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Chemical and Biological Technologies in Agriculture
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Exogenous melatonin strengthens saline-alkali stress tolerance in apple rootstock M9-T337 seedlings by initiating a variety of physiological and biochemical pathways

Chemical and Biological Technologies in Agriculture volume 11, Article number: 58 (2024) Cite this article

Abstract

Melatonin (MT) is an important plant growth regulator that significantly regulates the growth and development of plants. Previous studies confirmed the effectiveness of MT in improving plant stress tolerance. In this study, annual M9-T337 seedlings were selected as subjects, and five treatments were applied: control (CK), in which only half the concentration of Hoagland was applied; Saline-alkaline stress treatment (SA, 100 mmol·L−1 saline-alkaline solution); melatonin treatment (MT, CK + 200 μmol L−1 exogenous MT); Saline-alkaline + melatonin treatment (MS, SA + 200 μmol L−1 exogenous MT); and saline-alkaline stress + melatonin + inhibitor treatment (HS, additional 100 μmol L−1 p-CPA treatment to MS). The results showed that saline-alkaline stress negatively affected the growth of M9-T337 seedlings by reducing photosynthetic capacity, increasing Na+, promoting reactive oxygen species such as H2O2, and changing the osmotic content and antioxidant system. However, the application of exogenous MT effectively alleviated saline-alkaline damage and significantly promoted the growth of M9-T337 seedlings. It significantly increased plant height, diameter, root length, root surface area, volume and activity. Furthermore, MT alleviated osmotic stress by accumulating proline, soluble sugars, soluble proteins and starch. MT improved photosynthetic capacity by delaying chlorophyll degradation and regulating gas exchange parameters as well as fluorescence parameters in leaves. Additionally, MT reduced the Na+/K+ ratio to reduce ion toxicity by upregulating the expression of Na+ transporter genes (MhCAX5, MhCHX15, MhSOS1, and MhALT1) and downregulating the expression of K+ transporter genes (MhSKOR and MhNHX4). In addition, MT can increase antioxidant enzyme activity (superoxide dismutase (SOD), peroxidase(POD), catalase (CAT), ascorbic acid oxidase (AAO), ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR)) in the ASA-GSH cycle and increase ascorbic acid (AsA), reduced glutathione (GSH) and oxidized glutathione (GSSG) levels to counteract the accumulation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and Superoxide anion free radicals (O2−), reducing oxidative damage. Exogenous MT promotes M9-T337 seedlings growth under saline-alkaline stress by responding synergistically with auxin (IAA), gibberellin (GA3) and zeatin (ZT) to saline-alkaline stress. Our results confirm that MT has the potential to alleviate Saline-alkaline stress by promoting root growth, increasing biomass accumulation and photosynthetic capacity, strengthening the antioxidant defense system, maintaining ionic balance, the ascorbate–glutathione cycle and the Osmoregulation facilitates and regulates endogenous hormone levels in M9-T337 seedlings.

Graphical Abstract

Introduction

As a global environmental problem, soil salinization is a major factor hindering the sustainable development of agriculture [1]. With the intensification of the greenhouse effect and global warming, soil drought occurs, which leads to increased soil salinity [2]. Studies have shown that about 950 million hectares are affected by salinization worldwide, with China's saline land area being about 9.9 × 107 hectares, mainly distributing in the northwestern inland and eastern coastal areas [3]. However, apples, as an important crop in China, are subject to severe soil salinization in their main growing regions, which has a significant impact on the growth and development [4]. Therefore, it is crucial to improve the resistance of apple trees to saline-alkaline conditions to ensure sustainable development in the apple industry.

Saline-alkaline stress is one of the most important abiotic stress factors, and its damage to plants is mainly reflected in four aspects: osmotic stress, ion toxicity, high pH damage and reactive oxygen stress [5]. Osmotic stress mainly reflects that saline-alkaline stress increases the osmotic pressure of the environment of plant roots, so that the water potential of the environment of roots decreases, resulting in water leakage in plants [6]. The main manifestation of ion toxicity is that a large amount of Na+ accumulates in plants due to soil salinization, which greatly reduces the uptake of K+ ions by plants, resulting in a series of toxicities in plants, including destroying the cell membrane structure, inhibiting the synthesis of related enzymes, weakening photosynthesis and hindering signal transmission in vivo [7]. The harm of high pH is mainly reflected in the increase of the rhizosphere environment pH in plants, thereby damaging the root environment and reducing the root vitality. At the same time, there is also precipitation of a large amount of metal ions such as Ca2+ and Mg2+ in the soil, causing soil compaction, which ultimately leads to a reduction in plant absorption of Ca, Mg and other mineral nutrients, which affects the normal growth and development of plants impaired [6]. Active oxygen stress is mainly reflected in the accumulation of a large amount of ROS in plants under saline-alkaline stress, which aggravates the degree of lipid peroxidation of plant cell membrane and produces a large amount of the toxic substance malonaldehyde (MDA), ultimately leading to the destruction of plant cell membrane structure and protein synthesis, as well as the disruption of the electron transport chain in the photosynthetic respiratory pathway, which affects plant growth and development [8]. Under saline-alkaline stress, plants also alleviate salt-base damage through physiological and molecular regulatory mechanisms. The physiological mechanism is mainly manifested: the plant itself accelerates the synthesis of organic substances such as proline, betaine and polyols [9], increases the degree of Na+ efflux and Na+ compartmentalization, and promotes absorption Ca2+ [10], the secretion of organic acids in plants [11], the rapid response of enzyme protection systems and non-enzyme protection systems and accelerates the synthesis of endogenous hormones such as auxin (IAA), cytokinin (ABA) and gibberellin (GA3) in plants [12, 13]. The molecular mechanism is mainly manifested: plants first transmit salt-base stress signals through the ABA pathway, the protein kinase pathway and the SOS pathway, and then the transcription factors are accepted upstream to transmit saline-alkaline stress signals and regulate the expression of related salt-base tolerance genes downstream [14, 15], including the induction of plant osmoregulation, ion transport, antioxidants and other related genes [16,17,18]. Studies have found that the application of exogenous plant growth regulators under stress can improve plant resistance [19,20,21,22]. Melatonin, a hormone-like substance widely distributed in higher plants, plays an important role in alleviating abiotic stress. Studies have shown that the application of exogenous melatonin under drought conditions [23], saline-alkaline conditions [24] and low temperatures [25] is effective in promoting seed germination and dry matter accumulation in plants, alleviating leaf senescence and increasing chlorophyll content of the leaves. At the same time, it can also promote the synthesis of amino acids and osmotic regulatory substances in plants. Application of exogenous melatonin under manganese stress significantly increased MDA and hydrogen peroxide (H2O2) contents in tobacco seedlings, thereby increasing their antioxidant capacity [26]. Exogenous melatonin sprayed under high temperature stress can significantly promote the synthesis of endogenous hormones such as auxins, cytokinins and abscisic acid in cherries, thereby increasing their heat resistance [27]. Appropriate concentrations of melatonin can also effectively increase the content of photosynthetic pigments, osmotically regulating substances and antioxidant enzyme activity in rapeseed seedlings under salt stress, thereby alleviating the damage to seedlings caused by saline-alkaline stress [28]. In summary, the application of exogenous MT under various stresses has been reported in pepper [29], tomato [30], potato [31] and other plants. However, there are few reports on the effect of exogenous MT on the growth of apple plants under Saline-alkaline stress and its mechanism. Therefore, there is an urgent need to investigate whether it has the same regulatory mechanism as apple rootstocks.

Related studies have shown that exogenous MT treatment can reduce the adverse effects of abiotic stress on plants, indicating that these hormones are beneficial to increase agricultural production. However, under saline-alkali stress, the specific physiological mechanism by which exogenous MT enhances the saline-alkali resistance of apple dwarf rootstock M9-T337 (Malus domestica Borkh.) remains unclear. In this study, dwarf apple rootstock M9-T337 seedlings were utilized to investigate the effects of exogenous MT on plant growth under saline-alkaline stress. The regulatory mechanism of MT was elucidated across five key aspects: ion balance, osmotic regulation, antioxidant system, pH balance, and hormone regulation. The goal of this study was to establish a strong theoretical basis for the use of exogenous melatonin in enhancing the saline-alkaline tolerance of plants and also to provide technical support for the cultivation of apple rootstock saline-alkaline tolerance.

Materials and methods

Test materials

In May 2022, M9-T337 virus-free apple seedlings cultivated in tissue culture were procured from Shandong Huinong Horticulture Co., Ltd. The seedlings were initially nurtured for 1 week in the Laboratory of Fruit Tree Stress Physiology at the College of Horticulture, Gansu Agricultural University and then transplanted to a rain shelter at Gansu Agricultural University to facilitate standardized cultivation management procedures. Following acclimation for a period of 10 days, experimental treatments were initiated.

Treatment and experimental design

The experiment was carried out in the rain shelter of Gansu Agricultural University (N 36° 1′–37° 9′, E 106° 21′–107° 44′). Average temperature: 26 °C, relative humidity: 50%. The M9-T337 seedlings were transferred into flowerpots with the same size and nutrient soil weight (11.2 cm × 16.8 cm, containing 1 kg substrate), one seedling per pot. Unified management, regular weeding watering. Based on previous research conducted by the research group [32, 33], preliminary experiments were carried out with corresponding concentrations of MT before the formal experiment. Finally, the exogenous MT concentration were determined by verifying changes in plant phenotype, growth parameters, REC, MDA, and SPAD content (Additional file 4: Table S1). This experiment consisted of five treatments: (1) Control (CK): irrigation with half concentration of Hoagland nutrient solution; (2) Saline-alkaline stress (SA): application of a compound saline-alkaline solution with a concentration of 100 mmol·L−1 based on the control; (3) Melatonin treatment (MT): application of 200 μmol L−1 exogenous MT on a control basis; (4) saline-alkaline stress + melatonin treatment (MS): application of 200 μmol L−1 exogenous MT based on SA; (5) saline-alkaline stress + melatonin + p-CPA treatment (HS): application of 100 μmol L−1 p-CPA based on MS. Three replicates were used for each treatment and six plants were used for each replicate. Seedlings were evenly spaced for each treatment to ensure consistent light exposure. Melatonin was applied every 3 days from 7:00–8:00 p.m until the leaves dripped, for a total of 3 applications. Saline-alkaline stress was carried out by irrigating saline-alkaline solution (100 mmol · L−1NaCl + 100 mmol · L−1NaHCO3) once a day, 500 mL each time, for 3 days. On day 15, the functional leaves of M9-T337 seedlings were harvested for relevant measurements.

Growth parameter

Three seedlings of each treatment were randomly selected for the determination of plant height, stem diameter, leaf area, leaf circumference, fresh weight and dry weight.

Each plant was selected for processing and placed in the root system scanner (STD-4800 company, Canada) to capture images of the root system. Subsequently, the obtained root images were analyzed using WinRHIZO5.0 software (Regent Instruments Inc., Hydro Quebec, Canada) to determine various indices, including length, total root surface area, average root diameter, total root volume, and number of root tips.

The root tip tissue weighing 0.5 g was assessed for root activity using the triphenyltetrazolium chloride (TTC) method by Chu et al. [34]. Each treatment was repeated 3 times.

Photosynthetic measurements

The chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll a + b (Chl a + b), chlorophyll a/b (Chl a/b), and carotenoids contents of the leaves were measured according to the method described by Arnon et al. [35].

As described by Lin et al. [36], the net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO2 concentration (Ci), and stomatal conductance (Gs) of the third leaf from the top to bottom of each plant were quantified using a portable photofluorometric apparatus (CIRAS-2, PP-system, UK). Each treatment was repeated 3 times.

As described by Yuan et al. [37], M9-T337 seedlings were subjected to a 30-min period of darkness using a modulated leaf green fluorescence imager (image-PAM, WALZ, Germany). The third fully developed leaf was selected from the bottom of the plant for determining initial fluorescence (F0), maximum fluorescence (Fm), maximum photochemical quantum yield (Fv/Fm), and photochemical quenching coefficient (qP) following dark adaptation. Each treatment was repeated 3 times.

Antioxidant measurements

Fresh leaves (0.1 g) were homogenized with 1 mL of PBS solution with pH 7.8 on ice and centrifuged for 10 min at 4 °C and 12,000 rpm. The resulting supernatant was used to determine the activity levels of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) using a kit from Beijing Solarbio Technology Co., Ltd. used. Each treatment was repeated three times.

Lipid peroxidation

Relative electrical conductivity (REC) was measured using a DDS-307 conductivity meter, as described by Yuan et al. [38]. Malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) colorimetric method [39]. Each treatment was repeated 3 times.

ROS

Superoxide anion free radicals (O2−) were measured using the hydroxylamine oxidation method [40]. Fresh leaves (1 g) were weighed and placed in a mortar. Phosphate buffer (65 mmol·L−1, 3 mL) was added. Homogenization was carried out by grinding, and the resulting homogenate was transferred to a centrifuge tube. The homogenate was centrifuged at 10,000 r/min for 15 min, and the supernatant was used for measurement. The hydrogen peroxide (H2O2) content was determined using the xylenol orange method [41].

ASA-GSH cycle

Fresh leaves (0.1 g) were homogenized with 1 mL of extract on ice, and the resulting homogenate was placed into a centrifuge tube. The homogenate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was used to determine the levels of reduced glutathione (GSH), reduced ascorbic acid (AsA), oxidized glutathione (GSSG), ascorbic acid oxidase (AAO), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR) using assay kits from Beijing Soleibao Technology Co., Ltd. Each treatment was repeated 3 times.

Osmolytes

Soluble sugars (SS) was measured using the 3,5-dinitrosalicylic acid method, as described by Cut et al. [42]. Soluble proteins (SP) was measured using the Coomassie brilliant blue G-250 staining method, as described by Sevket et al. [43]. Free proline (Pro) was measured using the acid ninhydrin method, as described by Wang et al. [44], and starch (St) was measured using the anthrone colorimetric method, as described by Eros et al. [45].

Endogenous hormone

Endogenous hormone contents were determined according to method of Zheng et al. [46]. The endogenous hormone levels of auxin (IAA), gibberellin (GA3), abscisic acid (ABA), and zeatin (ZT) were determined in 0.5 g leaves using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC‒MS/MS). The chromatographic column was used a Symmetry C18 column (4.6 mm 250 mm, 5 μm) at a temperature of 30 °C, with a mobile phase consisting of a 1:9 ratio of methanol to 0.1% phosphoric acid at a flow rate of 1.0 mL/min. An injection volume of 10 μL was used.

Na+, K+, and Ca2+ contents

The leaf powder samples (5 g) were precisely weighed and dried, followed by digestion with H2SO4-H2O2. The concentrations of Na+, K+, and Ca2+ were determined using an inductively coupled plasma-optical emission spectrometer (Perkin Elmer, Waltham, Massachusetts, USA), following the methodology described by Kuang et al. [47]. Each treatment was repeated 3 times.

qRT‒PCR

Total RNA was extracted from leaves using the Plant RNA Extraction Kit and reverse transcribed with the RT Master Mix Reverse Transcription Kit, both provided by Takara Clontech Biochemicals in Shanghai. The apple actin gene (Accession No. MDPO000774288), was used as an internal reference gene. Nine apple saline-alkaline-responsive genes were selected and detected via fluorescence quantification with qRT-PCR. The reaction system, consisting of 20 μL, included 2 μL each of upstream and downstream primers, 2 μL of cDNA template, 10 μL of Trans Start Top Green qPCR Super Mix, and 6 μL of ddH2O. The qPCR primers were designed and synthesized by Shanghai Sangon Biotech, which were checked for specificity using BLAST in the Apple genome database (https://www.rosaceae.org). See Additional file 4: Table S2 for a list of the primers used. Each treatment was repeated 3 times.

Statistical analysis

Data processing was performed using Microsoft Office Excel 2019, and the graphs were plotted using Origin 2022 software. Statistical analysis was carried out using the IBM SPSS Statistics 25 program (SPSS Inc., Chicago, IL, USA). An analysis of the variance (ANOVA) was used to compare mean values between samplings. The Duncan post-hoc test was used to determine differences between treatments. Significance levels of 95% (P < 0.05) are indicated in figure legends.

Results

Effects of exogenous MT on leaves and roots of M9-T337 seedlings under saline-alkaline stress

After 15 days of saline-alkaline stress treatment, the phenotypic responses of M9-T337 seedlings to each treatment are illustrated in Fig. 1A. Compared to the control treatment (CK), the growth of seedlings subjected to the MT treatment did not exhibit significant advantages. Conversely, under saline-alkaline stress (SA), the leaves of seedlings displayed conspicuous chlorosis and wilting. Upon application of melatonin, the leaves of seedlings in MS treatment regreened and exhibited robust growth. However, the combined treatment of melatonin and p-CPA (HS) did not effectively alleviate the chlorosis and wilting of seedling leaves under saline-alkaline stress, nor did it demonstrate a discernible advantage over SA treatment. The root systems of seedlings under different treatments also exhibited marked differences (Fig. 1B). The total length, average diameter, volume, surface area, tip number, and overall activity of the roots under SA treatment were notably lower. Upon exogenous MT treatment, the damage to the root length, diameter, volume, surface area, tip number, and activity under saline-alkali stress was mitigated. In comparison with SA treatment, the increases were 43.22% in length, 10.71% in diameter, 96.06% in volume, 73.75% in surface area, and 9.70% in tip number, as shown in Additional file 4: Table S3. Nevertheless, the combined treatment of melatonin and p-CPA (HS) could not alleviate the saline-alkaline damage to the root growth of M9-T337 seedlings under saline-alkaline stress, as depicted in Fig. 1B.

Fig. 1
figure 1

Effects of exogenous MT on leaves and roots of M9-T337 seedlings under saline-alkaline stress. CK represents watering with a 1/2 concentration of Hoagland nutrient solution only; SA represents the application of 100 mmol·L−1 composite saline-alkaline solution based on the control; MT represents the spraying of 200 μmol L−1 exogenous MT based on the control; MS represents the spraying of 200 μmol L−1 exogenous MT based on SA; HS represents the spraying of 100 μmol L−1 p-CPA on the basis of MS. A Plant phenotype; B root phenotype. Vertical bars represent the standard errors of the means (three replicates. Data show the mean ± SE (n = 3). Different lowercase letters indicate significant differences between treatments with P < 0.05

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Effects of exogenous MT on growth parameters of M9-T337 seedlings under saline-alkaline stress

The growth rate of M9-T337 seedlings was significantly inhibited under saline-alkaline stress (Fig. 2). The plant height, stem diameter, leaf area, and leaf perimeter were notably lower than those of the control treatment (CK). However, after spraying exogenous MT (MS), these parameters showed significant increases compared to the SA treatment, with increments of 1.11 times for plant height, 1.59 times for stem diameter, 1.43 times for leaf area, and 1.33 times for leaf perimeter, respectively. Conversely, when both MT and p-CPA (HS) were simultaneously applied under saline-alkaline stress conditions, these mentioned parameters were significantly reduced without any advantage over the SA treatment. When exogenous MT was sprayed on top of the CK treatment as a supplement, there was a slight increase in plant height, stem diameter, leaf area and leaf perimeter of M9-T337 seedlings compared to the CK treatment; however, the difference between the two treatments was not statistically significant.

Fig. 2
figure 2

Effects of exogenous MT on growth parameters and biomass accumulation parameters of M9-T337 seedlings under saline-alkaline stress. CK represents watering with a 1/2 concentration of Hoagland nutrient solution only; SA represents the application of 100 mmol·L−1 composite saline-alkaline solution based on the control; MT represents the spraying of 200 μmol L−1 exogenous MT based on the control; MS represents the spraying of 200 μmol L−1 exogenous MT based on SA; HS represents the spraying of 100 μmol L−1 p-CPA on the basis of MS. A Leaf area; B Leaf perimeter; C Plant height; D Stem diameter. Vertical bars represent the standard errors of the means (three replicates. Data show the mean ± SE (n = 3). Different lowercase letters indicate significant differences between treatments with P < 0.05

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In addition, we also analyzed the biomass accumulation of M9-T337 seedlings. As shown in Additional file 1: Fig. S1, the fresh weight and dry weight of both aboveground and underground parts of the seedlings treated with CK and MT were significantly higher than those of the other treatments, but there was no significant difference between them. Saline-alkaline (SA) stress significantly reduced the fresh weight and dry weight of each part of the plant, but exogenous MT application mitigated this reduction caused by saline-alkaline stress. Compared to SA treatment alone, SA + MT (MS) treatment significantly increased the fresh weight and dry weight of each part of the seedlings (16.92% and 15.69% for aboveground; 80.56% and 70.97% for underground). When MT and p-CPA (HS) were simultaneously applied under saline-alkaline stress, the fresh weight and dry weight of both aboveground and underground parts were significantly lower than those in MS treatment, measuring only 1.45 g, 2.11 g, 0.53 g, and 0.35 g, respectively.

Effects of exogenous MT on photosynthesis in leaves of M9-T337 seedlings under saline-alkaline stress

The levels of Chl a (Fig. 3A), Chl b (Fig. 3B), total chlorophyll (Chl a + b) (Fig. 3C), and carotenoids (Car) (Fig. 3D) in the leaves of CK and MT treatments were significantly higher than those of the other treatments, with no significant difference between the two treatments. However, under SA treatment, the photosynthetic pigment content of leaves notably decreased, showing reductions of 36.25%, 53.19%, 41.09%, and 46.27% compared to CK treatment, respectively. On the contrary, under SA + MT (MS) treatment, the photosynthetic pigment content of leaves was significantly higher than that of SA treatment. Conversely, concurrent application of MT and p-CPA (HS) under saline-alkaline stress resulted in a significant decrease in the photosynthetic pigment content in leaves, without any significant difference from the SA treatment.

Fig. 3
figure 3

Effects of exogenous MT on photosynthetic pigment in leaves of M9-T337 seedlings under saline-alkaline stress. CK represents watering with a 1/2 concentration of Hoagland nutrient solution only; SA represents the application of 100 mmol·L−1 composite saline-alkaline solution based on the control; MT represents the spraying of 200 μmol L−1 exogenous MT based on the control; MS represents the spraying of 200 μmol L−1 exogenous MT based on SA; HS represents the spraying of 100 μmol L−1 p-CPA on the basis of MS. A Chl a; B Chl b; C Chl a + b; D Car. Vertical bars represent the standard errors of the means (three replicates. Data show the mean ± SE (n = 3). Different lowercase letters indicate significant differences between treatments with P < 0.05

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Additionally, after 15 days of treatment, the Pn, Tr, Gs, and Ci of leaves under different treatments were measured. There were no significant differences in photosynthetic parameters between the MT and CK treatments. The Pn, Tr, and Gs of leaves under SA treatment were the lowest, with reductions of 30.67%, 45.19%, and 33.07% compared to CK. Conversely, Ci significantly increased, reaching 1.67 times that of CK. However, when exogenous MT was applied under saline-alkaline stress conditions, the Pn, Tr, and Gs values of leaves in the MS treatment were higher by 30.66%, 39.93%, and 49.91% than those in the SA treatment, respectively (Fig. 4ACD). In contrast, the Ci value for leaves was lower by 23.52% compared to that in the SA treatment (Fig. 4B). Furthermore, it was observed that simultaneous application of MT and p-CPA (HS) did not result in significant changes in Pn, Tr, Gs, and Ci values for leaves under saline-alkaline stress conditions when compared with the SA treatment.

Finally, the chlorophyll fluorescence parameters (F0, Fm, Fv/Fm, and qP) of leaves subjected to different treatments were also measured. In comparison with CK treatment, the F0, Fm, Fv/Fm, and qP of the MT treatment did not show significant changes, while the SA treatment notably reduced these parameters in the leaves. However, the F0, Fm, Fv/Fm, and qP of seedling leaves in the MS treatment were significantly higher than those in the SA treatment, albeit still lower than those in the CK treatment (Additional file 2: Fig. S2). Furthermore, there were no significant changes in leaf F0, Fm, Fv/Fm, and qP, when MT and p-CPA (HS) were simultaneously applied under saline-alkaline stress in comparison with the SA treatment.

Fig. 4
figure 4

Effects of exogenous MT on photosynthetic gas parameters in leaves of M9-T337 seedlings under saline-alkaline stress. CK represents watering with a 1/2 concentration of Hoagland nutrient solution only; SA represents the application of 100 mmol·L−1 composite saline-alkaline solution based on the control; MT represents the spraying of 200 μmol L−1 exogenous MT based on the control; MS represents the spraying of 200 μmol L−1 exogenous MT based on SA; HS represents the spraying of 100 μmol L−1 p-CPA on the basis of MS. A Net photosynthetic rate (Pn); B Intercellular CO2 concentration (Ci); C Transpiration (Tr); D Stomatal conductance (Gs). Vertical bars represent the standard errors of the means (three replicates. Data show the mean ± SE (n = 3). Different lowercase letters indicate significant differences between treatments with P < 0.05

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Effect of exogenous MT on membrane lipid peroxidation degree of M9-T337 seedlings under saline-alkali stress

As depicted in Fig. 5A–D, no significant difference in H2O2 content, O2−, REC, and MDA content was observed in leaves treated with MT compared to CK. However, the H2O2 content, O2−, REC, and MDA content in leaves treated with SA significantly increased, reaching 1.38 times, 2.01 times, 1.61 times, and 2.02 times that of CK, respectively. Conversely, the increase in H2O2 content, O2−, REC, and MDA content in leaves was mitigated with the application of exogenous MT under saline-alkaline stress, which showed a reduction of 13.72%, 41.24%, 14.55%, and 26.99% compared to the SA treatment, respectively. However, concurrently applying MT and p-CPA (HS) under saline-alkaline stress did not inhibit the upward trend of H2O2 content, O2−, REC, and MDA content in the leaves and showed no significant difference as compared to the SA treatment.

Fig. 5
figure 5

Effects of exogenous MT on membrane lipid peroxidation degree in leaves of M9-T337 seedlings under saline-alkaline stress. CK represents watering with a 1/2 concentration of Hoagland nutrient solution only; SA represents the application of 100 mmol·L−1 composite saline-alkaline solution based on the control; MT represents the spraying of 200 μmol L−1 exogenous MT based on the control; MS represents the spraying of 200 μmol L−1 exogenous MT based on SA; HS represents the spraying of 100 μmol L−1 p-CPA on the basis of MS. A H202; B O2−; C Relative conductivity (REC); D Malonaldehyde (MDA); E SOD; F POD; G CAT. Vertical bars represent the standard errors of the means (three replicates. Data show the mean ± SE (n = 3). Different lowercase letters indicate significant differences between treatments with P < 0.05

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The enzyme activities of SOD, POD, and CAT in the leaves of M9-T337 seedlings were notably increased under SA treatment, being 2.13 times, 1.36 times, and 1.42 times higher than those in the control group (CK), as demonstrated in Fig. 5E–G. Moreover, when MT was applied under saline-alkali stress, the activities of SOD, POD, and CAT in the leaves of M9-T337 seedlings exhibited further increases of 36.76%, 12.45%, and 34.04% compared to the SA treatment. However, concurrent application of MT and p-CPA (HS) under saline-alkali stress resulted in the inhibition of the increasing trend of SOD, POD, and CAT activities in M9-T337 seedling leaves, showing reductions of 25.05%, 7.69%, and 22.46% compared to the SA treatment, respectively. Additionally, it was also observed that the application of MT on the basis of CK treatment did not lead to significant changes in SOD, POD, and CAT activities in M9-T337 seedling leaves.

Effects of exogenous MT on ASA-GSH cycle of M9-T337 seedlings under saline-alkaline stress

The activities of AAO, APX, GR, and MDHAR were measured to investigate the impact of exogenous MT on the activities of AsA-GSH cycle-related enzymes in seedling leaves, as shown in Fig. 6A–D. After 15 days of saline-alkaline stress, there was a significant decrease in AAO activity in the leaves. In contrast, the activities of APX, GR, and MDHAR were notably increased, showing significant differences compared to CK treatment. Upon the exclusive application of exogenous MT, the activities of AAO, APX, GR, and MDHAR in MT-treated leaves exhibited a marginal increase, with only AAO activity being significantly different from CK. However, following the spraying of exogenous MT under saline-alkali stress, the activities of AAO, APX, GR, and MDHAR in the leaves of MS-treated plants were 2.6 times, 1.89 times, 2.31 times, and 1.65 times compared to those in SA treatment. However, when MT and p-CPA (HS) were simultaneously applied under saline-alkaline stress, the activities of AAO, APX, GR, and MDHAR in the leaves of M9-T337 seedlings notably decreased by 56.41%, 44.07%, 53.73%, and 37.79%, respectively, compared to the SA treatment. These findings suggest that exogenous MT plays a crucial role in enhancing the activity of AsA-GSH cycle-related enzymes in M9-T337 seedlings under saline-alkaline stress.

Fig. 6
figure 6

Effects of exogenous MT on ASA-GSH cycle of M9-T337 seedlings under saline-alkaline stress. CK represents watering with a 1/2 concentration of Hoagland nutrient solution only; SA represents the application of 100 mmol·L−1 composite saline-alkaline solution based on the control; MT represents the spraying of 200 μmol L−1 exogenous MT based on the control; MS represents the spraying of 200 μmol L−1 exogenous MT based on SA; HS represents the spraying of 100 μmol L−1 p-CPA on the basis of MS. A APX; B AAO; C MDHAR; D GR; E: AsA; F GSH; G GSSG. Vertical bars represent the standard errors of the means (three replicates. Data show the mean ± SE (n = 3). Different lowercase letters indicate significant differences between treatments with P < 0.05

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Furthermore, the contents of AsA-GSH cycle-related substances (AsA, GSH, and GSSG) in the leaves of different treatments were also assessed. As shown in Fig. 6E–G, there were no significant differences in the contents of AsA, GSH, and GSSG in the leaves of seedlings treated with MT and CK. However, under saline-alkaline stress (SA) treatment, the contents of AsA and GSH in the leaves of M9-T337 seedlings notably decreased by 57.02% and 34.77%, respectively, compared to those treated with CK. In contrast, the content of GSSG significantly increased in the leaves reaching 2.69 times that of CK treatment. Upon the application of MT under saline-alkaline stress, the contents of AsA and GSH in leaves of MS treatment increased to different degrees, which were 2.98 times and 1.21 times that of SA treatment, respectively, while the GSSG content decreased significantly, being 47.23% lower than that of the SA treatment. However, when MT and p-CPA (HS) were simultaneously applied under saline-alkaline stress, there was an impediment to changes in AsA, GSH, and GSSG contents in the leaves of seedlings as indicated by a significant decrease in AsA and GSH contents and a significant increase in GSSG content.

Effects of exogenous MT on Osmotic adjustment of M9-T337 seedlings under saline-alkaline stress

Figure 7 illustrates that only after applying exogenous MT, the levels of Pro (Fig. 7A), SS (Fig. 7B), SP (Fig. 7C), and St (Fig. 7D) in the leaves of the MT treatment did not significantly differ from those of the CK treatment. Conversely, under saline-alkaline stress (SA) treatment, the contents of Pro, SS, SP, and St in the leaves of M9-T337 seedlings significantly increased to reach 2.15 times, 1.19 times, 1.26 times, and 0.48 times than those of the CK treatment, respectively. Subsequently, after the application of exogenous MT under saline-alkaline stress, the levels of Pro, SS, SP, and St in the leaves of M9-T337 seedlings further increased. The content achieved was 1.99 times higher for Pro, 1.37 times higher for SS, 1.14 times higher for SP, and 1.43 times higher for St compared to the SA treatment. However, when MT and p-CPA were simultaneously applied under saline-alkaline stress, the levels of Pro, SS, SP, and St in the leaves of M9-T337 seedlings were significantly lower than those in the MS treatment but showed no significant difference from the SA treatment.

Fig. 7
figure 7

Effects of exogenous MT on Osmotic adjustment of M9-T337 seedlings under saline-alkaline stress. CK represents watering with a 1/2 concentration of Hoagland nutrient solution only; SA represents the application of 100 mmol·L−1 composite saline-alkaline solution based on the control; MT represents the spraying of 200 μmol L−1 exogenous MT based on the control; MS represents the spraying of 200 μmol L−1 exogenous MT based on SA; HS represents the spraying of 100 μmol L−1 p-CPA on the basis of MS. A Pro; B Soluble sugar; C Soluble protein; D Starch. Vertical bars represent the standard errors of the means (three replicates. Data show the mean ± SE (n = 3). Different lowercase letters indicate significant differences between treatments with P < 0.05

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Effects of exogenous MT on endogenous hormone content in leaves of M9-T337 seedlings under saline-alkaline stress

The impact of exogenous MT on the endogenous hormone content in the leaves of M9-T337 seedlings is demonstrated in Fig. 8. After 15 days of saline-alkaline stress, the levels of ZT, GA3, IAA, and ABA notably increased in the leaves of M9-T337 seedlings compared to the CK treatment. However, there were no discernible differences in the ZT, GA3, IAA, and ABA contents between the CK and MT treatments. Upon the application of exogenous MT under saline-alkaline stress, the ZT, GA3, and IAA contents in the leaves of the MS treatment increased by 64.85%, 46.69%, and 42.90%, respectively, compared to the SA treatment (Fig. 8A–C), while the ABA content decreased by 20.66% (Fig. 8D). Conversely, simultaneous application of MT and p-CPA under saline-alkaline stress resulted in a reversal of changes in ZT, GA3, IAA, and ABA contents in the leaves. Specifically, there was a decrease in ZT, GA3, and IAA contents while the ABA content increased.

Fig. 8
figure 8

Effects of exogenous MT on endogenous hormone content in leaves of M9-T337 seedlings under saline-alkaline stress. CK represents watering with a 1/2 concentration of Hoagland nutrient solution only; SA represents the application of 100 mmol·L−1 composite saline-alkaline solution based on the control; MT represents the spraying of 200 μmol L−1 exogenous MT based on the control; MS represents the spraying of 200 μmol L−1 exogenous MT based on SA; HS represents the spraying of 100 μmol L−1 p-CPA on the basis of MS. A ZT; B GA; C IAA; D ABA. Vertical bars represent the standard errors of the means (three replicates. Data show the mean ± SE (n = 3). Different lowercase letters indicate significant differences between treatments with P < 0.05

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Effects of exogenous melatonin on Na+, K+, and Ca2+ contents in leaves of M9-T337 seedlings under saline-alkaline stress

Figure 9A illustrates that there were no significant differences in the levels of Na+, K+, Ca2+, and Na+/K+ in the leaves between the MT and CK treatments. However, under saline-alkaline stress (SA) treatment, the Ca2+ content in the leaves of M9-T337 seedlings was notably lower than that of the CK treatment. Conversely, compared to the CK treatment, there was a significant increase in the content of Na+ and K+, as well as an increase in the Na+/K+ ratio. When exogenous MT was applied under saline-alkaline stress, the contents of Ca2+ and K+ in the leaves of the MS treatment significantly increased to 1.18 times and 1.67 times compared with those of the SA treatment, respectively. Meanwhile, there was a significant decrease in Na+ content and Na+/K+ ratio by 20.45% and 53.33%, respectively, compared with the SA treatment.

Fig. 9
figure 9

Effects of exogenous melatonin on Na+, K+ and Ca2+ contents in leaves of M9-T337 seedlings under saline-alkaline stress. CK represents watering with a 1/2 concentration of Hoagland nutrient solution only; SA represents the application of 100 mmol·L−1 composite saline-alkaline solution based on the control; MT represents the spraying of 200 μmol L−1 exogenous MT based on the control; MS represents the spraying of 200 μmol L−1 exogenous MT based on SA; HS represents the spraying of 100 μmol L−1 p-CPA on the basis of MS. A Leaf Ca2+; B Leaf Na+; D Leaf K+; C Na+/K+. Vertical bars represent the standard errors of the means (three replicates. Data show the mean ± SE (n = 3). Different lowercase letters indicate significant differences between treatments with P < 0.05

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Effects of exogenous melatonin on saline-alkaline response gene expression in M9-T337 seedlings

As shown in Additional file 3: Fig. S3, the expression levels of leaf Na+ transport genes (MhCAX5, MhSOS, MhALT1, and MhCHX15), K+ transport genes (MhSKOR, MhNHX4), and antioxidant enzyme regulation genes (MhPOD, MhCAT, and MhSOD) in the MT treatment showed no significant change compared to CK. However, under saline-alkaline stress (SA) treatment, the expression levels of the four Na+ transporter genes and MhSKOR were notably down-regulated, while the expression levels of MhNHX4 and three antioxidant enzyme regulatory genes were significantly up-regulated. Remarkably, this phenomenon was reversed by MS treatment. However, the co-application of exogenous MT and p-PCA under saline-alkaline stress led to a state that the ability of MT to regulate the above genes was impeded.

Correlation and principal component analysis of various indexes of M9-T337 seedlings under different treatments

Figure 10A shows the results of a correlation analysis performed on 35 physiological indicators of M9-T337 seedlings after treatment. The results show that the Pn of M9-T337 seedlings has a highly significant positive correlation with Tr, Gs, F0, Fm, Fv/Fm, qP, Chl a, Chl b, Chl a + b, Cal, SP, ST, AsA, GSH and root activities (P < 0.01). Furthermore, it was significantly positively correlated with AAO (P < 0.05), but showed extremely negative correlation with Ci, MDA, REC, H2O2, O2−, GSSG and ABA (P < 0.01). Furthermore, it was negatively correlated with SOD, POD and IAA (P < 0.05).

Fig. 10
figure 10

Correlation and principal component analysis of various indexes of M9-T337 seedlings under different treatments

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To comprehensively evaluate the physiological response of M9-T337 seedlings to exogenous MT under saline-alkaline stress, we performed principal component analysis for 48 indices after treatment. Two principal components with eigenvalues greater than 1 were extracted. The differential contribution rates of the first and second principal components were 75.1% and 20.7%, respectively, and the cumulative variance contribution rate was 95.8%. Comprehensive ranking found that MS treatment was the most effective (Fig. 10B).

Discussion

Upon exposure to saline-alkaline stress, plant roots initially generate stress signals. Through self-regulation, these signals are transmitted to the aboveground sections of the plants, subsequently affecting their regular growth and development [48]. Such stress provokes the degradation of photosynthetic pigments within plant leaves, leading in turn to leaf senescence, shrinkage, and a reduction in photosynthetic capacity [49]. Research indicates that the application of exogenous melatonin (MT) can effectively decelerate the decline in chlorophyll content within plant leaves, thereby mitigating the inhibitory effect of saline-alkaline stress on plant photosynthetic capacity [50,51,52]. In this experiment, the content of Chl a, Chl b, Chl a + b and Car in the leaves of M9-T337 seedlings significantly decreased under saline-alkaline stress, with plant height, stem diameter, dry weight, and fresh weight all significant reductions. This aligns with the findings of Ashraf et al. [53] and He et al. [54], which may be because saline-alkaline stress may induce the increase of soil osmotic pressure, disrupt ion balance in plant cells, impede chlorophyll synthesis, and trigger oxidative stress, impacting photosynthetic pigment content, growth indices, and ultimately leading to diminished photosynthesis, growth, and development of M9-T337 seedlings [55]. Following treatment with exogenous MT, there was a significant increase in biomass accumulation, Chl a, Chl b, Chl a + b, and Car contents in the leaves, along with a considerable rise in root activity and promotion of root development. This enhancement is likely attributable to the ability of exogenous MT to regulate the expression levels of chlorophyll-related genes and promote chlorophyll synthesis, thereby boosting photosynthetic capacity, augmenting biomass accumulation, and stimulating root development, ultimately mitigating the damage to plants caused by saline-alkaline stress [56]. The stability of photosynthetic gas exchange parameters significantly influences plant photosynthesis [57]. Correlation analysis in this experiment revealed a significant positive correlation between Pn and Chl a, Chl b, Chl a + b, and Car, indicating that the decline in photosynthetic pigment content may be the key factor leading to reduced photosynthesis. Previous studies have indicated that both stomatal and non-stomatal constraints may contribute to a decrease in Pn in plant leaves [58]. A reduction in Gs of leaves with a stable or increased Ci in stomata indicates that non-stomatal factors, particularly the diminished photosynthetic assimilation capacity of mesophyll cells, are the primary causes of the decline in photosynthetic rate [59]. The results of this experiment indicate that the reduction in Pn of the leaves is caused by non-stomatal limiting factors, signifying decreased photosynthetic activity of mesophyll cells. This decrease could be attributed to saline-alkaline stress hindering photosynthetic electron transport rate and thylakoid protein synthesis, resulting in decreased stomatal conductance, slow CO2 assimilation, reduced leaf transpiration rate, and photosynthetic rate [60]. Following the application of exogenous MT, Pn, Tr, and Gs of M9-T337 seedlings increased significantly, while Ci decreased markedly, which may be because exogenous MT can inhibit the excessive decline in photosynthetic activity of mesophyll cells, maintain the stability of photosynthetic gas exchange parameters in plants, and enhance the photosynthetic efficiency of plants under stress, which is consistent with the findings of Zhang et al. [61] in cotton. Chlorophyll fluorescence parameters in plants serve as internal indicators of photosynthesis, offering insights into the absorption, transformation, and physiological changes of photosynthetic products in plants. These parameters not only impact the dynamic balance of the carbon cycle, but also play a crucial role in the growth and development of plants [62, 63]. This study uncovered a significant reduction in Fo, Fm, Fv / Fm, and qP in the leaves of M9-T337 seedlings under saline-alkaline stress, suggesting that the potential active sites of PS II in these seedlings were affected, leading to the degradation of PS II receptor-related electron transport proteins, ultimately resulting in decreased light energy utilization and light response inhibition [64,65,66]. Following exogenous MT treatment, this trend has been reversed, which aligns with the findings of Yan et al. [67]. This enhancement may be attributed to the ability of exogenous MT to diminish the relative efficiency of electron transfer and dissipate a significant amount of light intensity as heat, effectively alleviating the damage of saline-alkaline stress to PS II.

Saline-alkaline stress disrupts the equilibrium between photosynthetic electron transport and the Calvin cycle in plants, leading to the transfer of electrons from chloroplasts and mitochondria to oxygen molecules, consequently generating a significant amount of ROS, especially H2O2 and O2−. The accumulation of ROS triggers membrane lipid peroxidation, resulting in cell membrane damage, ultimately leading to cellular injury and possible death [68, 69]. REC and MDA content is used to assess the extent of cell membrane damage [70]. Under saline-alkaline stress, there was a noteworthy increase in H2O2 and O2− levels in the leaves of M9-T337 seedlings, along with a significant rise in REC and MDA levels, mirroring the findings of Weisany et al. [71] and High et al. [72]. Following exogenous MT treatment, this trend was reversed, which was consistent with the outcomes of Liang et al. [73]. This could be attributed to the induction of ROS scavenging genes by exogenous MT to participate in the ROS scavenging process in M9-T337 seedlings under saline-alkaline stress, thereby suppressing membrane lipid peroxidation and preserving the balance of photosynthetic electron transfer and the Calvin cycle [26].

When the concentration of reactive ROS in plants surpasses the normal threshold, the plant's antioxidant system eliminates the excess ROS, with SOD, POD, and CAT playing a crucial role in this process [74, 75]. The findings of this experiment revealed a significant increase in the activities of SOD, POD, and CAT in M9-T337 seedlings under saline-alkaline stress. This elevation may be attributed to the stimulation of the plant's internal defense system (enzymes and non-enzymes) in response to saline-alkaline stress, resulting in heightened Antioxidant enzyme activity in the leaves [76]. Subsequent to exogenous MT treatment, the activities of SOD, POD, and CAT in the leaves of M9-T337 seedlings were further elevated, surpassing those under saline-alkaline stress. This aligns with the results of the study by Chen et al. [77] in maize. This increase is likely due to the exogenous MT regulating the high expression of genes encoding antioxidant enzymes, thereby enhancing the activity of antioxidant enzymes in the leaves, and ultimately timely removing excess ROS [78,79,80].

The H2O2 in plants is predominantly eliminated in the AsA-GSH cycle, which is completed through the combined action of APX, GR, MDHAR, DHAR, AsA, and GSH, repairing the damage caused by free radicals [81]. APX reduces H2O2 to H2O, generating an unstable initial product MDHA, which is subsequently reduced to AsA by MDHAR. Additionally, GSH is oxidized to GSSG, then converted back to GSH by GR through the reduction of coenzyme II, thereby preserving the cellular redox balance [82, 83]. This study observed a significant reduction in AsA and GSH contents in the leaves of M9-T337 seedlings under saline-alkaline stress, alongside a notable increase in GSSG content. After exogenous MT treatment, the contents of AsA, GSH, and GSSG in leaves showed the opposite trend, aligning with the findings of Wu et al. [84]. This could be attributed to the promotion of the conversion of AsA to DHA by exogenous MT and the regulation of the exchange between GSH and GSSG, thus maintaining redox homeostasis and enhancing the saline-alkaline tolerance of M9-T337 seedlings. Furthermore, AAO and APX catalyze the conversion of AsA with H2O2 to form MDHA, which is then reduced to AsA by MDHAR. Simultaneously, GSSG is reduced to GSH by GR [85]. The results of this study demonstrated varying degrees of increase in the activities of APX, MDHAR, and GR in the leaves of M9-T337 seedlings under saline-alkaline stress, while AAO activity decreased significantly. Similar to the findings of Xu et al. [86]. Post exogenous MT treatment, the changes of APX, MDHAR, GR and AAO activities in leaves were reversed, consistent with the results of Wu et al. [84]. This may be due to the regulatory effect of exogenous MT on the expression of AsA-GSH cycle-related genes, thereby modulating the corresponding enzyme activity, fortifying antioxidant defense, and enhancing the antioxidant stress resistance of plants.

Osmotic regulators play a crucial role in alleviating the harmful effects of saline-alkaline stress. Soluble sugar, soluble protein, and proline can enhance the stability of the cell membrane by increasing the relative water content and cytoplasmic osmotic pressure in plants, thus enabling them to resist saline-alkaline stress [87,88,89]. The results of this study showed that under saline-alkaline stress, the levels of proline, soluble sugar, and soluble protein in the leaves of M9-T337 seedlings significantly increased. After exogenous MT treatment, the content of osmotic regulatory substances in the leaves further increased, which is consistent with the findings of Zhang et al. [90] in sugar beet. This could be attributed to the MT regulate the activities of proline synthase, glycolytic enzyme, and glycosidase, providing antioxidant protection. Additionally, it could modulate the equilibrium of plant hormones and bolster cell membrane stability. These alterations consequently facilitate the synthesis and accumulation of osmotic adjustment substances, thus enabling plants to more proficiently resist saline-alkaline stress.

Plant hormones, as important small-molecule osmotic substances in plants, play a crucial role in plant growth, development, and stress response, despite their low concentration [91]. Complex signaling pathways involve interactions between hormones, regulating plant development and stress responses [92, 93]. Studies have demonstrated that salt stress inhibits rice seed germination, and the application of exogenous gibberellin can mitigate this effect, thereby enhancing the salt tolerance of rice [24]. Additionally, research has indicated an increase in the expression of auxin-related genes in Arabidopsis thaliana under salt stress [94]. Furthermore, the results of a specific study revealed a significant increase in the contents of ZT, GA3, IAA, and ABA in M9-T337 seedling leaves under saline-alkaline stress. Following exogenous MT treatment, there was further increased in the contents of ZT, GA3, and IAA in the leaves, whereas ABA content decreased. These findings align with the research outcomes of Jia et al. [95] in cherry. It is suggested that exogenous melatonin could influence plant growth, development, cell division, extension, and response to environmental stress by regulating endogenous hormone levels, thus enhancing plant resistance to saline-alkaline stress. This process likely involves a range of physiological, biochemical, and signal transduction mechanisms, warranting further detailed investigation.

Under saline-alkaline stress, ion balance is of paramount importance for plants in maintaining membrane integrity [96]. Numerous studies have demonstrated that an excess of sodium ions infiltrates cells under this stress, leading to a cation imbalance that can cause cell damage and potentially result in cell death [97, 98]. This study’s findings revealed a significant down-regulation of the Na + transporter gene and a considerable reduction in the expression of the K+ transporter gene in plant leaves under saline-alkaline stress. These changes led to a notable increase in Na+ and Ca2+ content, coupled with a significant decrease in K+ content in the leaves, culminating in an ion imbalance [99]. Nevertheless, the application of exogenous melatonin reversed this trend, reducing Na+ content and significantly increasing K+ content, which aligns with the findings reported by Wei et al. [100]. This effect may result from exogenous MT promoting the high expression of Na+ transporter genes and concurrently down-regulating K+ transporter genes. Consequently, this facilitates the expulsion of Na+ from vacuoles, reduces the discharge of K+ and ultimately maintains the Na+/K+ balance within plant cells.

Conclusions

In this study, 200 μmol L−1 exogenous MT was used to test the effect of MT on the saline-alkaline tolerance of M9-T337 seedlings. The results demonstrated that exogenous MT treatment significantly enhanced the saline-alkaline tolerance of M9-T337 seedlings, which was evident in the promotion of growth, improvement in osmotic regulation ability, enhancement of antioxidant capacity, facilitation of endogenous reactive oxygen species scavenging, reduction in cell membrane damage, enhancement of photosynthetic rate, mitigation of ion toxicity, and elevation of endogenous hormone content. Furthermore, melatonin induced the biosynthesis of GSH, GSSG, and AsA in the AsA-GSH cycle, and significantly enhancing the activities of APX, DHAR, and MDHAR. Although recent studies have established the positive influence of exogenous MT on enhancing the salinity tolerance of M9-T337 seedlings, further research is required to elucidate the specific mechanism of MT and its synergistic effects with other compounds such as ZT, IAA, GA3, and ABA. In summary, understanding the intervention pathways and potential biochemical reactions of MT is essential for its improved utilization in boosting the tolerance of M9-T337 seedlings to saline-alkaline stress.

Availability of data and materials

All the data is present inside the manuscript. There is no supplementary file.

References

  1. Liang WJ, Ma XL, Wan P, Liu LY. Plant salt-tolerance mechanism: a review. Biochem Biophys Res Commun. 2017;495:286–91.

    Article  PubMed  Google Scholar 

  2. Zhao XY, Bian XY, Li ZX, Wang XW, Yang CJ, Ciu GF, Kentbayev Y, Kentbayeva B. Genetic stability analysis of introduced Betula pendula, Betula kirghisorum, and Betula pubescens families in saline-alkali soil of northeastern China. Scand J For Res. 2014;29(7):639–49.

    Article  Google Scholar 

  3. Wang FN, Sheng HC, Shi YJ, Yu DF, Li XY. Salt-alkali tolerance during germination and establishment of Leymus chinensis in the Songnen Grassland of China. Ecol Eng J Ecotechnol. 2016;95:763–9.

    Article  Google Scholar 

  4. Liang XL, Fang SM, Ji WB, Zheng DF. The positive effects of silicon on rice seedlings under saline-alkali mixed stress. Commun Soil Sci Plant Anal. 2015;46(17):2127–38.

    Article  CAS  Google Scholar 

  5. Lu CC, Tian Y, Hou X, Jia ZC, Li M, Hao MX, Jiang YK, Liu YG. Multiple forms of vitamin B6 regulate salt tolerance by balancing ROS and abscisic acid levels in maize root. Stress Biol. 2022;2(1):1–14.

    Article  Google Scholar 

  6. Capula R, Valdez A, Cartmill, Alia T. Supplementary calcium and potassium improve the response of Tomato (Solanum lycopersicum L.) to simultaneous alkalinity salinity and boron stress. Commun Soil Sci Plant Anal. 2016;47(4):505–11.

    Google Scholar 

  7. Hasegawa PM. Sodium (Na+) homeostasis and salt tolerance of plants. Environ Exp Bot. 2013;92:19–31.

    Article  CAS  Google Scholar 

  8. Singh N, Bhatla SC. Nitric oxide and iron modulate heme oxygenase activity as a long distance signaling response to salt stress in sunflower seedling cotyledons. Nitric Oxide. 2016;53:54–64.

    Article  CAS  PubMed  Google Scholar 

  9. Yamika WSD, Aini N, Setiawan A, Purwaningrahayu RD. Effect of gypsum and cow manure on yield, proline content, and K/Na ratio of soybean genotypes under saline conditions. J Degraded Mining Lands Manag. 2018;5(2):1047–53.

    Article  Google Scholar 

  10. An Y, Yang XX, Zhang L, Zhang J, Du B, Yao L, Li XT, Guo C. Alfalfa MsCBL4 enhances calcium metabolism but not sodium transport in transgenic tobacco under salt and saline–alkali stress. Plant Cell Rep. 2020;39:997–1011.

    Article  CAS  PubMed  Google Scholar 

  11. Deinlein U, Stephan AB, Horie T, Cuo W, Xu G, Schroeder JI. Plant salt-tolerance mechanisms. Trendsin Plant Science. 2014;19(6):371–9.

    Article  CAS  Google Scholar 

  12. Yang CW, Guo WQ, Shi DC. Physiological roles of organic acids in alkali-tolerance of the alkali-tolerant halophyte Chloris virgata. Agron J. 2010;102(4):1081–9.

    Article  CAS  Google Scholar 

  13. Yan SP, Chong PF, Zhao M. Effect of salt stress on the photosynthetic characteristics and endogenous hormones, and: a comprehensive evaluation of salt tolerance in Reaumuria soongorica seedlings. Plant Signal Behav. 2022;17(1):2031782–2031782.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Gong B, Wen D, Vandenlangenbery K, Wei M, Yang F, Shi Q, Wamh X. Comparative effects of NaCl and NaHCO3 stress on photosynthetic parameters, nutrient metabolism, and the antioxidant system in tomato leaves. Sci Hortic. 2013;157:1–12.

    Article  CAS  Google Scholar 

  15. Mikolajczyk M. Osmotic stress induces rapid activation of a salicylic acid-induced protein kinase and a homolog of protein kinase ASK1 in tobacco cells. Plant Cell. 2000;12(1):165–78.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Kobayashi NI, Yamaji N, Yamamoto H, Okubo K, Ueno H, Costa A, Horie T. OsHKT1;5 mediates Na+ exclusion in the vasculature to protect leaf blades and reproductive tissues from salt toxicity in rice. Plant J. 2017;91(4):657–70.

    Article  CAS  PubMed  Google Scholar 

  17. An D, Chen JG, Gao YQ, Li X, Chao DY. AtHKT1 drives adaptation of Arabidopsis thaliana to salinity by reducing floral sodium content. PLoS Genet. 2017;13(10): e1007086.

    Article  PubMed  PubMed Central  Google Scholar 

  18. María RS, Maria RP, Maria SF, Hu JP, Sandalio LM. Peroxisomes extend peroxules in a fast response to stress via a reactive oxygen species-mediated induction of the peroxin PEX11a. Plant Physiol. 2016;171(3):1665–74.

    Article  Google Scholar 

  19. Reeta K, Sonal B, Deepali, Neeti M, Amit V. Potential of organic amendments (AM fungi, PGPR, vermicompost and seaweeds) in combating salt stress- a review. Plant Stress. 2022;6: 100111.

    Article  Google Scholar 

  20. Zuo YJ, Li BR, Guan SX, Jia JY, Xu XJ, Zhamg ZL, Lu Z, Li Z, Li X, Pang XY. EuRBG10 involved in indole al-kaloids biosynthesis in Eucommia ulmoides induced by drought and salt stresses. J Plant Physiol. 2022;278:153813–153813.

    Article  CAS  PubMed  Google Scholar 

  21. Li BB, Fu YS, Li XX, Yin HN, Xi ZM. Brassinosteroids alleviate cadmium phytotoxicity by minimizing oxidative stress in grape seedlings: toward regulating the ascorbate-glutathione cycle. Sci Hortic. 2022;299: 111002.

    Article  CAS  Google Scholar 

  22. González VJ, Reyes DM, Tighe NR, Inostroza BC, Escobar AL, Bravo LA. Salicylic acid improves antioxidant defense system and photosynthetic performance in Aristotelia chilensis plants subjected to moderate drought stress. Plants. 2022;11(5):639–639.

    Article  Google Scholar 

  23. Omena-Garcia PR, Martins OA, Medeiros BD, Vallarino GJ, Ribeiro MD, Fernie RA, Araújo LW, Nunes-Nesi A. Growth and metabolic adjustments in response to gibberellin deficiency in drought stressed tomato plants. Environ Exp Bot. 2019;159:95–107.

    Article  CAS  Google Scholar 

  24. Li Q, Yang A, Zhang WH. Higher endogenous bioactive gibberellins and α-amylase activity confer greater tolerance of rice seed germination to saline-alkaline stress. Environ Exp Bot. 2019;162:357–63.

    Article  CAS  Google Scholar 

  25. Jiang M, Ye F, Liu FL, Brestic M, Li XG. Rhizosphere melatonin application reprograms nitrogen-cycling related micro-organisms to modulate low temperature response in barley. Front Plant Sci. 2022;13:998861–998861.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Gao HS, Huang LZ, Gong ZJ, Wang XT, Qiao XQ, Xiao F, Yang YT, Yu BH, Guo XT, Yu CY, Zhang HX. Exogenous melatonin application improves resistance to high manganese stress through regulating reactive oxygen species scavenging and ion homeostasis in tobacco. Plant Growth Regul. 2022;98(2):219–33.

    Article  CAS  Google Scholar 

  27. Jia CH, Zhang XJ, Liu M, Zou ZG, Ma P, Xu J, Chun Y. Application of melatonin-enhanced tolerance to high-temperature stress in cherry radish (Raphanus sativus L. var. radculus pers). J Plant Growth Regul. 2019;39:1–10.

    Google Scholar 

  28. Liu Z, Song CJ, Li JJ, Yuan LG, Li CS, Fu GP, Zhang XK, Ma HQ, Liu QY, Zou XL, Cheng Y. Exogenous ap-plication of a low concentration of melatonin enhances salt tolerance in rapeseed (Brassica napus L.) seedlings. J Integr Agric. 2016;17(2):328–35.

    Google Scholar 

  29. Li J, Xie JM, Yu JH, Lv J, Zhang JF, Ding DX, Li NH, Zhang J, Bakpa EP, Yang Y, Niu TH, Gao F. Melatonin enhanced low-temperature combined with low-light tolerance of pepper (Capsicum annuum L.) seedlings by regulating root growth, antioxidant defense system, and osmotic adjustment. Front Plant Sci. 2022;13:998293–998293.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Soumya M, Satish CB. Exogenous melatonin modulates endogenous H 2 S homeostasis and L-Cysteine desulfhydrase activity in salt-stressed tomato (Solanum lycopersicum L. var. cherry) seedling cotyledons. J Plant Growth Regul. 2020;40(6):1–13.

    Google Scholar 

  31. Eiyazied AA, Ibrahim MFM, Ibrahim MAR, Nasef IN, AlQahtani SM, AlHarbi NA. Melatonin mitigates drought induced oxidative stress in potato plants through modulation of osmolytes, sugar metabolism, ABA homeostasis and antioxidant enzymes. Plants. 2022;11(9):1151–1151.

    Article  Google Scholar 

  32. Jia X, Zhu Y, Hu Y, Zhang R, Cheng L, Zhu ZL, Zhao T, Zhang XY, Wang YX. Integrated physiologic, proteomic, and metabolomic analyses of Malus halliana adaptation to saline–alkali stress. Hortic Res. 2019;6(1):1–19.

    Article  Google Scholar 

  33. Gao LY, Liu B, Zhang R. Effects of melatonin on photosynthetic and physiological characteristics of Malus halliana under saline-alkali combined stress. J Gansu Agric Univ. 2020;55(2):90–7.

    Google Scholar 

  34. Chu Y, Bao Q, Li Y, Sun H, Liu Z, Shi J, Huang Y. Melatonin alleviates antimony toxicity by regulating the antioxidant response and reducing antimony accumulation in Oryza sativa L. Antioxidants. 2023;12(11):1917.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Arnon DI. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 1949;24:1–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lin L, Niu Z, Jiang C, Yu L, Wang H, Qiao M. Influences of open-central canopy on photosynthetic parameters and fruit quality of apples (Malus × domestica) in the Loess Plateau of China. Hortic Plant J. 2022;8(2):133–42.

    Article  CAS  Google Scholar 

  37. Yuan JH, Xu M, Duan W, Fan PG, Li SH. Effects of whole-root and half-root water stress on gas exchange and chlorophyll fluorescence parameters in apple trees. J Am Soc Hortic Sci. 2013;138(5):395–402.

    Article  Google Scholar 

  38. Yuan F, Liang X, Li Y, Yin SS, Wang BS. Methyl jasmonate improves tolerance to high salt stress in the recretohalophyte Limonium bicolor. Funct Plant Biol. 2018;46(1):82–92.

    Article  PubMed  Google Scholar 

  39. Hnilickova H, Kraus K, Vachova P, Hnilicka F. Salinity stress affects photosynthesis, malondialdehyde formation, and proline content in Portulaca oleracea L. Plants. 2021;10:845.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ullah A, Tian Z, Xu L, Abid M, Lei K, Khanzada A, Zeeshan M, Sun C, Yu J, Dai T. Improving the effects of drought priming against post-anthesis drought stress in wheat (Triticum aestivum L.) using nitrogen. Front Plant Sci. 2022;13: 965996.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Ge YH, Chen YR, Li CY, Wei ML, Li X, Tang Q, Duan B. Effect of trisodium phosphate treatment on black spot of apple fruit and the roles of anti-oxidative enzymes. Physiol Mol Plant Pathol. 2019;106:226–31.

    Article  CAS  Google Scholar 

  42. Cut NI, Rita A, Bakhtiar B, Sabaruddin Z, Efendi E. The relationship between relative water content of leaves, soluble sugars, accumulation of dry matter, and yield components of rice (Oryza sativa L.) under water-stress condition during the generative stage. Int J Adv Sci Eng Inf Technol. 2022;12(3):899–907.

    Article  Google Scholar 

  43. Şevket T, Melayib B. Enhanced soluble protein and biochemical methane potential of apple biowaste by different pre-treatment. Earth Syst Environ. 2018;2(1):85–94.

    Article  Google Scholar 

  44. Wang WL, Wang X, Lv ZS, Khanzada A, Huang M, Cai J, Zhou Q, Huo ZY, Jiang D. Effects of cold and salicylic acid priming on free proline and sucrose accumulation in winter wheat under freezing stress. J Plant Growth Regul. 2021;3:1–14.

    Google Scholar 

  45. Eros K, Shriravi PS. Deficiency in phytochrome A alters photosynthetic activity, leaf starch metabolism and shoot biomass production in tomato. J Photochem Photobiol, B. 2016;165:157–62.

    Article  Google Scholar 

  46. Zheng XD, Zhao Y, Shan DQ, Shi K, Wang L, Li QT, Wang N, Zhou JZ, Yao JZ, Xue Y, Fang S, Chu JF, Guo Y, Kong J. MdWRKY9 overexpression confers intensive dwarfing in the M26 rootstock of apple by directly inhibiting brassinosteroid synthetase MdDWF4 expression. New Phytol. 2018;217(3):1086–98.

    Article  CAS  PubMed  Google Scholar 

  47. Kuang L, Wang Z, Zhang J, Xu G, Li J. Factor analysis and cluster analysis of mineral elements contents in different blueberry cultivars. J Food Compos Anal. 2022;109: 104507.

    Article  CAS  Google Scholar 

  48. An Y, Gao Y, Tong SZ, Liu B. Morphological and physiological traits related to the response and adaption of Bolboschoenus planiculmis seedlings grown under salt-alkaline stress conditions. Front Plant Sci. 2021;12:567782–567782.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Wang YN, Jie WJ, Peng Y, Hua XY, Yan XF, Zhou ZQ, Lin JX. Physiological adaptive strategies of oil seed crop Ricinus communis early seedlings (cotyledon vs. true leaf) under salt and alkali stresses: from the growth, photosynthesis and chlorophyll fluorescence. Front Plant Sci. 2019;9:1939.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Liu N, Jin ZY, Wang SS, Gong B, Wen D, Wang XF, Wei M, Shi QH. Sodic alkaline stress mitigation with exogenous melatonin involves reactive oxygen metabolism and ion homeostasis in tomato. Sci Hortic. 2015;18:18–25.

    Article  Google Scholar 

  51. Li J, Liu J, Zhu T, Zhao C, Li L, Chen M. The role of melatonin in salt stress responses. Int J Mol Sci. 2019;20(7):1735.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Varghese N, Alyammahi O, Nasreddine S, Alhassani A, Gururani MA. Melatonin positively influences the photosynthetic machinery and antioxidant system of Avena sativa during salinity stress. Plants. 2019;8(12):610.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ashraf M, Orooj A. Salt stress effects on growth, ion accumulation and seed oil concentration in an arid zone traditional medicinal plant ajwain (Trachyspermum ammi [L.] Sprague). J Arid Environ. 2006;64(2):209–20.

    Article  Google Scholar 

  54. He H, Zhou W, Lü H, Liang B. Growth, leaf morphological and physiological adaptability of leaf beet (Beta vulgaris var. cicla) to salt stress: a soil culture experiment. Agronomy. 2022;12(6):1393.

    Article  CAS  Google Scholar 

  55. Taârit MB, Msaada K, Hosni K, Marzouk B. Physiological changes, phenolic content and antioxidant activity of Salvia officinalis L. grown under saline conditions. J Sci Food Agric. 2012;92(8):1614–9.

    Article  PubMed  Google Scholar 

  56. José LC, Carlos AB. Effect of exogenous melatonin on seed germination and seedling growth in melon (Cucumis melo L.) under salt stress. Hortic Plant J. 2019;5(2):79–87.

    Article  Google Scholar 

  57. Fan HF, Ding L, Xu YL, Du CX. Antioxidant system and photosynthetic characteristics responses to short-term PEG-induced drought stress in cucumber seedling leaves. Russ J Plant Physiol. 2017;64(2):162–73.

    Article  CAS  Google Scholar 

  58. Zhu LL, Li HC, Thorpe MR, Charles HH, Song X. Stomatal and mesophyll conductance are dominant limitations to photosynthesis in response to heat stress during severe drought in a temperate and a tropical tree species. Trees. 2021;35(5):1613–26.

    Article  CAS  Google Scholar 

  59. Mafakheri A, Siosemardeh A, Bahramnejad B. Effect of drought stress on yield, proline and chlorophyll contents in three chickpea cultivars. Aust J Crop Sci. 2010;4(8):580–5.

    CAS  Google Scholar 

  60. Dhokne K, Pandey J, Yadav RM, Ramachandran P, Rath JR, Subramanyam R. Change in the photochemical and structural organization of thylakoids from pea (Pisum sativum) under salt stress. Plant Physiol Biochem. 2022;177:46–60.

    Article  CAS  PubMed  Google Scholar 

  61. Zhang Y, Zhou XJ, Dong YT, Zhang F, He QL, Chen JH, Zhu SJ, Zhao TL. Seed priming with melatonin improves salt tolerance in cotton through regulating photosynthesis, scavenging reactive oxygen species and coordinating with phyto-hormone signal pathways. Ind Crops Prod. 2021;169:152–68.

    Article  Google Scholar 

  62. Harizanova A, Koleva-Valkova L. Effect of silicon on photosynthetic rate and the chlorophyll fluorescence parameters at hydroponically grown cucumber plants under salinity stress. J Central Eur Agric. 2019;20(3):953–60.

    Article  Google Scholar 

  63. Chen J, Burke JJ, John J, Xin ZG. Chlorophyll fluorescence analysis revealed essential roles of FtsH11 protease in regulation of the adaptive responses of photosynthetic systems to high temperature. BMC Plant Biol. 2018;18(1):1–13.

    Article  Google Scholar 

  64. Mehta P, Allakhverdiev SI, Jajoo A. Characterization of photosystem II heterogeneity in response to high salt stress in wheat leaves (Triticum aestivum). Photosynth Res. 2010;105(3):249–55.

    Article  CAS  PubMed  Google Scholar 

  65. Yan K, Mei H, Dong X, Zhou S, Cui J, Sun Y. Dissecting photosynthetic electron transport and photosystems performance in Jerusalem artichoke (Helianthus tuberosus L.) under salt stress. Front Plant Sci. 2022;13: 905100.

    Article  PubMed  PubMed Central  Google Scholar 

  66. Sengupta S, Majumder AL. Insight into the salt tolerance factors of a wild halophytic rice, Porteresia coarctata: a physio-logical and proteomic approach. Planta. 2009;229(4):911–29.

    Article  CAS  PubMed  Google Scholar 

  67. Yan FY, Zhang JY, Li WW, Ding YF, Zhong QY, Xu X, Wei HM, Li GH. Exogenous melatonin alleviates salt stress by improving leaf photosynthesis in rice seedlings. Plant Physiol Biochem. 2021;163:367–75.

    Article  CAS  PubMed  Google Scholar 

  68. Alharbi K, Al-Osaimi AA, Alghamdi BA. Sodium chloride (NaCl)-induced physiological alteration and oxidative stress generation in Pisum sativum (L.): a toxicity assessment. ACS Omega. 2022;7(24):20819–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Song Y, Zheng CF, Basne R, Li S, Chen JH, Jiang M. Astaxanthin synthesized gold nanoparticles enhance salt stress tolerance in rice by enhancing tetrapyrrole biosynthesis and scavenging reactive oxygen species in vitro. Plant Stress. 2022;6:100–22.

    Article  Google Scholar 

  70. Pu XH, An MY, Han LB, Zhang XZ. Protective effect of spermidine on salt stress induced oxidative damage in two Kentucky bluegrass (Poa pratensis L.) cultivars. Ecotoxicol Environ Saf. 2015;117:96–106.

    Article  Google Scholar 

  71. Weisany W, Sohrabi Y, Heidari G, Siosemardeh A, Ghassemi-Golezani K. Changes in antioxidant enzymes activity and plant performance by salinity stress and zinc application in soybean (Glycine max L.). Plant Omics. 2012;5(2):60–7.

    CAS  Google Scholar 

  72. High SI. Different oxidative stress and antioxidant responses in maize seedlings organs. Front Plant Sci. 2016;7:276.

    Google Scholar 

  73. Liang CZ, Zheng GY, Li WZ, Wang YQ, Hu B, Wang HR, Wu HK, Qian YW, Zhu XG, Tan DX, Chen SY, Chu CC. (2015) Melatonin delays leaf senescence and enhances salt stress tolerance in rice. J Pineal Res. 2015;59(1):91–101.

    Article  CAS  PubMed  Google Scholar 

  74. Ren Y, Zhang C. The excessive response: a preparation for harder conditions. Protein Cell. 2017;8(10):707–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Ali L, Shaheen MR, Ihsan MZ, Masood S, Zubair M, Shehzad F, Khalid AUH. Growth, photosynthesis and antioxidant enzyme modulations in broccoli (Brassica oleracea L. var. italica) under salinity stress. South Afr J Bot. 2022;148:104–11.

    Article  CAS  Google Scholar 

  76. Liang D, Ni Z, Xia H, Xie Y, Lv X, Wang J, Lin L, Deng Q, Luo X. Exogenous melatonin promotes biomass accumulation and photosynthesis of kiwifruit seedlings under drought stress. Sci Hortic. 2019;246:34–43.

    Article  CAS  Google Scholar 

  77. Chen YE, Mao JJ, Sun LQ, Huang B, Ding CB, Gu Y. Exogenous melatonin enhances salt stress tolerance in maize seedlings by improving antioxidant and photosynthetic capacity. Physiol Plant. 2018;164(3):349–63.

    Article  CAS  PubMed  Google Scholar 

  78. Hayat S, Hasan SA, Yusuf M, Hayat Q, Ahmad A. Effect of 28-homobrassinolide on photosynthesis, fluorescence and antioxidant system in the presence or absence of salinity and temperature in Vigna radiata. Environ Exp Bot. 2010;69(2):105–12.

    Article  CAS  Google Scholar 

  79. Zhang T, Wang Y, Ma X, Ouyang Z, Deng L, Shen S, Dong X, Du N, Dong H, Guo Z. Melatonin alleviates copper toxicity via improving ROS metabolism and antioxidant defense response in tomato seedlings. Antioxidants. 2022;11:758.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Goda H, Shimada Y, Asami T, Fujioka S, Yoshida S. Microarray analysis of brassinosteroid-regulated genes in Arabidopsis. Plant Physiol. 2002;130(3):1319–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Wang J, Wang D, Zhu M, Li F. Exogenous 6-benzyladenine improves waterlogging tolerance in maize seedlings by mitigating oxidative stress and upregulating the ascorbate-glutathione cycle. Front Plant Sci. 2021;12: 680376.

    Article  PubMed  PubMed Central  Google Scholar 

  82. Mirza H, Bhuyan MB, Taufika IA, Khursheda P, Kamrun N, Mahmud JA, Masayuki F. Regulation of ascorbate-glutathione pathway in mitigating oxidative damage in plants under abiotic stress. Antioxidants. 2019;8(9):384–384.

    Article  Google Scholar 

  83. Pravej A, Thamer H, Albalawi FH, Altalayan M, Mohammad AA, Vaseem R, Muhammad A, Parvaiz A. 24-Epibrassinolide (EBR) confers tolerance against NaCl stress in soybean plants by up-regulating antioxidant system, ascorbate-glutathione cycle, and glyoxalase system. Biomolecules. 2019;9(11):640.

    Article  Google Scholar 

  84. Wu Y, Hu LL, Liao WB, Mohammed MD, Lyu J, Xie JM, Feng Z, Calderón-Urrea A, Yu JH. Foliar application of 5-aminolevulinic acid (ALA) alleviates NaCl stress in cucumber (Cucumis sativus L.) seedlings through the enhancement of ascorbate-glutathione cycle. Sci Hortic. 2019;257:108761–108761.

    Article  CAS  Google Scholar 

  85. Kaya C. (2023) Nitric oxide and hydrogen sulfide work together to improve tolerance to salinity stress in wheat plants by upraising the AsA-GSH cycle. Plant Physiol Biochem. 2023;194:651–63.

    Article  CAS  PubMed  Google Scholar 

  86. Xu JW, Kang Z, Zhu KY, Zhao DK, Yuan YJ, Yang SC, Zhen WT, Hu XH. RBOH1-dependent H2O2 mediates spermine-induced antioxidant enzyme system to enhance tomato seedling tolerance to salinity–alkalinity stress. Plant Physiol Biochem. 2021;164:237–46.

    Article  CAS  PubMed  Google Scholar 

  87. Hosseini SM, Hasanloo T, Mohammadi S. Physiological characteristics, antioxidant enzyme activities, and gene expression in 2 spring canola (Brassica napus L.) cultivars under drought stress conditions. Turkish J Agric For. 2015;39:413–20.

    Article  CAS  Google Scholar 

  88. Tawfik MM, Badr EA, Ibrahim OM. Biomass and some physiological aspects of spartina patens grown under salt affected environment in south Sinai. Int J Agric Res. 2017;12(1):19–27.

    Article  CAS  Google Scholar 

  89. Cai MM, He ZH, Lin ZR, Nie GB, Li XY, Liu HS, Ma Q. Comparative physiological, transcriptome, and qRT-PCR analysis provide insights into osmotic adjustment in the licorice species Glycyrrhiza inflata under salt stress. Crop Sci. 2023;63(3):1442–57.

    Article  CAS  Google Scholar 

  90. Zhang PF, Liu L, Wang X, Wang ZY, Zhang H, Chen JT, Liu XY, Wang YB, Li CF. Beneficial effects of exogenous melatonin on overcoming salt stress in sugar beets (Beta vulgaris L.). Plants. 2021;10(5):886–886.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Wang HM, Liu XY, Yang P, Wu RZ, Wang SY, He SN, Zhou QH. Potassium application promote cotton acclimation to soil waterlogging stress by regulating endogenous protective enzymes activities and hormones contents. Plant Physiol Biochem. 2022;185:336–43.

    Article  CAS  PubMed  Google Scholar 

  92. Xu ZQ, Lei P, Pang X, Li HS, Feng XH, Xu H. Exogenous application of poly-γ-glutamic acid enhances stress defense in Brassica napus L. seedlings by inducing cross-talks between Ca2+, H2O2, brassinolide, and jasmonic acid in leaves. Plant Physiol Biochem. 2017;118:460–70.

    Article  CAS  PubMed  Google Scholar 

  93. Su L, Rahat S, Ren N, Kojima M, Takebayashi Y, Sakakibara H, Wang M, Chen X, Qi X. Cytokinin and auxin modulate cucumber parthenocarpy fruit development. Sci Hortic. 2021;282: 110026.

    Article  CAS  Google Scholar 

  94. Fu J, Zhu C, Wang C, Liu L, Shen Q, Xu D, Wang Q. Maize transcription factor ZmEREB20 enhanced salt tolerance in transgenic Arabidopsis. Plant Physiol Biochem. 2021;159:257–67.

    Article  CAS  PubMed  Google Scholar 

  95. Jia XM, Wang H, Svetla S, Zhu YF, Hu Y, Cheng L, Zhao T, Wang YX. Comparative physiological responses and adaptive strategies of apple Malus halliana to salt, alkali and saline-alkali stress. Sci Hortic. 2019;245:154–62.

    Article  CAS  Google Scholar 

  96. El-Badri AM, Batool M, Mohamed AAI, Wang Z, Khatab A, Sherif A, Ahmad H, Khan MN, Hassan HM, Elrewainy IM. Antioxidative and metabolic contribution to salinity stress responses in two rapeseed cultivars during the early seedling stage. Antioxidants. 2021;10(8):1227.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Gong Z, Xiong L, Shi H, Yang S, Herrera-Estrella LR, Xu G, Chao D, Li J, Wang P, Qin F, Li J, Ding Y, Shi Y, Wang Y, Yang Y, Guo Y, Zhu JK. Plant abiotic stress response and nutrient use efficiency. Sci China Life Sci. 2020;63(5):635–74.

    Article  PubMed  Google Scholar 

  98. Saima S. A DNA methylation reader with an affinity for salt stress. Plant Cell. 2020;32(11):3380–1.

    Article  Google Scholar 

  99. Parveen A, Ahmar S, Kamran M, Ali A, Riaz M. Abscisic acid signaling reduced transpiration flow, regulated Na + ion homeostasis and antioxidant enzyme activities to induce salinity tolerance in wheat (Triticum aestivum L.) seedlings. Environ Technol Innov. 2021;413: 101808.

    Article  Google Scholar 

  100. Wei R, Li C, Ming ZX, Peng XF. Combined transcriptome and metabolome analysis revealed pathways involved in improved salt tolerance of Gossypium hirsutum L. seedlings in response to exogenous melatonin application. BMC Plant Biol. 2022;22(1):552–552.

    Article  Google Scholar 

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Acknowledgements

This work was supported by the Special Fund for National Natural Science Foundation of China (Project Number 32160696).

Funding

This work was supported by the Special Fund for National Natural Science Foundation of China (Project Number 32160696) and the Grant from the Scientific Research Projects of Higher Education Institutions of Gansu Province, Grant Number: China (2022QB-075).

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  1. College of Horticulture, Gansu Agricultural University, Lanzhou, 730070, China

    Xulin Xian, Zhongxing Zhang, Shuangcheng Wang, Jiao Cheng, Yanlong Gao, Naiying Ma, Cailong Li & Yanxiu Wang

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  1. Xulin Xian
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  2. Zhongxing Zhang
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Contributions

Xulin Xian and Yanxiu Wang designed the research. Xulin Xian, Zhongxing Zhang, Jiao Cheng and Shuangcheng Wang performed the experiments. Yanlong Gao, Naiying Ma and Cailong Li performed the data analysis and inter-pretation. Xulin Xian and Yanxiu Wang prepared the figures and tables. Xulin Xian wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Yanxiu Wang.

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We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue. So, it is not applicable. Experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, must comply with relevant institutional, national, and international guidelines and legislation. No permission is required. Plants material was purchased.

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Supplementary Information

Additional file 1.

Effects of exogenous MT on biomass of M9-T337 seedlings

Additional file 2.

Effects of exogenous MT on chlorophyll fluorescence parameters of M9-T337 seedlings

Additional file 3.

Effects of exogenous MT on the expression of key genes in M9-T337 seedlings

Additional file 4:

Table S1. MT concentration screening. Table S2. Primer sequences for qRT‒PCR. Table S3. Effects of exogenous MT on root morphological parameters and root activity of M9-T337 seedlings under saline-alkali stress.

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Xian, X., Zhang, Z., Wang, S. et al. Exogenous melatonin strengthens saline-alkali stress tolerance in apple rootstock M9-T337 seedlings by initiating a variety of physiological and biochemical pathways. Chem. Biol. Technol. Agric. 11, 58 (2024). https://doi.org/10.1186/s40538-024-00577-x

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