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Exogenous melatonin alleviates sodium chloride stress and increases vegetative growth in Lonicera japonica seedlings via gene regulation

BMC Plant Biology volume 24, Article number: 790 (2024) Cite this article

Abstract

Melatonin (Mt) functions as a growth regulator and multifunctional signaling molecule in plants, thereby playing a crucial role in promoting growth and orchestrating protective responses to various abiotic stresses. However, the mechanism whereby exogenous Mt protects Lonicera japonica Thunb. (L. japonica) against salt stress has not been fully elucidated. Therefore, this study aimed to elucidate how exogenous Mt alleviates sodium chloride (NaCl) stress in L. japonica seedlings. Salt-sensitive L. japonica seedlings were treated with an aqueous solution containing 150 mM of NaCl and aqueous solutions containing various concentrations of Mt. The results revealed that treatment of NaCl-stressed L. japonica seedlings with a 60 µM aqueous solution of Mt significantly enhanced vegetative plant growth by scavenging reactive oxygen species and thus reducing oxidative stress. The latter was evidenced by decreases in electrical conductivity and malondialdehyde (MDA) concentrations. Moreover, Mt treatment led to increases in the NaCl-stressed L. japonica seedlings’ total chlorophyll content, soluble sugar content, and flavonoid content, demonstrating that Mt treatment improved the seedlings’ tolerance of NaCl stress. This was also indicated by the NaCl-stressed L. japonica seedlings exhibiting marked increases in the activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase) and in photosynthetic functions. Furthermore, Mt treatment of NaCl-stressed L. japonica seedlings increased their expression of phenylalanine ammonia-lyase 1 (PAL1), phenylalanine ammonia-lyase 2 (PAL2), calcium-dependent protein kinase (CPK), cinnamyl alcohol dehydrogenase (CAD), flavanol synthase (FLS), and chalcone synthase (CHS). In conclusion, our results demonstrate that treatment of L. japonica seedlings with a 60 µM aqueous solution of Mt significantly ameliorated the detrimental effects of NaCl stress in the seedlings. Therefore, such treatment has substantial potential for use in safeguarding medicinal plant crops against severe salinity.

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Introduction

High concentrations of salts such as sodium chloride (NaCl) in soil are a prominent and widespread source of stress that has an adverse effect on agricultural production worldwide [1]. For example, an excessive abundance of sodium ions (Na+) inhibits the uptake of potassium ions (K+), beneficial nutrients, and water, thereby adversely affecting plant biomass and productivity [2]. Plants under salt stress exhibit a marked increase in the compartmentalization of levels of reactive oxygen species (ROS), which are continuously generated in plastids, peroxisomes, mitochondria, the cytosol, and the apoplast. An imbalance between ROS generation and safe detoxification leads to the accumulation of ROS and thus oxidative stress, which is harmful to plants [3, 4]. Consequently, over a long period of evolution, plants have developed intricate antioxidant systems to cope with salt stress and other environmental stresses. For example, several enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), control ROS concentrations and are involved in responses to salt stress [5]. In addition, plant vacuoles are spacious compartments that are well suited for containment of Na+. Furthermore, numerous studies have confirmed that salt stress induces biosynthesis of endogenous hormones and sucrose, followed by a rapid increase in membrane peroxidation and in ROS levels, thereby indirectly activating genes encoding key proteins involved in lignin and flavonoid biosynthesis. Specifically, salt stress can trigger transcriptional activation of mediators (such as jasmonate, myelocytomatosis, and ethylene insensitive mediators) and transcription factors (TFs) (such as no apical meristem–Arabidopsis thaliana activating factor 1–cup-shaped cotyledon 2, v-myb avian myeloblastosis viral oncogene homolog (MYB), and basic helix–loop–helix (bHLH)). These mediators and TFs increase the expression levels of flavonoid-related genes (chalcone synthase (CHS), 4-coumarate: coenzyme A ligase (4CL), and phenylalanine ammonia-lyase (PAL)) and lignin-related genes (cinnamyl alcohol dehydrogenase (CAD), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase, and flavonoid 5′-hydroxylase), thereby alleviating oxidative stress and damage [6].

Salt stress can also increase the expression level of certain proteins. For example, tandem mass-tag-based and proteoform reaction monitoring-based proteomic analyses revealed that treatment of Sophora alopecuroides with a 150 mM aqueous solution of NaCl induced changes in this plant’s proteomic profile. That is, the concentrations of several transferases, oxidoreductases, and dehydrogenases involved in the biosynthesis of flavonoids, alkaloids, phenylpropanoids, and amino acid metabolism were increased 7 days after exposure to NaCl stress [7]. Such increases in protein production might have resulted from ethylene and jasmonate (JA) signals perturbing the biosynthesis and signal transduction processes of endogenous hormones, thereby negatively regulating the response of S. alopecuroides to NaCl stress. In addition, the biosynthesis of abscisic acid (ABA) and salicylic acid was found to be significantly upregulated in S. alopecuroides under NaCl stress [8]. The aforementioned results suggest that there is bidirectional regulation of endogenous hormones in plants under salt stress. Another representative study found that 52 differentially expressed genes in Sesuvium portulacastrum that are involved in lignin biosynthesis may be responsible for this plant’s salt tolerance. In particular, its salt tolerance was attributed to the differential expression of the genes 4CL, CAD, cinnamoyl-coenzyme A reductase, caffeic acid O-methyltransferase, ferulate-5-hydroxylase, cinnamate 4-hydroxylase, caffeoyl-coenzyme A 3-O-methyltransferase, and para-coumaroyl quinate/shikimate 3′-hydroxylase, which encode proteins that underpin the biosynthesis of para-coumaryl alcohol-rich lignin [9].

Melatonin (Mt) is a multifunctional signaling molecule that plays a vital role in the biological functions of humans and animals. It was originally identified in the bovine pineal gland and is a key regulator of sleep, the circadian rhythm, and anti-aging mechanisms, and exhibits antioxidant activities [10]. It was first identified in plants in 1995, which marked the first instance of its detection in non-animal organisms and subsequently led to the discovery that Mt and its derivatives are ubiquitous in plants [11]. Mt regulates various physiological processes in plants, such as circadian rhythms, blooming, leaf resilience, root maturity, and photosynthesis. In addition, it has antioxidant and growth-regulatory effects in plants. Furthermore, Mt alleviates the impacts of several environmental stressors on plant systems, encompassing but not limited to low temperature, drought, excessive concentrations of Na+, and exposure to heavy metal ions [12, 13]. As Mt is an amphiphilic small molecule, it can efficiently traverse cell membranes and thus can enter mitochondria and nuclei.

Numerous studies have investigated the mechanisms through which exogenous Mt can modulate plant development, growth, and stress resilience [14]. For example, it has been found that exogenous Mt can delay drought-induced senescence in apple leaves and increase the photosynthetic capacity of Bermuda grass exposed to low temperature stress [1, 15, 16]. In addition, exogenous Mt has been observed to enhance the activity of antioxidant enzymes, thereby mitigating the harmful effects of abiotic stress on plants. For example, exogenous Mt has been found to enhance photosynthetic efficiency and antioxidant potential in tomato plants under drought stress [17, 18]. Moreover, exogenous Mt can effectively reduce oxidative stress caused by NaCl in Arabidopsis thaliana [19, 20]. Such reductions in oxidative stress lead to a decrease in the concentration of ROS, an increase in the activity of antioxidant enzymes, and upregulation of the expression of genes encoding antioxidant enzymes [21]. In fact, Mt serves as both an antioxidant and a regulator of gene expression in various physiological processes. Thus, exogenous Mt was found to induce the activation of genes associated with photosystem activity, glucose metabolism, and fatty acid biosynthesis in soybean plants [10]. In addition, exogenous Mt markedly inhibited the transcription of genes associated with senescence in rice, thereby delaying the initiation of leaf senescence [22]. Moreover, exposure of Lonicera japonica Thunb. (L. japonica) to moderately saline conditions significantly increased the plant’s expression of genes associated with the biosynthesis of many phenolic compounds [23]. Mt also effectively mitigates the inhibitory effects of NaCl-induced stress on seed germination by coordinatively regulating ABA and gibberellic acid anabolism and catabolism [24]. Furthermore, Mt reduces concentrations of ROS such as hydrogen peroxide (H2O2) in plants, and H2O2 and Mt-induced stress tolerance exhibit an intricate reciprocal interaction. For example, the development of Mt-induced salt tolerance in Arabidopsis thaliana involves Arabidopsis thaliana respiratory burst oxidase homolog f (AtrbohF)-dependent signaling of ROS concentrations [25].

L. japonica is a member of the honeysuckle family (Caprifoliaceae) and is known as Jin Yin Hua in China, Korea, and Japan. Various parts of L. japonica (the roots, leaves, and fruit) are used as traditional medicines, due to the plant containing pharmacologically active compounds. The latest investigations have confirmed that the roots, stems, and flowers of L. japonica have similar chemical constituents. However, in clinical practice, L. japonica flowers are primarily used, and thus more than 500 traditional Chinese medicines contain Lonicerae Japonicae Flos. Thus, L. japonica is an important crop that is cultivated across a substantial area of land [26, 27]. Investigations of L. japonica have isolated more than 200 antimicrobial and antioxidant chemical compounds, including volatile oils, phenolic acids, terpenoids, and flavonoids, which have been demonstrated to improve human health. For instance, L. japonica extracts were used to treat flu virus (i.e., H1N1 and H7N9) and severe acute respiratory syndrome coronavirus 2 infections [28]. Moreover, L. japonica is also a valuable source of compounds for use in the food, beverage, cosmetic, and healthcare industries [29].

Given the above-mentioned utility of L. japonica, it was used as the experimental plant in the current study to investigate the effects of exogenous Mt. First, this study examined the impact of NaCl stress on various characteristics of L. japonica seedlings, namely their biomass, osmotic adjustment biochemicals, accumulation of active oxygen, chlorophyll contents, and antioxidant system. Second, this study examined the impact of exogenous Mt on the activation of salt-tolerant genes and salt overload sensitive (SOS) pathway-related genes (SOS1, SOS2, WRKY and plasma membrane H+-ATPase) in L. japonica seedlings subjected to NaCl stress [29, 30]. Third, this study examined the role of exogenous Mt in alleviating membrane peroxidation and increases in H2O2 concentrations caused by NaCl stress. The results revealed that Mt had a regulatory role that largely involved activating the expression of some antioxidant enzymes. Overall, this study yielded new information about how exogenous Mt can enhance a plant’s ability to deal with NaCl stress by altering its regulatory pathways.

Materials and methods

Plant cultivation and treatment

One-year-old L. japonica materials were collected from the Honeysuckle Breeding Base of Fengqiu County (Xinxiang, Henan). Bare-rooted plants were carefully cultivated in plastic pot trays (28 cm × 54 cm) containing horticultural substrate soil (peat and vermiculate mixture) [30]. Emerging seedlings were cultivated in a chamber with the photon flux density, day/night temperature, and humidity controlled at 200 µmol m− 2 s− 1 (12 h per day from 07:00 to 19:00), 25/18°C, and 65%, respectively. Subsequently, uniform 3-week-old seedlings were relocated to smaller pots (10 cm × 10 cm) and then subjected to various treatments and watered with Hoagland’s nutrient solution. After first leaf emergence, the seedlings were watered every 2 days for a total of three times. Irrigation with Mt was performed during the dark stage, as Mt is photosensitive. The seedlings were divided randomly into two groups: (i) a control group, which was treated with sterile water, and (ii) a 150 mM NaCl-treatment group, each plant of which was treated with 100 mL of an aqueous solution containing 150 mM of NaCl. Aqueous solutions containing different concentrations of Mt (20, 40, 60, 80 and 100 µM) were prepared by dissolving 2.32 mg of Mt in 1 L distilled water and then performing dilutions. Seedlings in the 150 mM NaCl-treatment group were treated with solutions containing various concentrations of Mt to evaluate its effect on several plant-growth characteristics Plants’ physiological growth parameters, such as fresh weight, were assessed on the 16th day after treatment. It was found that a solution containing 60 µM of Mt was the most suitable for further investigation [31].

Determination of H2O2 concentrations

Concentrations of H2O2 were determined using a previously reported methodology [32], with some adjustments made to suit the current study. First, 0.15 g of treated leaves were combined with 6 mL of tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 6.4) containing 250 mM of sucrose, 20 mM of nitric oxide diphosphate, and 20 mM of sodium butyrate. The spectrophotometer was used to measure the absorbance at a wavelength of 530 nm (A530) of the supernatant obtained after subjecting it to a vacuum for duration of 20–40 min.

Measurement of total chlorophyll contents, MDA concentrations, and relative conductivities

The total chlorophyll contents of samples were examined to determine the photosynthetic performance of seedlings subjected to the control treatment, 150 mM-NaCl treatment, and Mt treatment. For this purpose, 0.1 g of fresh leaf tissue in 95% ethanol was stood in the dark at room temperature for 10–12 h. Subsequently, an aliquot was taken and its chlorophyll content was determined using a multifunctional microplate reader (Infinite-200, Tecan, Switzerland) [33]. The concentration of MDA was measured using the thiobarbituric acid method reported by Draper and Hadley [16]. For this purpose, 0.1 g of fresh leaf tissue was ground with acetone and the resulting mixture was centrifuged at 1000 g and room temperature for 15 min. An aliquot of the supernatant of the resulting mixture was used for analysis.

Determination of antioxidant enzyme activities

To investigate antioxidant enzyme activities, 0.5 g of control and treatment leaves were separately weighed and then homogenized in 5 mL of 0.05 M phosphate buffer containing 1 mM ethylenediaminetetraacetic acid and 1% polyvinylpyrrolidone that had a pH of 7.4. Each homogenized mixture was then centrifuged at 12,000 g at 4 °C for 20 min. The supernatants of the centrifuged mixtures were analyzed using a previously established methodology [34] to quantify the activities of SOD, CAT, POD, and ascorbate peroxidase (APX).

Determination of gas exchange parameters

The transpiration rate (Tr), net photosynthetic rate (Pn), intercellular carbon dioxide (CO2) concentration (Ci), and stomatal conductance (Gs) of samples were analyzed using a gas exchange system (GFS-3000, Heinz Germany) at a humidity and temperature of 60% and 25 °C, respectively, and an air velocity and light intensity of 750 µmol s− 1 and 600 µmol m− 2, respectively. The leaf gas exchange measurements were carried out in the morning, i.e., at 08:00–11:00 am [30].

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) assays

Total RNA was extracted using a MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan) and then reverse transcribed to afford complementary DNA (cDNA). qRT-PCR was performed using SYBR Premix Ex TaqTM solution (TaKaRa) and the qRT-PCR amplification primers listed in Table S1. The qRT-PCR quantitative reaction profiling system was composed of SYBR Premix Ex TaqTM, 1 L of upstream primer, 1 L of downstream primer, 2 L of a cDNA template, and 8.5 L of double-distilled water. qRT-PCR assays were performed on a Bio-Rad 7500 Fast Fluorescence Quantitative PCR platform with TransStart® Top Green qPCR Supermix (TransGen Biotech Co., Ltd., Beijing, China), in accordance with the manufacturer’s protocol. The 2−ΔΔCt method was used to measure the relative expression levels of genes. The experiment consisted of three stages: a 30-second stage of denaturation at 95 °C, a 5-second stage of denaturation at 95 °C, and a 20-second stage of warming at 60 °C. These steps were repeated for a total of 40 cycles. Subsequently, a temperature gradient of 55 °C to 95 °C was applied for 81 cycles, with the temperature increasing by 0.5 °C every 30 s. To ensure precision, every sample was examined three times, and each gene was analyzed in three biological replicates.

Statistical and correlation analysis

Samples for analysis were collected from a minimum of six L. japonica seedlings subjected to each experimental treatment. Each analysis was replicated at least three times, using three separate and independent biological replicates. The data were subjected to a one-way analysis of variance in Statistical Product and Service Solutions 20.0. A post-hoc comparison was conducted using Duncan’s multi-range test, with the level of significance set to a P value of less than 0.05 to identify significant differences. The data were graphically depicted using GraphPad Prism 5.0. The SupCorrPlot tool obtained from TBtools software (v. 2.096) was used for Pearson correlation analysis of physiological and molecular traits.

Results

Physiological attributes

To investigate the effects of Mt and co-application of Mt and salt stress on L. japonica, 3-week-old seedlings were treated with a solution of Mt and then treated and not treated, respectively, with an aqueous solution containing 150 mM of NaCl for 5 days. Compared with the control treatment, the 150-mM NaCl treatment significantly reduced the fresh biomass and dry biomass of seedlings. As expected, compared with the 150-mM NaCl treatment seedlings, the Mt + 150-mM NaCl treatment seedlings had substantially greater shoot and root fresh weights (Fig. 1). In addition, compared with the 150-mM NaCl treatment seedlings, the Mt-only treatment seedlings had a greater shoot fresh weight (33.3%). Similarly, exogenous Mt induced significant increases in the fresh and dry weights of shoots and roots.

Fig. 1
figure 1

Fresh biomass of L. japonica seedlings under salt (S) and melatonin (Mt) treatment

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Effect of exogenous mt on oxidative stress

ROS serve as signaling molecules and, due to their hazardous properties, play a crucial role in coordinating responses to abiotic stresses in plants. Compared with the seedlings that received the control treatment, those that received the NaCl treatment exhibited increases in H2O2 and MDA concentrations and electrolyte leakage. The impact of exogenous Mt reduced oxidative stress indicators in comparison with salt-treated seedlings (Fig. 2). Furthermore, it is proposed that H2O2 plays a role in mediating this regulatory response to oxidative stress, because compared with the seedlings that received only the NaCl treatment, those that received the Mt treatment followed by the NaCl treatment (denoted the “Mt + NaCl treatment”) exhibited a marked reduction in H2O2 and O2 concentrations, as depicted in Fig. 2A and B. Taken together, the above-mentioned findings indicate that exogenous Mt can mitigate increases in H2O2 and O2 concentrations in L. japonica seedlings subjected to NaCl stress.

Fig. 2
figure 2

Oxidative stress parameters detected in L. japonica seedlings. (A) H2O2 activity, (B) O2 activity, (C) electrical activity, (D) MDA concentrations

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When subjected to high levels of salt stress, the membranes of plants undergo extensive oxidation, leading to the loss of electrolytes. Therefore, relative conductivity is a key indicator of the permeability of plant cell membranes. Moreover, as MDA is generated by the oxidation of membrane lipids, MDA concentrations can serve as an indicator of plants’ extent of salt-induced membrane damage. In this study, the NaCl treatment group exhibited significant increases in relative conductivity and MDA concentrations compared with the control group. However, the Mt + NaCl treatment group exhibited lower relative conductivities and MDA concentrations than the NaCl treatment group. Taken together, these findings suggest that the application of exogenous Mt prior to NaCl stress can mitigate NaCl-mediated oxidative damage in L. japonica seedlings (Fig. 2C and D).

Chlorophyll content analysis of L. Japonica seedlings under NaCl stress

It is vital to increase photosynthetic activity to reduce levels of abiotic stress in crops. As shown in Fig. 3, exposure to NaCl stress resulted in a reduction in overall chlorophyll content in L. japonica leaves. However, application of exogenous Mt alleviated the impact of NaCl stress on L. japonica leaves, resulting in a marked enhancement in the leaves’ overall contents of chlorophyll and carotenoids and in their chlorophyll fluorescence indices. Furthermore, Mt + NaCl treatment alleviated the reduction in overall total chlorophyll content and chlorophyll fluorescence parameters in L. japonica leaves compared with NaCl treatment and Mt treatment, respectively.

Fig. 3
figure 3

Chlorophyll content analysis of L. japonica seedlings treated with melatonin and salt. (A) Total chlorophyll content, (B) chlorophyll pigment content, (C) soluble sugar content, (D) flavonoid content

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Taken together, the above-mentioned findings indicate that application of Mt to L. japonica seedlings prior to exposure to NaCl stress can mitigate NaCl-mediated reductions in chlorophyll contents and the efficiency of photochemical reactions in the seedlings (Fig. 3). Hence, exogenous Mt enhanced the photosynthetic activity of L. japonica seedlings that were exposed to NaCl-induced stress.

Antioxidant enzyme activities of L. Japonica seedlings under NaCl stress

Plants have developed antioxidant systems that remove ROS and help prevent the cellular damage induced by excess concentrations of ROS under unfavorable conditions. Antioxidant enzymes play an important role by efficiently neutralizing excess ROS, thereby mitigating oxidative damage and safeguarding cellular integrity. This study assessed whether there were alterations in the activities of various antioxidant enzymes, namely CAT, POD, SOD, and APX, in response to NaCl-induced stress. This assessment was done primarily to determine whether there was an association between the mitigation of NaCl stress and the antioxidant defense induced by Mt. The results indicate that the exogenous Mt significantly enhanced the activity of all four antioxidant enzymes in the presence of NaCl-induced stress. Specifically, treatment with Mt and NaCl resulted in a marked enhancement in the concentrations of CAT (by 25.3%), SOD (by 28.9%), POD (by 19.6%), and APX (by 10.7%) compared with treatment with NaCl alone. In contrast, the efficacy of these antioxidant enzymes was markedly reduced by other treatments (Fig. 4).

Fig. 4
figure 4

Antioxidant enzyme activities in L. japonica seedlings under salt (S) and melatonin (Mt) treatment. (A) SOD activity, (B) POD activity, (C) CAT activity, (D) APX activity

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Taken together, the findings described in the previous sections demonstrate a robust correlation between the presence of H2O2 and the augmentation of antioxidant enzyme activity prompted by Mt in the context of NaCl-induced stress.

Gas exchange attributes under NaCl stress and with mt treatment

Mt plays vital roles in photosynthesis and photo-protection. Salt stress reduces the absorption of light energy and electron transport in photosystem II (PSII) by reducing chlorophyll content, thereby reducing the photochemical efficiency of PSII. In this study, NaCl treatment led to significant reductions in Pn, Gs, Tr, and Ci relative to the control treatment (Fig. 5). The Mt + NaCl treatment mitigated the adverse impacts of NaCl stress, but this mitigation varied significantly with the concentration of Mt. Nevertheless, Mt-only treatments increased Pn, Gs, Tr, and Ci compared with the NaCl-only treatment. Overall, the treatments showed significant interactive effects on Pn, Gs, Tr, and Ci (Fig. 5).

Fig. 5
figure 5

Leaf gas exchange alterations in L. japonica seedlings in response to melatonin treatment and salt stress. (A) Net photosynthetic rate (Pn), (B) stomatal conductance (Gs), (C) intercellular carbon dioxide (CO2) concentration (Ci), (D) transpiration rate (Tr). Bars represent values as means ± standard deviations

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Expression of key genes in response to NaCl stress and mt treatment

Phenolic compounds have high antioxidant-scavenging capacities and thus their accumulation under stress is related to reductions in oxidative stress. In this study, we chose to quantify the expression of genes involved in the biosynthesis of phenolic compounds in an effort to elucidate the mechanisms of Mt-mediated NaCl-stress tolerance in L. japonica seedlings. The abundances of PAL1, PAL2, CHS, FLS, CAD, and CPK transcripts were analyzed, and the results revealed that the NaCl treatment caused a significant reduction in the expression levels of the aforementioned genes compared with the control treatment (Fig. 6). However, 60-µM Mt treatment increased the expression levels of PAL1 and PAL2 compared with the control treatment.

Fig. 6
figure 6

qPCR analysis of salt tolerance genes of L. japonica seedlings. (A) PAL1, (B) PAL2, (C) CPK, (D) CAD, (E) FLS, and (F) CHS

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The correlation analysis of physiological indicators and gene expression treated with mt and NaCl

To further clarify the relationship between NaCl treatment and key factors in the process of Mt alleviating salt stress, we performed Pearson correlation analysis on all physiological indices, enzyme activities, and gene expression (Fig. 7). The results showed that photosynthesis-related physiological parameters were positively correlated with each other. Antioxidant enzymes also showed high positive correlations with each other. In addition, antioxidant enzymes were highly correlated with the key genes FLS and CHS for flavonoid biosynthesis, which suggested their synergistic effect on the antioxidant process. Soluble sugar, flavonoids, H2O2 level, O2 level, MDA, and APX activity were highly negatively correlated with photosynthesis-related parameters. Similarly, the expression of CPK, CAD, FLS, and CHS was also negatively correlated with these photosynthetic parameters, while the expression of PAL2 was positively correlated with these indicators. In addition, soluble sugar was highly correlated with the CPK gene. Flavonoids were highly correlated with the FLS and CHS genes, with correlation coefficients of 0.99 and 0.97, respectively (Fig. S1).

Fig. 7
figure 7

Correlation analysis between physiological indices and molecular traits under salt (S) and melatonin (Mt) treatments

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Discussion

Mt plays a crucial role in the stimulation of various physiological processes in plants, including growth, development, and the ability to withstand a wide range of environmental challenges [35]. In addition, the principal function of Mt in the human body is to protect against both internal and external oxidative stress [36, 37]. Exogenous and endogenous Mt have both been found to improve plant resilience to abiotic stimuli, including salinity, cold, and drought, while also delaying leaf senescence [1, 38]. In the current study, L. japonica was employed as a model plant to investigate the influence of NaCl stress on growth, photosynthetic pigment composition, and photosynthesis. The aim was to determine whether Mt can improve the hormetic responses of L. japonica to abiotic stress. It has been shown that the stem and shoot tissues of L. japonica have a high tolerance to and accumulation capacity for cadmium (Cd) and a high tolerance to environmental stresses [39,40,41]. The current study demonstrated that NaCl stress caused cellular damage in L. japonica seedlings. However, it also showed that Mt pre-treatment mitigated the detrimental effects of NaCl on the physiological and biochemical processes of seedlings, thereby augmenting their resistance to NaCl stress. In summary, compared with NaCl-alone treatment, Mt + NaCl treatment promoted L. japonica seedling growth by increasing fresh and dry biomass (Fig. 8). Similarly, Mt plays a prominent role in regulating vegetative growth and development of crops under different abiotic stresses [42, 43]. In addition, the efficacy of Mt in improving a condition is contingent upon the concentration of Mt used. Furthermore, Mt was previously shown to enhance root and shoot growth of Zoysia japonica under water-deficient conditions [44].

Fig. 8
figure 8

Schematic diagram of mechanism whereby exogenous melatonin (Mt) endows plants with resistance to salt stress. Mt enhances the activity of antioxidant enzymes by scavenging ROS. Salt stress activates stress response signals and the generation of ROS in the cytoplasm and mitochondria. In response, Mt directly enhances the expression of genes encoding antioxidant enzymes and thereby maintains ionic homeostasis. Mt also triggers overexpression of salt-related genes, leading to reductions in oxidative stress

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The protective effects of Mt against oxidative stress can be ascribed to two mechanisms: counteraction of ROS production and augmentation of antioxidant enzymatic activity, leading to a decrease in ROS concentrations [45]. Moreover, application of Mt to wheat plants under low-temperature stress was found to increase the activity of antioxidant enzymes and decrease the concentration of ROS in the plants [46]. In the current study, exogenous Mt increased vegetative growth and root-to-shoot ratios and decreased ROS production in L. japonica seedlings. Research has indicated that Mt possesses oxidant-scavenging properties and exhibits a wide range of antioxidant activities. In addition, recent investigations proposed that Mt possesses a diversity of functions in addition to antioxidant functions. For example, ROS such as H2O2 were demonstrated to be key factors in the development of Mt-induced stress tolerance in plants [47]. In addition, Mt was found to serve as a downstream signaling molecule in the tomato stress response, facilitated by respiratory burst oxidase homolog-mediated production of H2O2. This finding underscores the role of Mt in enhancing stress resistance through the promotion of H2O2 biosynthesis [48]. Moreover, the ability of Mt to enhance salt tolerance is contingent upon the activation of AtrbohF-dependent ROS signaling, as NADPH oxidase-generated H2O2 is essential to this process [49].

In the current study, cell membrane integrity was evaluated by measuring MDA concentrations and relative electrical conductivities, which are crucial physiological markers [50]. In particular, variations in MDA concentrations and relative conductivities can be used to determine the extent of a plant’s adaptation to environmental fluctuations and the magnitude of any damage it has incurred [51]. In the current study, L. japonica seedlings treated with Mt showed a marked reduction in NaCl-induced electrolyte leakage and MDA production (Fig. 2C-D). Similarly, in a previous report, the application of Mt to tomato plants affected by Cd toxicity resulted in a decrease in the plants’ relative conductivities and MDA concentrations [52]. Furthermore, in the current study, it was shown that prior application of Mt effectively mitigated the toxic effect of NaCl stress in L. japonica seedlings. Taken together, the above-mentioned findings illustrate the potential of Mt to be used to support L. japonica plants by mitigating the adverse effects of NaCl-induced cellular membrane stress.

As mentioned, Mt is a prominent plant growth regulator that protects plants against various environmental stresses [53]. Under salt stress, chlorophyll biosynthesis is inhibited and it is readily degraded [54]. The results of the current study indicate that NaCl stress decreased the chlorophyll content, Pn, Gs, and Ci of L. japonica seedling leaves, suggesting that stomatal limitation was the primary cause of the decrease in the Pn of the leaves. However, Mt treatment of seedlings significantly increased the Pn and decreased the degradation of chlorophyll in their leaves under NaCl stress. These results are consistent with the fact that photosynthesis is a key biological process that is important for the life and productivity of plants. Environmental challenges have been observed to cause a reduction in photosynthesis, which subsequently hampers plant growth and leads to a loss in output. Other research suggests that Mt has the potential impact to enhance photosynthesis and delay leaf senescence. For example, pre-treatment of tomato plants with Mt resulted in significant enhancements in the plants’ total chlorophyll contents [19]. Furthermore, Mt treatment resulted in an increase in fresh biomass and photosynthetic activity [55]. Furthermore, it has been shown that when plants are exposed to salt stress, exogenous Mt mitigates the toxic effect of salt and promotes net photosynthetic activity and total chlorophyll [55, 56]. Moreover, Mt pre-treatment can reduce chlorophyll loss and suppress PSII activity in apple leaves to enhance disease resistance, such as by mitigating the negative effects of apple leaf spot infection [57]. The findings of the current study showed that Mt effectively mitigated the adverse impact of NaCl stress on various physiological parameters of L. japonica seedlings, including fresh biomass and total chlorophyll content (determined by measuring fluorescence of chlorophyll pigments). These effects of Mt led to seedlings exhibiting increased accumulation of photosynthetic pigments, enhanced photosynthetic efficiency, and improved resistance to PSII damage.

H2O2 has been found to enhance the activation of genes involved in the generation of endogenous Mt in the presence of both biotic and abiotic stressors [10, 58]. Similarly, exogenous Mt can be used to mitigate oxidative stress induced by various stressors. As Mt is amphiphilic, it can traverse cell membranes and infiltrate intracellular compartments with considerable efficiency. Furthermore, the unique properties of Mt as a novel growth regulator, including its effectiveness at low concentrations and its environmentally friendly nature, mean that the use of exogenous Mt is a promising strategy for mitigating stress-related damage in crops. In particular, exogenous Mt has the potential to enhance both production and quality of horticultural crops [59]. To fully harness the application potential of Mt, a thorough investigation of the mechanisms underlying its activity is necessary.

Severe stress alters the regulation of Gs and photosynthesis in plants [60, 61]. In response to an increased uptake of salts, plants may release K+ to close their stomata and thereby minimize water lost via transpiration. However, the closure of stomata can reduce CO2 intake, ultimately affecting plant growth and photosystem activity [62]. In the current study, gas exchange parameters (Pn, Ci, Gs, and Tr) of L. japonica seedlings decreased when the seedlings were exposed to NaCl stress conditions (Fig. 5). However, Mt alleviates the adverse effect of salt stress in crops by regulating stomatal conductance and gas exchange [63, 64]. The findings of the current study also align with those of previous research, that is, supplementation with Mt enhanced photosynthetic functions in Ranunculus asiaticus, tomato plants, and Beta vulgaris under salt stress conditions [64,65,66].

The mechanism underpinning plants’ salt tolerance is supported by Mt directly or indirectly modulating stress-related gene expression [12]. Among the genes whose expression is controlled by Mt are those encoding TFs that are essential to cold and drought tolerance [67]. In addition, Mt enhances the expression of genes encoding TFs that are essential to abiotic stress responses in Arabidopsis and medicinal plants. The aforementioned genes include WRKY, MYB, and those encoding various other TFs [68]. The expression of dehydration response element-binding, WRKY, and MYB TFs in Bermuda grass is unregulated by Mt [69, 70]. In the current study, pre-treatment of L. japonica seedlings with Mt led to increases in the expression of PAL and FLS in the seedlings. Exogenous Mt also increased the expression of genes encoding the TFs MYB, bHLH, and WD40. These salt-related TFs can activate genes involved in the abiotic stress response by increasing the transcription of anthocyanin biosynthesis genes (PAL, dihydroflavonol 4-reductase, leucoanthocyanidin dioxygenase, flavonoid-3-O-glucosyltransferase, and glutathione S-transferase), leading to improved antioxidant capacities in cabbage [71]. Phenols, including some lignins and flavonoids, have strong antioxidant effects. Mt mediates membrane peroxidation and ROS accumulation caused by salt stress mainly by increasing the activity of antioxidant enzymes and phenol synthases. In the current study, there were increases in the expression of PAL, FLS, and CHS following exogenous Mt administration, implying that this treatment induces the export of Na+ through the salt defense mechanism [28]. Thus, Mt alleviates salt stress by activating the expression of genes encoding TFs and by activating related signal transduction pathways. Our findings and those of previous studies suggest that there is an intricate relationship between Mt and additional signal transduction pathways that regulate a plant’s response to stress, as depicted in Fig. 8.

Conclusion

In this study, we confirmed that NaCl stress reduces photosynthetic capacity and inhibits growth and biomass production in L. japonica seedlings. In addition, we found that exogenous Mt augmented the NaCl tolerance of L. japonica seedlings. Moreover, exogenous Mt enhanced the production of fresh biomass, increased the activity of antioxidant enzymes, and reduced oxidative damage in L. japonica seedlings by activating genes encoding proteins that alleviated salt stress in the seedlings. Mt also enhanced chlorophyll-related parameters, decreased MDA concentrations, and prevented electrolyte leakage in L. japonica seedlings exposed to NaCl stress. Furthermore, exogenous Mt increased concentrations of SOD, POD, CAT, and APX and increased photosynthetic functions in L. japonica seedlings, thereby enhancing their salt tolerance.

Overall, the findings of this study indicate that exogenous Mt increased transcription of defense-response genes in L. japonica seedlings under NaCl stress. This suggests that exogenous Mt plays a crucial role in the Mt-mediated defense mechanism in plants, indicating that there is a theoretical foundation for using exogenous Mt to reduce salt stress in plants. Further investigations are needed to explore the molecular mechanisms whereby Mt regulates the growth of L. japonica seedlings under NaCl stress.

Data availability

All data and materials used in the study are described in the main text. The transcripts used for RT-PCR analysis were retrieved from transcriptome raw reads deposited in the Sequence Read Archive of the National Center for Biotechnology Information under the accession number SRP417164.

Abbreviations

ROS:

Reactive oxygen species

MDA:

Malondialdehyde

SOS:

Salt overload sensitive

SOD:

Superoxide dismutase

CAT:

Catalase

POD:

Peroxidase

APX :

Ascorbate peroxidase

Mt:

Melatonin

NaCl:

Sodium chloride

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Funding

This work was supported by the National Health Commission Scientific Research Fund (SBGJ202301010), the Joint Construction Project of Henan Provincial Medical Science and Technology Research Program (LHGJ20190550), the High-level Talents Research Initiation Fund of West Anhui University (WGKQ2022025), and the Open Fund of Anhui Engineering Research Center for Eco-agriculture of Traditional Chinese Medicine (WXZR202318).

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  1. College of Biological and Pharmaceutical Engineering, West Anhui University, Lu’an, 237012, China

    Cheng Song

  2. Department of Plant Science, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 201109, China

    Muhammad Aamir Manzoor

  3. The First Affiliated Hospital, College of Clinical Medicine of Henan, University of Science and Technology, Luoyang, 471003, China

    Yanshuang Ren, Jingjing Guo & Yingyu Zhang

  4. Henan Denuoanke Occupational Health Evaluation Co., Ltd, Anyang, 473000, China

    Pengfei Zhang

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Contributions

CS and YYZ designed the study. YSR and JJG conducted the experiments. CS and MAM analyzed the data. CS wrote the draft manuscript. CS, MAM, and YYZ edited the manuscript. PFZ conducted the sampling. YYZ and CS acquired the funding. All the authors have read and approved the final version of the manuscript.

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Correspondence to Yingyu Zhang.

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12870_2024_5506_MOESM1_ESM.jpg

Supplementary Material 1: Fig. S1 Correlation coefficients between physiological indices and molecular traits under salt (S) and melatonin (Mt) treatments

Supplementary Material 2: Table S1 The primers used for qRT-PCR analysis

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Song, C., Manzoor, M.A., Ren, Y. et al. Exogenous melatonin alleviates sodium chloride stress and increases vegetative growth in Lonicera japonica seedlings via gene regulation. BMC Plant Biol 24, 790 (2024). https://doi.org/10.1186/s12870-024-05506-6

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