Exogenous melatonin improves seed germination in Limonium bicolor under salt stress

ABSTRACT

Melatonin involves in improving tolerance to abiotic and biotic stresses by regulating various biological processes. However, little is known about the underlying mechanism. Here, we investigated the effects of exogenous melatonin on seed germination in the halophyte Limonium bicolor under salt stress. Specifically, we examined the effect of salt stress on seed germination, melatonin concentration, and changes in the concentrations of nutrients, amylase activity, and hormones in L. bicolor seeds with and without pre-treatment with melatonin. Seed germination was significantly suppressed under a 200 mM NaCl treatment, but pre-treatment with melatonin significantly improved seed germination under salt stress. During seed germination, seeds pre-treated with melatonin contained high levels of melatonin and gibberellic acid (GA), low levels of abscisic acid (ABA), and high levels of amylase and alpha-amylase activity. Melatonin treatment upregulated the expression of key genes involved in GA biosynthesis (GA20ox and GA3ox), downregulated key genes involved in ABA biosynthesis (LbNCED1 and LbNCED3), and upregulated ABA 8ʹ-hydroxylase genes (LbCYP707A1 and LbCYP707A2), which mediate the changes in GA and ABA levels in seeds during germination. A high melatonin concentration in seeds promotes the utilization of nutrients and the synthesis of new proteins to enhance seed germination.

KEYWORDS:

• Limonium bicolor
• melatonin
• salinity
• germination
• hormone

Introduction

Seed germination is a critical stage of the plant’s lifecycle in which static seeds are converted into highly active, growing seedlings.^1,2 Seed germination begins with water uptake by macromolecular substances in seeds and is attenuated by embryo and endosperm expansion, which terminate in two successive stages: seed coat and endosperm rupture.^3,4 Due to their sessile nature, most plant species remain at the same place throughout their lifecycles, where their seeds germinate. Therefore, endogenous and environmental cues must precisely mediate the timing of seed germination to ensure the reproduction of offspring.⁵

Germination is a highly dynamic process in which macromolecules are re-synthesized to build new structures and are converted into micromolecules. Therefore, the germination process involves the activation of enzymatic pathways (e.g., amylases and proteases), energy acquisition, the building of new structures (e.g., via the phenylpropanoid pathway and laccase), and the metabolism of functional compounds such as hormones and regulators.⁶ Many hypotheses about the regulation of seed dormancy and germination have been proposed and rejected,⁷ and the regulatory mechanisms underlying seed dormancy and germination-induced changes remain unclear. Most of the proposed mechanisms involve certain plant hormones, especially abscisic acid (ABA) and gibberellic acid (GA). ABA functions as a positive regulator that induces and maintains seed dormancy and as a negative regulator of seed germination. GA positively regulates seed germination and can release dormancy. ABA and GA interact to regulate seed germination.^1,6

Salinity (mainly referring to Na⁺) is a major abiotic stress in plants.^8–10 Most plant species are sensitive to salt stress during seed germination and early seedling establishment.¹¹ High Na⁺ concentrations in saline soils inhibit seed germination, mainly because salt reduces the soil water potential, which impairs the ability of seeds to absorb water from the soil, thus suppressing seed imbibition and embryo growth.^1,12,13 Moreover, high Na⁺ concentrations appear to inhibit seed germination by restricting the availability of energy and metabolites. Though halophytes can complete their life cycle under above 200 mM NaCl, the germination was also affected under salinity.¹⁴

Increasing evidence indicates that melatonin (N-acetyl-5-methoxytryptamine) plays vital roles in plant growth and development by improving tolerance to abiotic and biotic stresses by regulating various biological processes.^15–21 The endogenous melatonin content of plants differs among species and can be modulated through genetic transformation.^22,23 Melatonin application to the seeds of various plant species (Brassica oleracea, Cucumis sativus, Zea mays, and Vigna radiata) promoted seed germination and seedling development under environmental tress.^24–27

To date, much of the work examining the roles of melatonin in plants has focused on its physiological effects on plant growth and development, and little is known about its influence on seed germination mechanisms. Limonium bicolor, a salt-secreting halophyte, discharges excess salt from the plant body through salt glands located on the leaf blades. L. bicolor has a high medicinal value and can improve salinity due to its salt tolerance. However, its seed germination is sensitive to salt and increasing its seed germination rate is very important for improving the use of saline-alkali land. In this study, we exogenously treated the seeds of L. bicolor with melatonin under salt stress and observed their germination and associated physiological parameters. The results of this study improve our understanding about the mechanism by which melatonin enhances plant tolerance to salt stress.

Materials and methods

Plant materials

All experiments were performed in a greenhouse at Shandong Normal University, Jinan (36°67΄N, 116°98΄E), China. The Limonium bicolor seeds were collected in Dongying city, Shandong Province (37°43΄N, 118°67΄E), China. The seeds were soaked in 0, 1, 50, 100, 200, 300, or 500 µM melatonin for 12 hours and dried to their initial weight.

Seed germination

Sterilized seeds were plated on sterilized filter paper in 9 cm × 9 cm Petri dishes and incubated at 22°C under a 16:8 h light:dark photoperiod. The seeds were watered daily with 0, 100 mM, or 200 mM NaCl dissolved in 1/5 strength Hoagland nutrient solution. Germination was quantified based on radicle emergence from three replicate experiments, with 100 seeds per replicate.

The calculation formula for each indicator is as follows:

$\begin{array}{rl}& \mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\mathrm{\%}\right)=\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{g}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s}/\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\\ & \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s}\times 100.\\ & \mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{p}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\left(\mathrm{\%}\right)=(\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s}\mathrm{g}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\\ & \mathrm{a}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{t}\mathrm{h}\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{a}\mathrm{y}\mathrm{s}/\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{e}\mathrm{x}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s})\times 100\\ & \mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}\left(\mathrm{G}\mathrm{I}\right)=\sum \left(\mathrm{G}\mathrm{t}/\mathrm{D}\mathrm{t}\right)\end{array}$

In the formula, Gt is the number of seeds germinated at a given time (Dt) measured in days after the start of the germination test.

Determination of Na⁺ and Cl⁻ concentrations

0.1 grams of seeds (seed coats or embryo) were crushed in 1.5 mL of distilled water. And then were boiled in distilled water (10 mL) for 3 h, and then supplemented evaporated water to 10 mL and the solution was filtered. The concentrations of Na⁺ and Cl⁻ in the filtrate were determined with the Dionex ICS-1100 Ion Chroma-tography System (Dionex Corp., Sunnyvale, CA, USA).

Extraction and analysis of sugars

To determine sugar levels in L. bicolor seeds, 15 mg (FW) seed samples were homogenized in 2 mL of 80% (v/v) ethanol solution with a mortar and pestle. And then add 80% (v/v) ethanol solution until the volume is 10 ml. The homogenate was heated in a water bath at 75°C for 10 min and centrifuged at 5,000 g for 10 min to remove the insoluble residue. The supernatants (diluted with 80% ethanol to 10 ml) were further processed prior to soluble sugar measurements according to Liu.²⁸

Preparation of protein extracts

Protein extraction for native PAGE was performed as described in the “Enzyme Extraction and Assay” section.²⁹ To increase the protein concentration in the extracts and for protein purification, the supernatants were concentrated on Amicon ULTRA-0.5 mL 10 kDa Ultracel-PL membrane–centrifugal filters (Millipore, UFC501096) at 20,000 g for 30 min at 10°C. Protein concentration in the extracts was determined using the Bradford (1976) method.

Amylase and α-amylase activity assays

Amylase activity was measured as described by Jacobsen and Varner (1967).³⁰ 100 sterilized seeds were pre-incubated in various concentrations of melatonin at 36°C for 12 h. The seeds were placed on filter paper moistened with different concentrations of NaCl and were incubated for 12 h at 27°C. And then seeds were minced in 2 ml pre-chilled PBS (phosphate buffer saline) solution. A crude enzyme solution was prepared by clarifying the supernatant via centrifugation at 5000 g for 10 min. 1 mL aliquot of diluted supernatant was used in the amylase and α-amylase assays. Amylase and α-amylase activity analysis was carried out using a plant amylase and plant α-amylase Enzyme-linked Immunoassay Kit (double antibody sandwich method, Huding Biological Technology Co., Shanghai, China), respectively. The enzyme units were determined as described by Jacobsen and Varner (1967).

Extraction and determination of GA and ABA concentrations

Each seed sample (15 mg) was homogenized in 1 mL of distilled water and shaken at 4°C overnight prior to the determination of endogenous GA and ABA concentrations. The homogenate was centrifuged at 12,000 g for 10 minutes at 4°C, and the supernatant was aspirated and collected. GA and ABA analysis was carried out using a Plant Gibberellin (GA) and Plant Abscisic Acid (ABA) Enzyme-linked Immunoassay Kit (double antibody sandwich method, Huding Biological Technology Co., Shanghai, China), respectively.

Quantitative real-time PCR analysis

The RNA was extracted from the seeds treated with NaCl and melatonin for two days and reverse transcribed into cDNA. Genes for GA synthesis (GA20ox, GA3ox), ABA synthesis genes (LbNCED1, LbNCED3) and decomposition genes (LbCYP707A1, LbCYP707A2) were selected for qRT-PCR validation. Primers for these 6 genes were designed using Beacon Desiger (Supplementary table). The tubulin gene of the species was used as an internal standard. Real-time PCR was performed with Ace Universal AYBR qPCR Master Mix kit and on a real-time quantitative PCR instrument (Bio-Rad, California, USA). The 2-△△Ct method was used to calculate the relative expression of each gene.³¹

Statistical analysis

The SPSS software package (IBM, SPSS statistics 19.0) was used in the statistical analyzes in the study. Analysis of variance (ANOVA) was performed for determination of the statistical significances. Duncan’s multiple range tests were carried out to identify significant differences between values at P < .05.

Results

The effects of salt stress and melatonin on L. bicolor seed germination

Salt stress inhibited the germination of L. bicolor seeds. As the salt concentration increased, the germination rate significantly decreased (Figure 1a). The germination rate, germination potential (Figure 1b), and germination index (Figure 1c) of the seeds were significantly reduced under a 200 mM NaCl treatment. The germination ability of the seeds initially increased, followed by a decrease, in response to increasing melatonin concentration. Under a 0 mM NaCl (CK) treatment, the germination ability of L. bicolor seeds significantly increased in response to a 1 µM melatonin treatment, whereas under a 100 and 200 mM NaCl treatment, the germination ability of the seeds significantly increased in response to a 200 µM melatonin treatment. Melatonin concentrations ranging from 50–300 µM, especially 200 µM, significantly alleviated the inhibited germination rate (Figure 1a), germination potential (Figure 1b), and germination index (Figure 1c) of L. bicolor seeds in response to salt treatment. We therefore used 200 µM melatonin treatment for subsequent experiments.

Figure 1. Germination of L. bicolor seeds after 7 days of treatment with various concentrations of melatonin (MT) (0 µM, 1 µM, 50 µM, 100 µM, 200 µM, 300 µM, and 500 µM) and NaCl (CK (0 mM), 100 mM, and 200 mM). A: germination rate; B: germination potential; C: germination index. Values are mean ± standard deviation of three biological replicates. Bars labeled with different letters are significantly different at P < .05 according to Duncan’s multiple range tests.

Melatonin concentration in L. bicolor seeds

The melatonin concentration significantly increased in response to a 200 mM NaCl treatment. When exogenous melatonin was applied to L. bicolor seeds under salt stress, the melatonin concentration significantly increased to ~1.5 times that observed in the 200 mM NaCl treatment (Figure 2). Under a 0 mM NaCl (CK) treatment, exogenous melatonin did not increase the melatonin concentration in seeds, but under a 200 mM NaCl treatment, exogenous melatonin significantly increased the melatonin concentration in seeds.

Figure 2. Melatonin concentration of L. bicolor seeds after two days of treatment with different concentrations of melatonin (MT) (0 µM, 200 µM) under 0 and 200 mM NaCl conditions. Values are mean ± standard deviation of three biological replicates. Bars labeled with different letters are significantly different at P < .05 according to Duncan’s multiple range tests.

Germination rates of L. bicolor seeds under various treatments

Whereas 89% of the seeds germinated at 7 d of incubation under normal conditions (CK), only 62.2, 80, and 67.8% of the seeds germinated under 200 mM NaCl, 20 mM LiCl, and 400 mM mannitol treatment, respectively. Treatment with 200 µM melatonin significantly alleviated the inhibition of seed germination by NaCl and mannitol (Figure 3).

Figure 3. Germination rates of L. bicolor seeds after 7 days of treatment with CK, 200 mM NaCl, 20 mM LiCl, and 400 mM mannitol after pre-treatment with 0 and 200 µM melatonin (MT). Values are mean ± standard deviation of three biological replicates. Bars labeled with different letters are significantly different at P < .05 according to Duncan’s multiple range tests.

Effects of melatonin on Na⁺ and Cl ^– contents in seeds, seed coats, and embryos

The Na⁺ concentration in the seed (Figure 4a), seed coat (Figure 4c), and embryo (Figure 4e) and the Cl ^– concentration in the seed (Figure 4b) and seed coat (Figure 4d) were significantly greater under a 200 mM NaCl treatment, and pretreatment with melatonin did not alter the concentrations of these ions in seeds, seed coats, or embryos. The increase in ion concentrations in the seeds was mainly due to the increase in ion concentrations in the seed coat.

Figure 4. Na⁺ and Cl ^– concentration in seeds, seed coats, and embryos of L. bicolor after 2 days of treatment with different concentrations of melatonin (MT) (0 µM, 200 µM) under 0 and 200 mM NaCl conditions. A: Na⁺ concentration in seeds; B: Cl ^– concentration in seeds; C: Na⁺ concentration in seed coats; D: Cl ^– concentration in seed coats; E: Na⁺ concentration in embryos; F: Cl ^– concentration in embryos. Values are mean ± standard deviation of three biological replicates. Bars labeled with different letters are significantly different at P < .05 according to Duncan’s multiple range tests.

Effects of melatonin on soluble sugar and total protein concentrations

Treatment with 200 mM NaCl led to an increase in soluble sugar concentration and a decrease in protein concentration. Under a 200 µM melatonin treatment, a marked decrease in soluble sugar concentration and an increase in protein concentration were detected (P < .05) (Figure 5). Therefore, melatonin treatment decreases the soluble sugar concentration and increases the protein concentration in L. bicolor seeds under salt stress.

Figure 5. Soluble sugar concentration and total protein concentration in L. bicolor seeds after 2 days of treatment with different concentrations of melatonin (MT) (0 µM, 200 µM) under 0 and 200 mM NaCl conditions. A: soluble sugar concentration; B: total protein concentration. Values are mean ± standard deviation of three biological replicates. Bars with different letters are significantly different at P < .05 according to Duncan’s multiple range tests.

Effects of melatonin on amylase and α-amylase activity

Treatment with 200 mM NaCl significantly inhibited amylase and α-amylase activity in L. bicolor seeds. In contrast, the activities of these enzymes significantly increased in response to treatment with 200 µM melatonin under 200 mM NaCl. Thus, melatonin increases amylase (Figure 6a) and α-amylase (Figure 6b) activity in L. bicolor seeds under salt stress.

Figure 6. Amylase and α-amylase activity in L. bicolor seeds after 1 day of treatment with different concentrations of melatonin (MT) (0 µM, 200 µM) under 0 and 200 mM NaCl conditions. A: amylase activity; B: α-amylase activity. Values are mean ± standard deviation of three biological replicates. Bars with different letters are significantly different at P < .05 according to Duncan’s multiple range tests.

Effects of melatonin on gibberellic acid (GA) and abscisic acid (ABA) concentrations

200 mM NaCl treatment significantly decreased the GA concentration under 0 µM melatonin treatment in germinating seeds after two days. 200 µM melatonin treatment significantly increased the GA concentration in seeds (Figure 7a). In contrast, the ABA concentration in germinating seeds after two days significantly increased in response to treatment with 200 mM NaCl and significantly decreased in response to treatment with 200 µM melatonin (Figure 7b). Thus, melatonin appears to play a role in regulating GA and ABA concentrations during seed germination.

Figure 7. GA and ABA concentrations in L. bicolor seeds after 2 days of treatment with different concentrations of melatonin (MT) (0 µM, 200 µM) under 0 and 200 mM NaCl conditions. A: GA concentration; B: ABA concentration. Values are mean ± standard deviation of three biological replicates. Bars with different letters are significantly different at P < .05 according to Duncan’s multiple range tests.

Effects of melatonin on the expression of GA and ABA biosynthesis and ABA hydroxylase genes

Both GA 20-oxidase (GA20ox) and GA 3-oxidase (GA3ox), which are involved in key steps of GA biosynthesis in seeds, were significantly downregulated in response to a 200 mM NaCl treatment, but significantly upregulated in response to 200 µM melatonin and 200 µM melatonin combined with 200 mM NaCl (Figure 8). These effects contribute to the increase in GA content during the subsequent germination periods.

Figure 8. Effects of 200 µM melatonin on the transcript levels of key GA biosynthesis, ABA biosynthesis, and hydroxylase genes during L. bicolor seed germination under NaCl stress. Tubulin was used as an internal standard. Values are mean ± standard deviation of three biological replicates. Bars with different letters are significantly different at P < .05 according to Duncan’s multiple range tests.

Both genes were significantly upregulated by a 200 mM NaCl treatment on the second day of seed germination, but significantly downregulated by treatment with 200 µM melatonin or 200 µM melatonin plus 200 mM NaCl (Figure 8). These results indicate that melatonin reduces ABA biosynthesis under salt stress. The activity of the ABA 8ʹ-hydroxylases LbCYP707A1 and LbCYP707A2 causes a rapid decrease in ABA levels during seed germination. LbCYP707A1 was downregulated by the 200 mM NaCl treatment but was significantly upregulated by treatment with 200 µM melatonin or 200 µM melatonin plus 200 mM NaCl. Finally, LbCYP707A2 was significantly upregulated by treatment with 200 µM melatonin or 200 µM melatonin plus 200 mM NaCl.

Discussion

Exogenous melatonin significantly promoted the germination of L. bicolor seed under salt stress. Melatonin is considered to be a biostimulator, as it is a non-toxic substance of natural origin that can improve and stimulate plant health.^32,33 Endogenous melatonin concentrations differ not only among plant species but also among varieties of the same species.³⁴ An analysis of endogenous melatonin contents in different plant organs and seeds revealed that the highest levels of this compound are found in reproductive organs, particularly seeds,^33,35 which might be related to its role in the process of seed germination. In addition, increasing evidence suggests that changes in melatonin contents might result from various types of environmental stress.³⁶

Salt stress is a major abiotic stress.^8–10 Both halophytes and non-halophytes are highly sensitive to salt stress during seed germination and early seedling establishment.^7,10,11 Seed germination is one of the most critical stages of the plant’s lifecycle and is greatly affected by salinity.³⁷ Many studies have shown that high salinity levels completely inhibit seed germination and that low levels induce seed dormancy.³⁸ In addition, salinity disrupts the hormonal balance in plants^14,39 and reduces the ability to use stored substances in seeds.⁴⁰ In the current study, we found that high salt stress impaired seed germination, decreased the GA concentration, increased the ABA concentration, and reduced the utilization of seed reserves in L. bicolor (Figures 1, 5, and 6). High salt levels have two major effects on plants: the induction of osmotic stress and ion toxicity.¹⁴ The current results indicate that the low germination rate of L. bicolor seeds under salt treatment was mainly due to the osmotic effect of high salt levels (Figure 3).

Melatonin promotes L. bicolor seed germination under salt stress (Figure 1). The inhibitory effect of high salinity on L. bicolor seed germination was alleviated by the application of 200 µM melatonin, which is consistent with its previously reported effects on seed germination.⁴¹ We found that the endogenous melatonin concentration in L. bicolor seeds increased under salt stress. Moreover, exogenous melatonin treatment significantly increased melatonin levels in seeds, while there were no changes in melatonin levels under control conditions (Figure 2). Therefore, in this study, we demonstrated that exogenous melatonin increases the melatonin concentration in L. bicolor seeds and promotes seed germination under salt stress.²¹

The high melatonin concentration in seeds upregulated the expression of key genes (GA20ox and GA3ox, Figure 8) involved in GA biosynthesis, ⁴² which led to an increase in GA concentration during the subsequent germination periods. Nine-cis-epoxycarotenoid dioxygenase (NCED) is a key regulatory enzyme involved in ABA biosynthesis.⁴³ The key genes responsible for ABA biosynthesis in L. bicolor, LbNCED1 and LbNCED3 were downregulated during seed germination in melatonin-pretreated seeds under salt stress. The transcriptional levels of the ABA 8ʹ-hydroxylase genes LbCYP707A1 and LbCYP707A2, which play major roles in the rapid decrease in ABA content during seed germination, ⁴⁴ were upregulated during seed germination in melatonin-pretreated seeds under salt stress. It appears that the downregulated expression of LbNCED1 and LbNCED3 and the upregulated expression of LbCYP707A1 and LbCYP707A2 are responsible for the decrease in ABA content in these seeds.

The quantity and utilization of seed reserves directly affect early seedling growth.⁷ Seed germination in L. bicolor is negatively affected by salinity stress, as observed in other plants.^45,46 Seed germination involves two distinct metabolic processes: (1) enzymatic hydrolysis of seed storage proteins and (2) the formation of new cellular structures.^47,48 The reduced germination rate of L. bicolor is due to reduced nutrient availability under salt stress (Figure 5). Melatonin promotes soluble sugar utilization and the synthesis of new proteins, thereby increasing the germination rate of L. bicolor seeds under salt stress. However, the mechanism by which melatonin promotes nutrient utilization in seeds is not clear. Perhaps the increase in GA levels in seeds promotes the synthesis of hydrolases (especially α-amylase) (Peng and Harberd, 2002).⁴⁹ To explore how melatonin improves nutrient utilization during seed germination, we measured amylase activity in seeds. During seed germination, amylase activity rapidly decreased under NaCl stress compared to the control (Figure 6). This reduction in amylase activity was less when plants were pretreated with melatonin, confirming that melatonin protects enzymes from high salinity-induced damage. Indeed, melatonin stimulates the secretion of pancreatic amylase and l-tryptophan (the precursor of melatonin) as well as enzyme secretion in rats.⁵⁰ However, the mechanism by which melatonin affects amylase activity in plants is unclear.

In this study, we showed that exogenous melatonin improves L. bicolor seed germination under salt stress. Melatonin promotes ABA catabolism and GA biosynthesis under salt stress conditions and regulates seed germination by promoting nutrient utilization and protein synthesis in seeds. This is the first study exploring the mechanism by which melatonin mitigates the inhibitory effects of salt stress on seed germination in halophytes. It may provide a new idea for MT modulation in the regulation of plant salt defenses. This study represents a step toward defining the role of melatonin in the response to adverse conditions in halophytes. Moreover, these results provide a theoretical basis for MT to alleviate salt stress in L. bicolor.

Authors’ Contribution Statement

Junpeng Li and Chen Zhao wrote this manuscript; Mingjing Zhang and Chen Zhao performed experiments; Junpeng Li and Mingjing Zhang collected data, carried out all analyses and were involved in preparing figures; Fang Yuan and Min Chen conceptualized the idea. All authors read and approved the final manuscript.

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

Supplemental data for this article can be accessed on publisher’s website.

Additional information

Funding

This work was supported by Shandong Province Natural Science Foundation (ZR2019MC065), the major projects of science and technology in Shandong province (2017CXGC0313), National Natural Science Research Foundation of China (NSFC, project No. 31600200), the Shandong Province Key Research and Development Plan (2015ZDJS03002) and the Program for Scientific Research Innovation Team in Colleges and Universities of Shandong Province.