root height (19.23% and 37.43%), shoot fresh weight (20.18% and 22.15%), shoot dry weight (20.07% and 21.76%), root fresh weight (14.58% and 20.68%) and root dry weight (14.74% and 15.04%) than salt-treated biter melons with 50 and 100 mM (p < 0.05) (Table 1).

Table 1 Effect of application of melatonin (0, 75 and 150 µM) on growth parameters of bitter melon (Momordica charantia) under salt stress (0, 50 and 100 mM NaCl) conditions

Effect of exogenous melatonin on photosynthetic parameters under salinity stress condition

High concentration of salt significantly reduced SPAD, Fv/Fo and Y (II) and increased Y (NO) and Y (NPQ), in comparison to the control (Table 2). Being consistent with the former reports [41], MT (150 µM) reduced the adverse impacts of high level of salinity by increasing the values of SPAD, Fv/Fo, Y(II) and by decreasing Y(NO) and Y(NPQ), in comparison to either unprimed or salt-stressed plants (p < 0.05).

Table 2 Effect of application of melatonin (0, 75 and 150 µM) on photosynthetic parameters of bitter melon (Momordica charantia) under salt stress (0, 50 and 100 mM NaCl) conditions

Effect of exogenous melatonin on proline and RWC under salinity stress condition

High concentration of salt stress caused significant increments in proline content, while it decreased RWC. Once compared with 50 and 100 mM NaCl stress, pre-treatments with MT (150 µM) increased proline and RWC (p < 0.05) (Fig. 1a, b).

Fig. 1
figure 1

Effect of application of melatonin (0, 75 and 150µM) on Proline (a) and RWC (b) in bitter melon under salt stress (0, 50 and 100 mM NaCl) conditions. Data are the average of 3 replicas ± standard error. Different letters show significant difference according to Duncan’s multiple range test at p ≤ 0.05

Effect of exogenous melatonin on oxidative stress indicators under salinity stress condition

In regard to common cellular damage indicators, 100 mM NaCl increased the level of MDA, H2O2 and electrolyte leakage, in comparison to the control. Along with the pre-treatment of 150 µM MT, significant reductions were observed in levels of MDA, H2O2 and electrolyte leakage, in comparison to either the unprimed or 50 and 100 mM NaCl treatments (Fig. 2a-c).

Fig. 2
figure 2

Effect of application of melatonin (0, 75 and 150µM) on MDA (a), H2O2 (b) and Relative Permeability (%) (c) content in bitter melon under salt stress (0, 50 and 100 mM NaCl) conditions. Data are the average of 3 replicas ± standard error. Different letters show significant difference according to Duncan’s multiple range test at p ≤ 0.05

Effect of exogenous melatonin on antioxidative enzymes activities under salinity stress condition

In accordance with the increased stress-related parameters, significant increases in activities of POD and SOD were observed at 100 mM NaCl, relative to the control. However, pretreatment with 150 µM MT further increased the activity of POD and SOD more than salinity alone, in comparison with 50 and 100 mM NaCl (Fig. 3a, b).

Fig. 3
figure 3

Effect of application of melatonin (0, 75 and 150µM) on POD (a) and SOD (b) enzyme activity in bitter melon under salt stress (0, 50 and 100 mM NaCl) conditions. Data are the average of 3 replicas ± standard error. Different letters show significant difference according to Duncan’s multiple range test at p ≤ 0.05

Effect of exogenous melatonin on nutrient concentration under salinity stress condition

In relation to the control, sharp decreases in K+, P and Ca2+ content by 50.88%, 40.97%, and 55.69%, respectively, and increases in Na+ and Cl of up to 60.83% and 60.22% were observed in roots under 100 mM NaCl. However, pretreatment with 150 µM MT increased K+ (21.98% and 30.29%), P (24.18% and 29.21%) and Ca2+ (26.77% and 21.67%) content and decreased Na+ (27.07% and 18.68%) and Cl (25.97% and 15.46%) content, in relation to 50 and 100 mM NaCl (Fig. 4a-e).

Fig. 4
figure 4

Effect of application of melatonin (0, 75 and 150µM) on K+ (a), P (b), Ca2+ (c), Na+ (d) and Cl (e) content of bitter melon roots under salt stress (0, 50 and 100 mM NaCl) conditions. Data are the average of 3 replicas ± standard error. Different letters show significant difference according to Duncan’s multiple range test at p ≤ 0.05

Effect of exogenous melatonin on defense-related genes expression under salinity stress conditions

To evaluate the effects of MT on the ion homeostasis in roots of bitter melon under salinity conditions, the transcription level of SOS1, SKOR, and PM H+-ATPase were also investigated. Accordingly, 100 mM NaCl caused significant inductions in the transcriptions level of all three genes (Fig. 5a-c), in comparison to the control. However, pre-treatment with 150 µM MT further increased expressions level of SKOR, SOS1, and PM H+-ATPase in relation to 50 and 100 mM NaCl treatments (Fig. 6a-c).

Fig. 5
figure 5

Effect of application of melatonin (0, 75 and 150µM) on relative expression level of WRKY1 (a), SOAR1 (b) and Mc5PTase7 (c) genes in the bitter melon leaves. Data are the average of 3 replicas ± standard error. Different letters show significant difference according to Duncan’s multiple range test at p ≤ 0.05

Fig. 6
figure 6

Effect of application of melatonin (0, 75 and 150µM) on relative expression level of SOS1 (a), SKOR (b) and PM H+-ATPase (c) genes in bitter melon roots. Data are the average of 3 replicas ± standard error. Different letters show significant difference according to Duncan’s multiple range test at p ≤ 0.05

Under 100 mM salinity, WRKY1, SOAR1, and Mc5PTase7 were significantly up-regulated in the shoot of bitter melon plant once compared with non-saline conditions (Fig. 7a-c). However, pre-treatment with 150 µM MT showed significant up-regulation in expressions level of the three genes in shoot tissues, in comparison to the stressed plants (Fig. 5a-c).

Fig. 7
figure 7

Effect of application of melatonin (0, 75 and 150µM) on relative expression level of PAL (a), MAP30 (b), a-mmc (c) and polypeptide-p (d) genes in bitter melon leaves. Data are the average of 3 replicas ± standard error. Different letters show significant difference according to Duncan’s multiple range test at p ≤ 0.05

In addition, current findings revealed that transcript levels of PAL, MAP30, α-MMC, and Polypeptide-P were significantly up-regulated under 100 mM salinity stress. As the case of other estimated parameters, pre-treatment with MT 150 µM significantly inducted PAL, MAP30, α-MMC and polypeptide-P compared with unprimed, salt-stressed plants under both NaCl concentrations (Fig. 7a-d).

Discussion

In the current study, exogenous effects of melatonin on salt stress-submitted bitter melon plants were investigated through an array of agronomic, physiological and biochemical attributes. The relevant findings were collectively visualized and presented in Fig. 8. As expected, high levels of salinity critically decreased growth and photosynthetic parameters such as SPAD index, Fv/Fo, Y(II) and increased Y(NO) and Y(NPQ). These findings are in accordance with the observations of Wu et al. [42] and Gohari et al. [43] for cucumber and Moldavian balm plants, respectively. As expected, the application of MT led to increases in SPAD index, Fv/Fo, Y(II) and decreases in values of Y(NO) and Y(NPQ). Photosynthesis is an important process due to its pivotal role in plant survival and productivity. For that reason, reduction in photosynthesis is commonly translated into reduced growth and development. Numerous reports have shown the protective role of MT on photosynthetic apparatus of crop plants exposed to salt stress. For instance, employing MT significantly increased chlorophyll pigments, carotenoids concentration and Fm, Fv/Fm, ETR, Y(II), and qP in cucumber plants [44]. Similarly, MT priming increased photosynthetic quantum yield (φPSII), the total content of chlorophyll, as well as RbcL and RbcS genes expression in Phaseolus vulgaris L. [45], while relative chlorophyll content and genes involved in photosynthesis (including ATPF0A, ATPF0B, ATPF1B and LHCB) genes were induced in melatonin-primed rubber tree (Hevea brasiliensis) grown under salt stress [46]. In addition, Xie et al. [47] reported that MT decreased Y(NO) and Y(NPQ) in tomato seedlings under calcium nitrate stress.

Fig. 8
figure 8

Schematic presentation of the findings of the study

As one of the adopted strategies for combating the stress, plants accumulate osmotic regulators for maintaining intra-cellular stability and protecting their cells from the toxicity of salt stress [48]. Ferchichi et al. [49] reported that proline presents multiple roles such as regulation of salt stress-responsive gene expression, redox homeostasis, as well as stabilization of membrane and proteins. In the present experimental setup, the application of MT significantly improved proline’s concentration and RWC in bitter melons plant during salt stress, as the cases observed in several other plant species treated with MT and salt stress [50, 51]. Specifically, MT amplified proline, total soluble carbohydrate content as well as pyrroline-5-carboxylate synthase (P5CS) activity in tomatoes [52]. Furthermore, Chen et al. [50] reported that soluble sugar and soluble protein contents in cotton seeds were enhanced following MT application under salinity stress.

Current findings showed that 100 mM NaCl caused critical increments in levels of free radicals (H2O2), MDA, and relative conductivity. These findings are similar with the observations of Zhang et al. [23]. In the same way, Chen et al. [53] and Li et al. [54] reported that MT generally protects the crop plants from oxidative-induced detrimental stress by detoxification of the ROS and owing to increased activities of antioxidative enzymes. Similarly, findings of the present study revealed that MT application lowered free radicals (H2O2), MDA content and relative conductivity level by increasing activities of POD and SOD enzymes. In agreement with our findings, the application of MT increased antioxidative enzyme activities and transcriptions level of the associated genes that encode the antioxidative enzyme expressions, while decreasing free radicals (H2O2, O2), MDA, and relative conductivity in cucumber [44] and Phaseolus vulgaris L. [45] under salinity stress.

Maintaining ionic homeostasis in plant tissues has been linked with the status of the antioxidant enzymes, membrane integrity and osmotic potential of the cells, affecting cellular turgor which is translated into plant growth and development. In this regard, Shabala and Cuin [55] reported that maintaining a high K+/Na+ ratio and a low cytosolic Na+ content were essential factors for plants to maintain homeostasis of their cellular metabolism as Na+ and Cl were metabolically toxic at high concentrations [56]. Kurusu et al. [57] reported that Ca2+ is a key signaling component in a plant’s salt stress response. It has been reported that Ca2+ reduces the negative effects of salt stress in plants [58] by stabilizing cell wall structures [59], maintaining functional and structural integrities of membrane in plants [58], regulating ion selectivity and transport and controlling ion-exchange behavior [60]. Stressors such as cold shock [61], heat shock [62], salinity [63] and drought [61] induce cytosolic Ca2+ accumulation, which acts as in the form of secondary messenger during the stressful conditions signaling [64].

The SOS2-SOS3 complex activities SOS1 (Na+/H+ anti-porter), which in turn regulates cytosolic Na+ concentration [65]. It has been reported that SOS2 affects CAX1, thus further connecting cell Ca2+ with Na+ transportation [66]. Phosphorus (P) is an essential element of the macro-category that is involved in a variety of processes in the plants such as transfer of the energy where it is required, photosynthesis, respiration, signaling transduction cascades, and macromolecular biosynthesis [67]. The availability of P can affect salt tolerance of plants [68]. Current results showed that salt stress-imposition enhanced the Na+ and Cl and reduced K+, Ca2+ and P, while application of MT significantly increased K+, Ca2+ and P contents and substantially reduced Na+ and Cl in roots of bitter melon under salinity stress. Furthermore, MT increased SKOR, SOS1, and PM H+-ATPases transcripts level in the root cells of salt-stressed bitter melons in comparison to either Unprimed or salt-stressed plants. Application of MT increased transcription levels of SOS pathway genes (SOS1-3) in cucumber [44], PM H+–ATPases activities, and the homeostasis of K+/Na+ in the seedlings of sweet potatoes [69], genes expression associated with key potassium channels and transporters (OsHAK1, OsAKT1, OsGORK and OsHAK5) and K+ content [70], the content of Ca2+ [71] and reduced Na+ and Cl [50] under salt stress. Regarding current findings, MT significantly induced WRKY1, SOAR1 and Mc5PTase7 expression under salinity stress. The WRKY1 transcription factor is a key component of stress-related signal transduction pathways and is a factor in the improvement of plant tolerance to stress [72]. A wide range of downstream genes [72] including jasmonic acid-responsive genes [73], and genes associated with signal transduction of salicylic acid [74] and regulators of secondary metabolism are controlled by WRKY1 [72]. In agreement with our findings, MT induced up-regulation of various genes expression such as MYB, WRKY, and other (genes) transcription in Arabidopsis and cucumber [75, 76] and DREB, WRKY, and MYB in Bermuda grass [77].

SOAR1 is a downstream of the ABA receptor and upstream of an important ABA-responsive bZIP transcription factor [11]. Ma et al. [78] stated that two isoforms of Arabidopsis eIF4G, eIFiso4G1 and eIFiso4G2 interacted with SOAR1 in order to regulate ABA signaling negatively. In addition, Bi et al. [79] reported that both USB1 and SOAR1 were required genes for transcripts splicing of numerous genes such as the genes associated with salinity responses and signaling of ABA pathways. The over-expression of SOAR1 also increased proline levels, expression levels of SOS1, SOS2, and P5CS1 and growth of plants under salinity while it decreased the level of electrolyte leakage [10]. Our results showed that MT increased growth, transcript levels of SOAR1 and SOS1, proline, and decreased electrolyte leakage under salinity stress conditions.

Huang et al. [7] reported that certain ROS production has been found associated with NADPH oxidase-which is further linked with tolerance of salinity in different plants. The main apoplastic ROS generation source is burst oxidase homolog (RBOH). Torres et al. [80] described that RBOH generated superoxide, which was then dismutated to H2O2 [81].

Kaye et al. [82] found that AtRbohJ plays a key role in production of ROS in plants under salinity stress and the production of ROS in AtRbohJ mutants was significantly lower under salinity stress. Other reports revealed that the transcriptions of AtRbohJ in At5ptase7 mutants were significantly decreased during salt stress [82]. Those findings suggest that At5ptase7 plays an imperative function in production of ROS and NADPH oxidase activity under salinity stress. It was observed that At5PTase7 mutants showed failure in the induction of RD22 and RD29A, that contains numerous ROS-reliant components with regard to their certain promoters [82]. NADPH oxidase activity increased rosette fresh weight, K+ concentration, K+/Na+ ration, total chlorophyll content, chlorophyll fluorescence (Fv/Fm), CAT, APX, GR and SOD enzymatic activities, and decreased Na+, H2O2 and MDA concentrations in Arabidopsis thaliana under salt stress [83]. Present findings showed that application of MT increased transcript levels of Mc5PTase7, fresh weight, K+ concentration, SPAD, Fv/Fo, Y(II), POD and SOD enzymatic activities, and decreased Na+, H2O2 and MDA concentrations in salt-stressed plants. Similar to our conclusion, Chen et al. [84] reported that MT may improve tolerance against certain stresses through modulation of ROS-signaling which is well co-ordinated by NADPH oxidase.

Several important proteins and peptides such as 30 kD (MAP-30) which is an anti-HIV protein, α-momorcharin (α-MMC) and polypeptide-P were isolated from bitter melon. MAP30 and α-MMC is a single chain RIP (type I ribosome-inactivating proteins) and their molecular mass are 30 kD. MAP30 and α-MMC prevent many types of cancers such as blood, brain, breast, colon, liver, and lung cancer [85]. In addition, polypeptide-P, a hypoglycemic peptide, has an imperative function in the recognition of cells and certain reactions required for the adhesion purpose [30]. In this study, the application of MT increased MAP30, α-MMC, polypeptide-P and PAL gene expression levels under control and saline conditions. The current molecular profiles are in accordance with earlier reports that MT treatment positively induced transcription of flavonoid biosynthetic genes such as C4H, PAL, LAR, CHS, F3H, ANR, and UFGT in kiwifruit [86], genes related to the biosynthesis of rosmarinic acid (PAL and RAS) in Dracocephalum kotschyi [87], phenylpropanoid pathway genes (PAL, STS) in grape berries [88] and biosynthesis associated genes of anthocyanin (C4H, PAL, CHI, CHS, DFR, LDOX, F3H, F3′H, GST and UFGT) in red cabbage and white cabbage [27].

Conclusions

Exogenous MT enhanced salinity stress tolerance in bitter melon through different mechanisms. Exogenously applied MT (150 µM) in salt-stressed plants improved growth and photosynthetic parameters, increased osmoprotectant through higher proline content, lowered oxidative stress by up-regulating antioxidant enzymatic activity, regulated ionic homeostasis and importantly, resulted in the transcriptional regulation of multiple defense-related genes. Furthermore, MT induced the transcription levels of genes linked to the secondary metabolites. Overall, it can be concluded that MT can be successfully employed as an effective priming agent for the amelioration of salt stress in bitter melon plants.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

  1. Kaya C, Higgs D, Ashraf M, Alyemeni MN, Ahmad P. Integrative roles of nitric oxide and hydrogen sulfide in melatonin-induced tolerance of pepper (Capsicum annuum L.) plants to iron deficiency and salt stress alone or in combination. Physiol Plant. 2020;168(2):256–77. https://doi.org/10.1111/ppl.12976.

    CAS  Article  PubMed  Google Scholar 

  2. Singh A, Roychoudhury A. Gene regulation at transcriptional and post-transcriptional levels to combat salt stress in plants. Physiol Plant. 2021;173(4):1556–72. https://doi.org/10.1111/ppl.13502.

    CAS  Article  PubMed  Google Scholar 

  3. Song J, Shi W, Liu R, Xu Y, Sui N, Zhou J, Feng G. The role of the seed coat in adaptation of dimorphic seeds of the euhalophyte Suaeda salsa to salinity. Plant Species Biol. 2017;32(2):107–14. https://doi.org/10.1111/1442-1984.12132.

    Article  Google Scholar 

  4. Van Zelm E, Zhang Y, Testerink C. Salt tolerance mechanisms of plants. Annu Rev Plant Biol. 2020;71:403–33. https://doi.org/10.1146/annurev-arplant-050718-100005.

    CAS  Article  PubMed  Google Scholar 

  5. Almeida DM, Oliveira MM, Saibo NJ. Regulation of Na+ and K+ homeostasis in plants: towards improved salt stress tolerance in crop plants. Genet Mol Biol. 2017;40:326–45. https://doi.org/10.1590/1678-4685-GMB-2016-0106.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Hasanuzzaman M, Bhuyan MH, Zulfiqar F, Raza A, Mohsin SM, Mahmud JA, Fujita M, Fotopoulos V. Reactive oxygen species and antioxidant defense in plants under abiotic stress: Revisiting the crucial role of a universal defense regulator. Antioxidants. 2020;9(8):681. https://doi.org/10.3390/antiox9080681.

    CAS  Article  PubMed Central  Google Scholar 

  7. Huang Y, Cao H, Yang L, Chen C, Shabala L, Xiong M, Niu M, Liu J, Zheng Z, Zhou L, Peng Z. Tissue-specific respiratory burst oxidase homolog-dependent H2O2 signaling to the plasma membrane H+-ATPase confers potassium uptake and salinity tolerance in Cucurbitaceae. J Exp Bot. 2019;70(20):5879–93. https://doi.org/10.1093/jxb/erz328.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Wang Z, Zhu Y, Wang L, Liu X, Liu Y, Phillips J, Deng X. A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter. Planta. 2009;230(6):1155–66. https://doi.org/10.1007/s00425-009-1014-3.

    CAS  Article  PubMed  Google Scholar 

  9. Luo X, Li C, He X, Zhang X, Zhu L. ABA signaling is negatively regulated by GbWRKY1 through JAZ1 and ABI1 to affect salt and drought tolerance. Plant Cell Rep. 2020;39(2):181–94. https://doi.org/10.1007/s00299-019-02480-4.

    CAS  Article  PubMed  Google Scholar 

  10. Jiang SC, Mei C, Liang S, Yu YT, Lu K, Wu Z, Wang XF, Zhang DP. Crucial roles of the pentatricopeptide repeat protein SOAR1 in Arabidopsis response to drought, salt and cold stresses. Plant Mol Biol. 2015;88(4):369–85. https://doi.org/10.1007/s11103-015-0327-9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Mei C, Jiang SC, Lu YF, Wu FQ, Yu YT, Liang S, Feng XJ, Portoles Comeras S, Lu K, Wu Z, Wang XF. Arabidopsis pentatricopeptide repeat protein SOAR1 plays a critical role in abscisic acid signalling. J Exp Bot. 2014;65(18):5317–30. https://doi.org/10.1093/jxb/eru293.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Li H, Fu Y, Sun H, Zhang Y, Lan X. Transcriptomic analyses reveal biosynthetic genes related to rosmarinic acid in Dracocephalum tanguticum. Sci Rep. 2017;7(1):1–10. https://doi.org/10.1038/s41598-017-00078-y.

    CAS  Article  Google Scholar 

  13. Sharma A, Shahzad B, Rehman A, Bhardwaj R, Landi M, Zheng B. Response of phenylpropanoid pathway and the role of polyphenols in plants under abiotic stress. Molecules. 2019;24(13):2452. https://doi.org/10.3390/molecules24132452.

    CAS  Article  PubMed Central  Google Scholar 

  14. Zhao C, Nawaz G, Cao Q, Xu T. Melatonin is a potential target for improving horticultural crop resistance to abiotic stress. Sci Hortic. 2022;291: 110560. https://doi.org/10.1016/j.scienta.2021.110560.

    CAS  Article  Google Scholar 

  15. Jahan MS, Guo S, Sun J, Shu S, Wang Y, Abou El-Yazied A, Alabdallah NM, Hikal M, Mohamed MH, Ibrahim MF, Hasan MM. Melatonin-mediated photosynthetic performance of tomato seedlings under high-temperature stress. Plant Physiol Biochem. 2021;167:309–20. https://doi.org/10.1016/j.plaphy.2021.08.002.

    CAS  Article  PubMed  Google Scholar 

  16. Yan F, Zhang J, Li W, Ding Y, Zhong Q, Xu X, Wei H, Li G. Exogenous melatonin alleviates salt stress by improving leaf photosynthesis in rice seedlings. Plant Physiol Biochem. 2021;163:367–75. https://doi.org/10.1016/j.plaphy.2021.03.058.

    CAS  Article  PubMed  Google Scholar 

  17. Altaf MA, Shahid R, Ren MX, Naz S, Altaf MM, Khan LU, Tiwari RK, Lal MK, Shahid MA, Kumar R, Nawaz MA. Melatonin Improves Drought Stress Tolerance of Tomato by Modulating Plant Growth, Root Architecture, Photosynthesis, and Antioxidant Defense System. Antioxidants. 2022;11(2):309. https://doi.org/10.3390/antiox11020309.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Hu Z, Fan J, Xie Y, Amombo E, Liu A, Gitau MM, Khaldun AB, Chen L, Fu J. Comparative photosynthetic and metabolic analyses reveal mechanism of improved cold stress tolerance in bermudagrass by exogenous melatonin. Plant Physiol Biochem. 2016;100:94–104. https://doi.org/10.1016/j.plaphy.2016.01.008.

    CAS  Article  PubMed  Google Scholar 

  19. Zhang HM, Zhang Y. Melatonin: a well-documented antioxidant with conditional pro-oxidant actions. J Pineal Res. 2014;57(2):131–46. https://doi.org/10.1111/jpi.12162.

    CAS  Article  PubMed  Google Scholar 

  20. Reiter RJ, Mayo JC, Tan DX, Sainz RM, Alatorre-Jimenez M, Qin L. Melatonin as an antioxidant: under promises but over delivers. J Pineal Res. 2016;61(3):253–78. https://doi.org/10.1111/jpi.12360.

    CAS  Article  PubMed  Google Scholar 

  21. Arnao MB, Hernández-Ruiz J. Functions of melatonin in plants: a review. J Pineal Res. 2015;59(2):133–50. https://doi.org/10.1111/jpi.12253.

    CAS  Article  PubMed  Google Scholar 

  22. Agathokleous E, Zhou B, Xu J, Ioannou A, Feng Z, Saitanis CJ, Frei M, Calabrese EJ, Fotopoulos V. Exogenous application of melatonin to plants, algae, and harvested products to sustain agricultural productivity and enhance nutritional and nutraceutical value: A meta-analysis. Environ Res. 2021;200: 111746. https://doi.org/10.1016/j.envres.2021.111746.

    CAS  Article  PubMed  Google Scholar 

  23. Zhang HJ, Zhang NA, Yang RC, Wang L, Sun QQ, Li DB, Cao YY, Weeda S, Zhao B, Ren S, Guo YD. Melatonin promotes seed germination under high salinity by regulating antioxidant systems, ABA and GA 4 interaction in cucumber (Cucumis sativus L.). J Pineal Res. 2014;57(3):269–79. https://doi.org/10.1111/jpi.12167.

    CAS  Article  PubMed  Google Scholar 

  24. Wang Q, An B, Wei Y, Reiter RJ, Shi H, Luo H, He C. Melatonin regulates root meristem by repressing auxin synthesis and polar auxin transport in Arabidopsis. Front Plant Sci. 2016;7:1882. https://doi.org/10.3389/fpls.2016.01882.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Meng JF, Xu TF, Song CZ, Yu Y, Hu F, Zhang L, Zhang ZW, Xi ZM. Melatonin treatment of pre-veraison grape berries to increase size and synchronicity of berries and modify wine aroma components. Food Chem. 2015;185:127–34. https://doi.org/10.1016/j.foodchem.2015.03.140.

    CAS  Article  PubMed  Google Scholar 

  26. Xu T, Chen Y, Kang H. Melatonin is a potential target for improving post-harvest preservation of fruits and vegetables. Frontiers in Plant Science. 2019;10:1388. https://doi.org/10.3389/fpls.2019.01388.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Zhang N, Sun Q, Li H, Li X, Cao Y, Zhang H, Li S, Zhang L, Qi Y, Ren S, Zhao B. Melatonin improved anthocyanin accumulation by regulating gene expressions and resulted in high reactive oxygen species scavenging capacity in cabbage. Front Plant Sci. 2016;7:197. https://doi.org/10.3389/fpls.2016.00197.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Saeed F, Afzaal M, Niaz B, Arshad MU, Tufail T, Hussain MB, Javed A. Bitter melon (Momordica charantia): a natural healthy vegetable. Int J Food Prop. 2018;21(1):1270–90. https://doi.org/10.1080/10942912.2018.1446023.

    CAS  Article  Google Scholar 

  29. Grover JK, Yadav SP. Pharmacological actions and potential uses of Momordica charantia: a review. J Ethnopharmacol. 2004;93(1):123–32. https://doi.org/10.1016/j.jep.2004.03.035.

    CAS  Article  PubMed  Google Scholar 

  30. Jia S, Shen M, Zhang F, Xie J. Recent advances in Momordica charantia: functional components and biological activities. Int J Mol Sci. 2017;18(12):2555. https://doi.org/10.3390/ijms18122555.

    CAS  Article  PubMed Central  Google Scholar 

  31. Li C, Wang P, Wei Z, Liang D, Liu C, Yin L, Jia D, Fu M, Ma F. The mitigation effects of exogenous melatonin on salinity-induced stress in Malus hupehensis. J Pineal Res. 2012;53(3):298–306. https://doi.org/10.1111/j.1600-079X.2012.00999.x.

    CAS  Article  PubMed  Google Scholar 

  32. Back K. Melatonin metabolism, signaling and possible roles in plants. Plant J. 2021;105(2):376–91. https://doi.org/10.1111/tpj.14915.

    CAS  Article  PubMed  Google Scholar 

  33. Yin Z, Lu J, Meng S, Liu Y, Mostafa I, Qi M, Li T. Exogenous melatonin improves salt tolerance in tomato by regulating photosynthetic electron flux and the ascorbate–glutathione cycle. J Plant Interact. 2019;14(1):453–63. https://doi.org/10.1080/17429145.2019.1645895.

    CAS  Article  Google Scholar 

  34. Nanjo T, Kobayashi M, Yoshiba Y, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K. Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. Febs Lett. 1999;461(3):205–10. https://doi.org/10.1016/S0014-5793(99)01451-9.

    CAS  Article  PubMed  Google Scholar 

  35. Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water-stress studies. Plant Soil. 1973;39(1):205–7. https://doi.org/10.1007/BF00018060.

    CAS  Article  Google Scholar 

  36. Sairam RK, Srivastava GC. Changes in antioxidant activity in sub-cellular fractions of tolerant and susceptible wheat genotypes in response to long term salt stress. Plant Sci. 2002;162(6):897–904. https://doi.org/10.1016/S0168-9452(02)00037-7.

    CAS  Article  Google Scholar 

  37. Heath RL, Packer L. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys. 1968;125(1):189–98. https://doi.org/10.1016/0003-9861(68)90654-1.

    CAS  Article  PubMed  Google Scholar 

  38. Allen LA. 13. Studies in the ripening of cheddar cheese1 I. amethod of following the course of ripening of cheddar cheese. J Dairy Res. 1930;2(1):38–67. https://doi.org/10.1017/S0022029900000157.

    CAS  Article  Google Scholar 

  39. Hemeda HM, Klein BP. Effects of naturally occurring antioxidants on peroxidase activity of vegetable extracts. J Food Sci. 1990;55(1):184–5. https://doi.org/10.1111/j.1365-2621.1990.tb06048.x.

    CAS  Article  Google Scholar 

  40. Giannopolitis CN, Ries SK. Superoxide dismutases: I. Occurrence in higher plants Plant Physiol. 1977;59(2):309–14. https://doi.org/10.1104/pp.59.2.309.

    CAS  Article  PubMed  Google Scholar 

  41. Lazar D, Murch SJ, Beilby MJ, Al KS. Exogenous melatonin affects photosynthesis in characeae Chara australis. Plant Signal Behav. 2013;8(3):e23279. https://doi.org/10.4161/psb.23279.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Wu Y, Jin X, Liao W, Hu L, Dawuda MM, Zhao X, Tang Z, Gong T, Yu J. 5-Aminolevulinic acid (ALA) alleviated salinity stress in cucumber seedlings by enhancing chlorophyll synthesis pathway. Front Plant Sci. 2018;9:635. https://doi.org/10.3389/fpls.2018.00635.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Gohari G, Mohammadi A, Akbari A, Panahirad S, Dadpour MR, Fotopoulos V, Kimura S. Titanium dioxide nanoparticles (TiO2 NPs) promote growth and ameliorate salinity stress effects on essential oil profile and biochemical attributes of Dracocephalum moldavica. Sci Rep. 2020;10(1):1–14. https://doi.org/10.1038/s41598-020-57794-1.

    CAS  Article  Google Scholar 

  44. Zhang T, Shi Z, Zhang X, Zheng S, Wang J, Mo J. Alleviating effects of exogenous melatonin on salt stress in cucumber. Sci Hortic. 2020;262:109070. https://doi.org/10.1016/j.scienta.2019.109070.

    CAS  Article  Google Scholar 

  45. ElSayed AI, Rafudeen MS, Gomaa AM, Hasanuzzaman M. Exogenous melatonin enhances the reactive oxygen species metabolism, antioxidant defense-related gene expression, and photosynthetic capacity of Phaseolus vulgaris L. to confer salt stress tolerance. Physiol Plant. 2021;173(4):1369–81. https://doi.org/10.1111/ppl.13372.

    CAS  Article  PubMed  Google Scholar 

  46. Yang H, Dai L, Wei Y, Deng Z, Li D. Melatonin enhances salt stress tolerance in rubber tree (Hevea brasiliensis) seedlings. Ind Crops Prod. 2020;145:111990. https://doi.org/10.1016/j.indcrop.2019.111990.

    CAS  Article  Google Scholar 

  47. Xie Q, Luo H, Cheng X, Li Z, Lu W, He Z, Zhou X. Effects of melatonin on growth, non-photochemical quenching and related components in tomato seedlings under calcium nitrate stress. In IOP Conference Series: Earth and Environmental Science. 2021;vol.621, no.1, p.012104. IOP Publishing. https://doi.org/10.1088/1755-1315/621/1/012104

  48. Sheikhalipour M, Esmaielpour B, Behnamian M, Gohari G, Giglou MT, Vachova P, Rastogi A, Brestic M, Skalicky M. Chitosan–selenium nanoparticle (Cs–Se NP) foliar spray alleviates salt stress in bitter melon. Nanomaterials. 2021;11(3):684. https://doi.org/10.3390/nano11030684.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. Ferchichi S, Hessini K, Dell’Aversana E, D’Amelia L, Woodrow P, Ciarmiello LF, Fuggi A, Carillo P. Hordeum vulgare and Hordeum maritimum respond to extended salinity stress displaying different temporal accumulation pattern of metabolites. Funct Plant Biol. 2018;45(11):1096–109. https://doi.org/10.1071/FP18046.

    CAS  Article  PubMed  Google Scholar 

  50. Chen L, Liu L, Lu B, Ma T, Jiang D, Li J, Zhang K, Sun H, Zhang Y, Bai Z, Li C. Exogenous melatonin promotes seed germination and osmotic regulation under salt stress in cotton (Gossypium hirsutum L.). Plos One. 2020;15(1):e0228241. https://doi.org/10.1371/journal.pone.0228241.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Yan F, Wei H, Ding Y, Li W, Liu Z, Chen L, Tang S, Ding C, Jiang Y, Li G. Melatonin regulates antioxidant strategy in response to continuous salt stress in rice seedlings. Plant Physiol Biochem. 2021;165:239–50. https://doi.org/10.1016/j.plaphy.2021.05.003.

    CAS  Article  PubMed  Google Scholar 

  52. Siddiqui MH, Alamri S, Al-Khaishany MY, Khan MN, Al-Amri A, Ali HM, Alaraidh IA, Alsahli AA. Exogenous melatonin counteracts NaCl-induced damage by regulating the antioxidant system, proline and carbohydrates metabolism in tomato seedlings. Int J Mol Sci. 2019;20(2):353. https://doi.org/10.3390/ijms20020353.

    CAS  Article  PubMed Central  Google Scholar 

  53. Chen Y, Zhang Y, Nawaz G, Zhao C, Li Y, Dong T, Zhu M, Du X, Zhang L, Li Z, Xu T. Exogenous melatonin attenuates post-harvest decay by increasing antioxidant activity in wax apple (Syzygium samarangense). Front Plant Sci. 2020;1411. https://doi.org/10.3389/fpls.2020.569779

  54. Li Y, Zhang L, Zhang L, Nawaz G, Zhao C, Zhang J, Cao Q, Dong T, Xu T. Exogenous melatonin alleviates browning of fresh-cut sweetpotato by enhancing anti-oxidative process. Sci Hortic. 2022;297:110937. https://doi.org/10.1016/j.scienta.2022.110937.

    CAS  Article  Google Scholar 

  55. Shabala S, Cuin TA. Potassium transport and plant salt tolerance. Physiol Plant. 2008;133(4):651–69. https://doi.org/10.1111/j.1399-3054.2007.01008.x.

    CAS  Article  PubMed  Google Scholar 

  56. Teakle NL, Tyerman SD. Mechanisms of Cl- transport contributing to salt tolerance. Plant Cell Environ. 2010;33(4):566–89. https://doi.org/10.1111/j.1365-3040.2009.02060.x.

    CAS  Article  PubMed  Google Scholar 

  57. Kurusu T, Kuchitsu K, Tada Y. Plant signaling networks involving Ca2+ and Rboh/Nox-mediated ROS production under salinity stress. Front Plant Sci. 2015;6:427. https://doi.org/10.3389/fpls.2015.00427.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Tuna AL, Kaya C, Ashraf M, Altunlu H, Yokas I, Yagmur B. The effects of calcium sulphate on growth, membrane stability and nutrient uptake of tomato plants grown under salt stress. Environ Exp Bot. 2007;59(2):173–8. https://doi.org/10.1016/j.envexpbot.2005.12.007.

    CAS  Article  Google Scholar 

  59. Neves-Piestun BG, Bernstein N. Salinity-induced inhibition of leaf elongation in maize is not mediated by changes in cell wall acidification capacity. Plant Physiol. 2001;125(3):1419–28. https://doi.org/10.1104/pp.125.3.1419.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  60. Zhao MG, Tian QY, Zhang WH. Nitric oxide synthase-dependent nitric oxide production is associated with salt tolerance in Arabidopsis. Plant Physiol. 2007;144(1):206–17. https://doi.org/10.1104/pp.107.096842.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  61. Cheong YH, Kim KN, Pandey GK, Gupta R, Grant JJ, Luan S. CBL1, a calcium sensor that differentially regulates salt, drought, and cold responses in Arabidopsis. Plant Cell. 2003;15(8):1833–45. https://doi.org/10.1105/tpc.012393.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  62. Gong M, van der Luit AH, Knight MR, Trewavas AJ. Heat-shock-induced changes in intracellular Ca2+ level in tobacco seedlings in relation to thermotolerance. Plant Physiol. 1998;116(1):429–37. https://doi.org/10.1104/pp.116.1.429.

    CAS  Article  PubMed Central  Google Scholar 

  63. Tattini M, Traversi ML. Responses to changes in Ca2+ supply in two Mediterranean evergreens, Phillyrea latifolia and Pistacia lentiscus, during salinity stress and subsequent relief. Ann Bot. 2008;102(4):609–22. https://doi.org/10.1093/aob/mcn134.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  64. Halfter U, Ishitani M, Zhu JK. The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3. PNAS. 2000;97(7):3735–40. https://doi.org/10.1073/pnas.97.7.3735.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  65. Mahajan S, Pandey GK, Tuteja N. Calcium-and salt-stress signaling in plants: shedding light on SOS pathway. Arch Biochem Biophys. 2008;471(2):146–58. https://doi.org/10.1016/j.abb.2008.01.010.

    CAS  Article  PubMed  Google Scholar 

  66. Cheng NH, Pittman JK, Zhu JK, Hirschi KD. The protein kinase SOS2 activates the Arabidopsis H+/Ca2+ antiporter CAX1 to integrate calcium transport and salt tolerance. Int J Biol Chem. 2004;279(4):2922–6. https://doi.org/10.1074/jbc.M309084200.

    CAS  Article  Google Scholar 

  67. Raghothama KG, Karthikeyan AS. Phosphate acquisition. Plant Soil. 2005;274(1):37–49. https://doi.org/10.1007/s11104-004-2005-6.

    CAS  Article  Google Scholar 

  68. Zribi OT, Barhoumi Z, Kouas S, Ghandour M, Slama I, Abdelly C. Insights into the physiological responses of the facultative halophyte Aeluropus littoralis to the combined effects of salinity and phosphorus availability. J Plant Physiol. 2015;189:1–10. https://doi.org/10.1016/j.jplph.2015.08.007.

    CAS  Article  Google Scholar 

  69. Yu Y, Wang A, Li X, Kou M, Wang W, Chen X, Xu T, Zhu M, Ma D, Li Z, Sun J. Melatonin-stimulated triacylglycerol breakdown and energy turnover under salinity stress contributes to the maintenance of plasma membrane H+–ATPase activity and K+/Na+ homeostasis in sweet potato. Front Plant Sci. 2018;9:256. https://doi.org/10.1111/jpi.12167.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  70. Liu J, Shabala S, Zhang J, Ma G, Chen D, Shabala L, Zeng F, Chen ZH, Zhou M, Venkataraman G, Zhao Q. Melatonin improves rice salinity stress tolerance by NADPH oxidase-dependent control of the plasma membrane K+ transporters and K+ homeostasis. Plant Cell Environ. 2020;43(11):2591–605. https://doi.org/10.1111/pce.13759.

    CAS  Article  PubMed  Google Scholar 

  71. Zhang Y, Fan Y, Rui C, Zhang H, Xu N, Dai M, Chen X, Lu X, Wang D, Wang J, Wang J. Melatonin improves cotton salt tolerance by regulating ROS scavenging system and Ca2+ signal transduction. Front Plant Sci. 2021;12:1239. https://doi.org/10.3389/fpls.2021.693690.

    Article  Google Scholar 

  72. Iranbakhsh A, OraghiArdebili Z, Molaei H, OraghiArdebili N, Amini M. Cold plasma up-regulated expressions of WRKY1 transcription factor and genes involved in biosynthesis of cannabinoids in Hemp (Cannabis sativa L.). Plasma Chem Plasma Process. 2020;40(2):527–37. https://doi.org/10.1007/s11090-020-10058-2.

    CAS  Article  Google Scholar 

  73. Marchive C, Léon C, Kappel C, Coutos-Thévenot P, Corio-Costet MF, Delrot S, Lauvergeat V. Over-expression of VvWRKY1 in grapevines induces expression of jasmonic acid pathway-related genes and confers higher tolerance to the downy mildew. PLoS ONE. 2013;8(1):e54185. https://doi.org/10.1371/journal.pone.0054185.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  74. Bakshi M, Oelmüller R. WRKY transcription factors: Jack of many trades in plants. Plant Signal Behav. 2014;9(2):e27700. https://doi.org/10.4161/psb.27700.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  75. Weeda S, Zhang N, Zhao X, Ndip G, Guo Y, Buck GA, Fu C, Ren S. Arabidopsis transcriptome analysis reveals key roles of melatonin in plant defense systems. PLoS ONE. 2014;9(3):e93462. https://doi.org/10.1371/journal.pone.0093462.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  76. Zhang N, Zhang HJ, Zhao B, Sun QQ, Cao YY, Li R, Wu XX, Weeda S, Li L, Ren S, Reiter RJ. The RNA-seq approach to discriminate gene expression profiles in response to melatonin on cucumber lateral root formation. J Pineal Res. 2014;56(1):39–50. https://doi.org/10.1111/jpi.12095.

    CAS  Article  PubMed  Google Scholar 

  77. Shi H, Jiang C, Ye T, Tan DX, Reiter RJ, Zhang H, Liu R, Chan Z. Comparative physiological, metabolomic, and transcriptomic analyses reveal mechanisms of improved abiotic stress resistance in bermudagrass [Cynodon dactylon (L.) Pers.] by exogenous melatonin. J Exp Bot. 2015;66(3):681–94. https://doi.org/10.1093/jxb/eru373.

    CAS  Article  PubMed  Google Scholar 

  78. Ma Y, Zhang S, Bi C, Mei C, Jiang SC, Wang XF, Lu ZJ, Zhang DP. Arabidopsis exoribonuclease USB1 interacts with the PPR-domain protein SOAR1 to negatively regulate abscisic acid signaling. J Exp Bot. 2020;71(19):5837–51. https://doi.org/10.1093/jxb/eraa315.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  79. Bi C, Ma Y, Jiang SC, Mei C, Wang XF, Zhang DP. Arabidopsis translation initiation factors eIF iso4G1/2 link repression of mRNA cap-binding complex eIF iso4F assembly with RNA-binding protein SOAR 1-mediated ABA signaling. New Phytol. 2019;223(3):1388–406. https://doi.org/10.1111/nph.15880.

    CAS  Article  PubMed  Google Scholar 

  80. Torres MA, Jones JD, Dangl JL. Pathogen-induced, NADPH oxidase–derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana. Nat Genet. 2005;37(10):1130–4. https://doi.org/10.1038/ng1639.

    CAS  Article  PubMed  Google Scholar 

  81. Suzuki N, Koussevitzky SH, Mittler RO, Miller GAD. ROS and redox signaling in the response of plants to abiotic stress. Plant Cell Environ. 2012;35(2):259–70. https://doi.org/10.1111/j.1365-3040.2011.02336.x.

    CAS  Article  PubMed  Google Scholar 

  82. Kaye Y, Golani Y, Singer Y, Leshem Y, Cohen G, Ercetin M, Gillaspy G, Levine A. Inositol polyphosphate 5-phosphatase7 regulates the production of reactive oxygen species and salt tolerance in Arabidopsis. Plant Physiol. 2011;157(1):229–41. https://doi.org/10.1104/pp.111.176883.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  83. Rejeb KB, Benzarti M, Debez A, Bailly C, Savouré A, Abdelly C. NADPH oxidase-dependent H2O2 production is required for salt-induced antioxidant defense in Arabidopsis thaliana. J Plant Physiol. 2015;174:5–15. https://doi.org/10.1016/j.jplph.2014.08.022.

    CAS  Article  PubMed  Google Scholar 

  84. Chen Z, Xie Y, Gu Q, Zhao G, Zhang Y, Cui W, Xu S, Wang R, Shen W. The AtrbohF-dependent regulation of ROS signaling is required for melatonin-induced salinity tolerance in Arabidopsis. Free Radic Biol Med. 2017;108:465–77. https://doi.org/10.1016/j.freeradbiomed.2017.04.009.

    CAS  Article  PubMed  Google Scholar 

  85. Sur S, Ray RB. Bitter melon (Momordica charantia), a nutraceutical approach for cancer prevention and therapy. Cancers. 2020;12(8):2064. https://doi.org/10.3390/cancers12082064.

    CAS  Article  PubMed Central  Google Scholar 

  86. Liang D, Shen Y, Ni Z, Wang Q, Lei Z, Xu N, Deng Q, Lin L, Wang J, Lv X, Xia H. Exogenous melatonin application delays senescence of kiwifruit leaves by regulating the antioxidant capacity and biosynthesis of flavonoids. Front Plant Sci. 2018;9:426. https://doi.org/10.3389/fpls.2018.00426.

    Article  PubMed  PubMed Central  Google Scholar 

  87. Vafadar F, Amooaghaie R, Ehsanzadeh P, Ghanadian M, Talebi M, Ghanati F. Melatonin and calcium modulate the production of rosmarinic acid, luteolin, and apigenin in Dracocephalum kotschyi under salinity stress. Phytochemistry. 2020;177:112422. https://doi.org/10.1016/j.phytochem.2020.112422.

    CAS  Article  PubMed  Google Scholar 

  88. Xu L, Yue Q, Bian FE, Sun H, Zhai H, Yao Y. Melatonin enhances phenolics accumulation partially via ethylene signaling and resulted in high antioxidant capacity in grape berries. Front Plant Sci. 2017;8:1426. https://doi.org/10.3389/fpls.2017.01426.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We would like to thank University of Mohaghegh Ardebili, Ardebil, Iran and University of Tabriz, Iran for their support during this project.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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All authors have contributed to carry out this research. G.G., B.E. and S.A.M., supervisor and designed the experimental setup. M.S. and E.Z., performed greenhouse experiments, biochemical and gene expression analysis. M.S., M.N., M.K.B. and M.K., analyzed data and results, M.S., E.Z., S.A. and G.G., wrote the first draft of manuscript while G.G., M.K. and V.F., wrote and edited the final version of manuscript. All authors read and approved the final manuscript.

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Correspondence to Gholamreza Gohari.

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Sheikhalipour, M., Mohammadi, S.A., Esmaielpour, B. et al. Exogenous melatonin increases salt tolerance in bitter melon by regulating ionic balance, antioxidant system and secondary metabolism-related genes. BMC Plant Biol 22, 380 (2022). https://doi.org/10.1186/s12870-022-03728-0

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  • DOI: https://doi.org/10.1186/s12870-022-03728-0

Keywords

  • Momordica charantia L.
  • Abiotic stress
  • Salinity
  • Defense transcripts