Melatonin-Induced Resilience Strategies against the Damaging Impacts of Drought Stress in Rice

Abstract

Drought is a severe abiotic stress that imposes a serious threat to rice productivity. Although previous studies have found that melatonin can promote seed germination, the physiological regulation mechanism of drought tolerance in rice seed germination mediated by exogenous melatonin is still unclear. In order to overcome these challenges, polyethylene glycol 6000 (PEG6000) at concentrations of 20% and 35% was used to simulate osmotic stress. Rice seeds were treated with different concentrations of melatonin (i.e., 0, 20, 100, and 500 μM) to study the effects on germination characteristics, growth and development, superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, malondialdehyde (MDA) content, and soluble protein content. The results showed that the seed soaking treatment using melatonin at a concentration of 100 μM under drought stress effectively promoted the germination rate and improved the biomass of rice seed shoots and roots. Meanwhile, this treatment reduced MDA content to alleviate the oxidative damage of rice seeds caused by drought stress. The two-way ANOVA showed that the effect of single melatonin soaking treatment on rice seed germination was more significant than that of single drought stress and the interaction of drought stress and melatonin (p < 0.05). Using the membership function method, it was shown that the critical gradient of rice seeds under drought stress was 35%, and the critical treatment of interactive treatment was 35% + 100 μM. Through grey correlation analysis, it was found that germination rate (7 d) had the highest grey correlation with melatonin seed soaking treatment to evaluate the mitigation effect of melatonin on drought stress. This study provides a theoretical basis for light and simple cultivation technology for the dry direct seeding of rice.

1. Introduction

Rice (Oryza sativa L.) is an important food crop in the world. As one of the most important abiotic stresses restricting rice production, drought can not only destroy metabolic processes at the cellular level and reduce the production of ATP, but also reduce respiration and slow the germination and growth of rice seeds [1]. Seed germination is a complex process that is regulated by a series of physical and metabolic events in the life cycle of plants [2]. As the initial stage of seedling establishment, rice seed germination is extremely sensitive to drought stress. Drought stress causes the excessive production of reactive oxygen species (ROS) in plant cells, which leads to oxidative damage and cell death [3]. However, rice seeds can effectively improve the antioxidant defense mechanism by regulating the activities of antioxidant enzymes and the regulation of non-enzymatic antioxidants to reduce the oxidative damage induced by ROS [4]. Therefore, it is particularly important to study the physiological mechanism of drought resistance during rice seed germination.

Antioxidants are vital scavenging components of ROS in rice. The increase in antioxidant activities can improve the drought tolerance of rice [5]. The ROS include superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (OH⁻), ozone (O₃), and singlet oxygen (¹O₂), and they cause lipid peroxidation, cellular oxidative damage, disruption in cellular homeostasis, DNA mutations, and protein denaturation [6]. The antioxidant system for eliminating ROS can be divided into the enzyme system and non-enzyme system. The enzyme system includes superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol peroxidase (GPX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and ascorbate-glutathione cycle enzyme, while glutathione (GSH) and ascorbate (AsA) are part of the non-enzyme system within the cell [7]. The antioxidant activities in the enzyme system and non-enzyme system of rice can be activated under drought stress. The increase in these antioxidant enzyme activities can explain the protective effect of oxidative damage induced by drought stress [8].

Melatonin is a natural biological stimulant that can regulate plant growth and development and improve plant resistance to various abiotic stresses [9]. Seed soaking with 500 μM melatonin by reducing the lipid peroxidation of the cell membrane and increasing the activities of antioxidant enzymes such as SOD, POD, CAT, and APX to minimize the excessive generation of ROS promoted the germination of soybean seeds under water stress [10]. Melatonin can regulate the response of crops to water deficit by regulating the biosynthesis of secondary metabolites such as phenylpropanoids, flavonoids, and steroids, and ultimately alleviate the adverse effects of drought stress on crop growth [11]. Exogenous spraying with 50 μM melatonin can significantly enhance the expression of melatonin synthesis-related genes, increase antioxidant enzyme activity, reduce oxidative damage, and improve salt tolerance in purple alfalfa [12]. Melatonin can effectively alleviate the negative effects of low-temperature stress on the antioxidant system and photosynthetic system of rice seedlings [13]. Melatonin can promote the growth and development of melon under copper stress by inhibiting the biosynthesis of jasmonic acid [14]. Melatonin can alleviate the damage of heat stress to Pinellia ternata by regulating ROS scavenging enzymes, hormone signal transduction, photosynthesis, and other transcription factors such as HSFs [15]. Melatonin treatment can improve the starch metabolism and related enzyme activities of maize under low-temperature stress to improve the germination rate of maize seeds [16]. Melatonin pretreatment could significantly reduce the electrolyte leakage, malondialdehyde (MDA) content and H₂O₂ content, increase the activities of SOD, POD, and CAT, and upregulated of genes related to melatonin and antioxidant enzyme biosynthesis to improve the salt tolerance of alfalfa plants [12]. A 100 μM melatonin application as soil drenching could enhance plant growth, and alleviate ROS-induced oxidative damages by increasing the photosynthetic pigments, antioxidant enzyme activities, relative water content, and osmo-protectants of maize seedlings [17].

At present, melatonin has been studied in crop seed germination and grain filling, but there has been no report on the resilience of rice seed germination under drought stress. Therefore, this experiment used the rice variety ‘XZX45′ to study the effect of melatonin on the germination and physiological characteristics of rice seeds under different drought stress conditions. Four melatonin concentrations (0, 20, 100, and 500 μM) were used to screen out the most optimal concentration for rice seed germination and growth to explore its physiological regulation mechanism under drought stress. This study may provide a foundation for the molecular mechanism involved in melatonin regulation of rice seed germination under drought stress.

2. Materials and Methods

2.1. Experiment Materials

All rice seeds used in this study were of the ‘XZX45′ rice cultivar that were harvested in July 2020. ‘XZX45′ is a rice cultivar that is widely grown in the Yangtze Valley double-crop rice region of China. Melatonin (N-acetyl-5-methoxytryptamine, MT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). All chemicals were of analytical grade in this study.

2.2. Experimental Design

Selected plump and uniform rice seeds of the same size were sterilized with 5% sodium hypochlorite solution for 40 min and then rinsed four times with sterilized distilled water. After the seeds were sterilized, they were soaked with different concentrations of melatonin for 24 h, each 100 seeds were sown in repetition, and each treatment had six repetitions for the determination of physiological and biochemical indicators. Aerating the solution was used during soaking. The following melatonin solution treatments were used: M0 (0 μM melatonin, i.e., distilled sterile purified water only), M20 (20 μM melatonin), M100 (100 μM melatonin), and M500 (500 μM melatonin). In this experiment, polyethylene glycol 6000 (PEG6000) was used to simulate drought stress and the concentrations were 25% (T25) and 35% (T35). The seeds were evenly sown in a germination box covered with sterilized germination paper, and a 7 mL PEG6000 solution with different concentrations was added, and then they were placed in a light incubator. The light conditions were as follows: fluorescent light with intensity expressed as a PPFD of 150 mmol m⁻² s⁻¹ for 14 h/day, and 70% relative humidity (RLD-1000E-4, Ningbo Ledian Instrument Manufacturing Co. Ltd., Ningbo, Zhejiang, China). During the germination of rice seeds, the number of germinated seeds was observed on the 7th, 10th, and 14th days. The shoots and the roots of the rice seeds were taken on the 14th day, respectively, to determine the relevant indicators.

2.3. Determination Items and Methods

2.3.1. Determination of Seed Germination Rate

The germination rate was measured on the 7th, 10th, and 14th days after seed germination. A seed was considered to be germinated when the total shoot length exceeded half the length of the seed and when the root length exceeded the length of the seed.

Germination rate (GR, %) = (Germinated seedlings/number of total seeds) × 100

(1)

2.3.2. Determination of Growth Indices of Seed Shoots and Roots

On the 14th day after sowing, five plants from each treatment were taken to measure the growth of the shoot and the root, which were divided into five replicates. The measurement of the shoot length was determined by the base of the root to the growth point; the measurement of the root length was based on the length of the main root.

2.3.3. Determination of Physiological and Biochemical Indices

Superoxide dismutase (SOD) activity was determined by the nitrogen blue tetrazole method. In a cuvette, 0.4 mL deionized water, 0.25 mL phosphate buffer, 0.1 mL of 0.1% Triton-X, 0.1 mL of 13 mM L-methionine, 0.05 mL nitroblue tetrazolium, 50 μL of 1.3 μM riboflavin, and 50 μL enzyme extract were added. The OD of the samples was noted at 560 nm after 15 min [18]. Peroxidase (POD) activity by the guaiacol method was determined by preparing a reaction mixture (100 μL of 0.5% H₂O₂, 100 μL of 0.5% guaiacol, 1.8 mL phosphate buffer, and 100 μL enzyme extract). The OD of this mixture was noted at 470 nm for a 30 s time internal up to 3.0 min [19]. Catalase (CAT) activity was carried out by the UV absorption method. To 100 μL of the extract, 1 mL of 5.9 mM H₂O₂ and 1.9 mL of 50 mM phosphate buffer were added. The absorbance of each treated sample was noted at 240 nm for 180 s [20]. Malondialdehyde (MDA) content was conducted by the thiobarbituric acid color method. A total of 2 mL extraction solution and 2 mL 0.6% TBA were mixed vigorously, and boiled in a water bath for 15 min. The supernatant was taken to measure the OD at 450 nm, 532 nm, and 600 nm [21]. Soluble protein content was conducted by the Coomassie brilliant blue G-250 staining method. To a 100 μL aliquot of the sample, 2.0 mL of the Bradford reagent was mixed, and the OD was read at 590 nm [22].

2.4. Statistical Analysis

The data were sorted and calculated by Microsoft Excel 2017 software. The SPSS statistics 20.0 data processing system was used for statistical analysis. GraphPad Prism 8.4 was used for drawing. Duncan’s new complex difference method was used for the difference test at the level of p ≤ 0.05. The grey correlation degree refers to the method of Su et al. [23]. The membership function method was used to comprehensively evaluate the tolerance of rice seeds to melatonin, drought single stress, and interactive stress [24]. The specific membership function value calculation formula of each index of each sample is as follows:

Xu = (X − Xmin)/(Xmax − Xmin)

(2)

Xu = 1 − (X − Xmin)/(Xmax − Xmin)

(3)

In the formula, X is the measured value of a resistance index of the test sample, and Xmax and Xmin are the maximum and minimum values of the index in all samples, respectively. If the measured indices were positively correlated with the tolerance of rice seeds, Equation (2) was used to calculate the membership value, and Equation (3) was used for negative correlation. Finally, the membership function values of each index for each sample were accumulated, and the average value was taken.

3. Results

3.1. Germination Rate

The germination rate of rice seeds decreased gradually with increasing drought stress concentration (

Table 1. Effects of exogenous melatonin on the germination rate of rice seeds under drought stress. Means ± SEs with different letters in each parameter indicate significant differences (LSD, 0.05).

Drought Stress Treatment	Melatonin Treatment	Germination Rate (7 d)/%	Germination Rate (10 d)/%	Germination Rate (14 d)/%
T25	M0	19.36 ± 1.45 d	71.01 ± 1.85 c	81.39 ± 2.91 b
	M20	30.94 ± 0.42 c	78.93 ± 3.31 b	88.5 ± 0.21 a
	M100	42.48 ± 1.14 a	90.56 ± 0.69 a	92.68 ± 1.29 a
	M500	38.39 ± 0.9 b	82.61 ± 1.38 b	93.94 ± 0.64 a
T35	M0	0	52.2 ± 3.23 c	75.41 ± 1.91 b
	M20	0	61 ± 2.9 bc	75.66 ± 2.12 b
	M100	0	67.95 ± 4.38 ab	85.16 ± 0.28 a
	M500	0	75.53 ± 3.56 a	86.89 ± 0.23 a

). In the drought stress T25 treatment, the germination rate on the 7th day, 10th day, and 14th day of each concentration of melatonin were significantly higher than that of the control M0. Among them, the M100 had the most obvious promoting effect on the germination rate. In the drought stress T35 treatment, the rice seeds of each treatment were inhibited by drought stress and could not germinate on the 7th day of seed germination. After recovery treatment, the germination rate of M100 and M500 on the 10th and 14th day was significantly higher than that of the control M0, which increased by 30.17%, 44.69% and 12.93%, 15.22%, respectively.

3.2. Biomass of Rice Seed Shoots and Roots

In the drought stress T25 treatment, the shoot length, root length, fresh weight of shoots, fresh weight of roots, dry weight of shoots, and dry weight of roots treated with the M100 were significantly higher than those of the control M0, increasing by 12%, 56.45%, 25.71%, 102.55%, 10%, and 104.62%, respectively (Figure 1A–F). Melatonin with M20 compared with the control M0 also promoted the shoot length, fresh weight of shoots, and dry weight of shoots, which increased by 6.18%, 23.96%, and 4.71%, respectively. In the drought stress T35 treatment, the M20 and M100 significantly increased the shoot length, root length, fresh weight of shoots, fresh weight of roots, dry weight of shoots, and dry weight of roots of rice seeds. The M100 had the most obvious promoting effect compared with the control M0, increasing by 38.14%, 78%, 24.64%, 187.32%, 21.15%, and 153.14%, respectively (Figure 1A–F).

3.3. SOD Activity in Rice Seed Shoots and Roots

In the drought stress T25 treatment, the SOD activity of shoots treated with M100 and M500 increased by 85.81% and 89.65%, respectively, compared with the control M0 while there was no significant difference in the SOD activity of roots between the melatonin of each concentration (Figure 2A,B). In the drought stress T35 treatment, the M20, M100, and M500 increased the SOD activity of rice seed shoots and roots by 12.62~34.82% and 79.02~101.94%, respectively, compared with the control (Figure 2A,B).

3.4. POD Activity of Rice Seed Shoots and Roots

In the drought stress T25 treatment, melatonin had a more obvious effect on the POD activity of rice roots. The M20 and M500 significantly increased the POD activity of rice seed roots as they were 63.98% and 22.17% higher, respectively, than that of the control M0 (Figure 3A,B). In the drought stress T35 treatment, the POD activity of rice seed shoots treated with the M500 was 40.39% higher than that of the control M0, and the POD activity of rice seed roots treated with M20 was 26.24% higher than that of the control M0 (Figure 3A,B).

3.5. CAT Activity of Rice Seed Shoots and Roots

In the drought stress T25 treatment, the M20 and M100 could significantly increase the CAT activity of rice seed shoots by increasing 66.37% and 55.94%, respectively, and the CAT activity of rice seed roots by increasing 108.28% and 92.45%, respectively, when compared with the control M0 (Figure 4A,B). In the drought stress T35 treatment, the M100 significantly increased the CAT activity of rice seed roots, which was 71.25% higher than that in the control M0 (Figure 4A,B).

3.6. MDA Content in Rice Shoots and Roots

In the drought stress T25 treatment, compared with the control M0, the MDA content of rice seed shoots and roots treated with the M100 decreased by 28.44% and 26.69%, respectively (Figure 5A,B). In the drought stress T35 treatment, M20, M100 and M500 could significantly reduce the MDA content of rice seed shoots and roots, which was 30.22~41.04% and 18.92~41.19% lower than the control M0, respectively (Figure 5A,B).

3.7. Soluble Protein Content of Rice Seed Shoots and Roots

In the drought stress T25 treatment, M20, M100, and M500 significantly increased the soluble protein content of rice seed shoots, which was 7.09~62.89% higher than that of the control M0. Melatonin at the M100 significantly increased the soluble protein content of rice roots, which was 35.49% higher than that of the control M0 (Figure 6A,B). In the drought stress T35 treatment, the soluble protein content of the roots of rice seeds treated with the M100 increased by 20.28% compared with the control M0 (Figure 6A,B).

3.8. Correlation Analysis of Exogenous Melatonin Concentration on Rice Seed Germination and Seedling Growth under Drought Stress

According to modern agricultural grey system theory, closer changing trends of the experimental array and reference array indicate a closer mutual relation. The analysis of the grey correlation degree between rice seed germination, seedling physiological indices, and melatonin seed soaking concentration under drought stress is shown in Figure 7. The germination rate (7 d), MDA content in the shoots, and MDA content in the roots were the comprehensive indicators of the antioxidant system in rice seeds and were closely related to the melatonin seed soaking concentration. These could be used as indicators to measure the mitigation effect of melatonin on rice seed germination under drought stress. In general, the grey correlation between the germination rate (7 d) and the melatonin seed soaking concentration was the highest, which showed that the germination rate (7 d) was the index that could best evaluate the mitigation effect of melatonin on drought stress.

Correlation analysis showed that there was a very significant positive correlation between the germination rate at 7 d, 10 d, and 14 d (p < 0.01) (Figure 8). The germination rate (7 d) was positively correlated with shoot fresh weight and shoot dry weight (p < 0.01), but positively correlated with shoot length (p < 0.05), indicating that shoot growth had a certain positive effect on the germination rate of rice seeds. The germination rate (7 d) was positively correlated with shoot dry weight (p < 0.01) and with root fresh weight (p < 0.05), indicating that there was consistency between the germination rate (7 d) and shoot weight. The MDA content of shoots was significantly negative correlated with root length and root dry weight (p < 0.01), while it was significantly negatively correlated with root fresh weight (p < 0.05). The MDA content of roots was negatively correlated with root length and root dry weight (p < 0.01).

3.9. Comparison of Differences and Comprehensive Evaluation of Tolerance between Drought Stress and Exogenous Melatonin Interaction

The two-way ANOVA results of the effects of interactive stress on rice seed germination parameters and physiological indicators are shown in

Table 2. Two-factor analysis of variance for the effects of drought stress and exogenous melatonin concentration on rice seed germination and physiological indices.

Indices	Drought Stress Treatment			Melatonin Treatment			Interactive Treatment
	Df	F	p	Df	F	p	Df	F	p
Germination rate (7 d)	1	3938.563	<0.001	3	94.312	<0.001	3	94.312	<0.001
Germination rate (10 d)	1	65.489	<0.001	3	16.942	<0.001	3	2.643	0.085
Germination rate (14 d)	1	59.215	<0.001	3	27.403	<0.001	3	1.994	0.156
Shoot length	1	57.112	<0.001	3	67.067	<0.001	3	13.534	<0.001
Root length	1	0.005	0.943	3	16.687	<0.001	3	0.837	0.493
Shoot fresh weight	1	44.097	<0.001	3	17.274	<0.001	3	0.557	0.651
Root fresh weight	1	21.701	<0.001	3	34.146	<0.001	3	3.846	0.03
Shoot dry weight	1	335.614	<0.001	3	28.181	<0.001	3	2.608	0.087
Root dry weight	1	33.087	<0.001	3	34.936	<0.001	3	7.38	0.003
SOD activity of shoot	1	82.163	<0.001	3	19.495	<0.001	3	6.462	0.005
SOD activity of root	1	9.871	0.006	3	5.277	0.01	3	3.7	0.034
POD activity of shoot	1	34.983	<0.001	3	6.18	0.005	3	3.729	0.033
POD activity of root	1	14.308	0.002	3	17.848	<0.001	3	3.013	0.061
CAT activity of shoot	1	61.537	<0.001	3	17.758	<0.001	3	9.196	0.001
CAT activity of root	1	71.022	<0.001	3	36.756	<0.001	3	4.746	0.015
MDA content of shoot	1	4.263	0.056	3	16.494	<0.001	3	11.673	<0.001
MDA content of root	1	1.666	0.215	3	11.224	<0.001	3	8.819	0.001
Soluble protein content of shoot	1	154.346	<0.001	3	58.075	<0.001	3	46.606	<0.001
Soluble protein content of root	1	3.486	0.08	3	9.752	0.001	3	2.543	0.093

. Under the single drought stress treatment, there were very significant differences in the indices (p < 0.01), except for the root length of rice seeds, the MDA content of shoots, the MDA content of roots, and the soluble protein content of roots. Under the treatment of a single melatonin concentration, the dry weight and soluble protein content of rice seed shoots were significantly different (p < 0.05). There were very significant differences in various germination parameters and physiological indices of rice seeds (p < 0.01). Under the interactive treatment of drought stress and melatonin seed soaking concentration, there was no significant difference in germination rate on the 10th day, germination rate on the 14th day, shoot length, shoot fresh weight, shoot dry weight, root dry weight, root SOD activity, shoot POD activity, root POD activity, and root soluble protein content, but there were very significant differences in the other indices (p < 0.01).

Through the above analysis of seed germination and changes in seedling physiological indicators, it was found that rice seeds have certain adaptive mechanisms under single and interactive stress. There was a critical stress concentration under different stresses. When the stress concentration was exceeded, the adaptability of rice seed to this stress decreased (Figure 9). The membership function was used to comprehensively evaluate the drought stress concentration, melatonin soaking concentration, and tolerance under the two interactive treatments. The results showed that under drought stress alone, the mean value of the membership function of the T35M0 was the largest. Under the interactive treatment of drought stress and melatonin, the membership function value of the T35M100 was the largest. T35M100 was the critical concentration of interactive stress, and its membership function value was higher than that of T35M0, which performed best under a single drought treatment.

4. Discussion

Drought stress can affect seed germination and inhibit seedling growth and development [25]. Studies have shown that the germination rate, germination potential, seed vigor index, root length, shoot length, and dry matter quality of plant seeds reflect the drought resistance of seeds to a certain extent [26]. Melatonin (100 μM) pretreatment could efficiently improve the seedlings’ growth, root characteristics, leaf photosynthesis, and antioxidant machinery under drought stress, thereby increasing the seedlings’ adaptability to drought stress [27]. The mitigating potential of 100 μM melatonin with an optimum level of nitrogen (250 kg N ha⁻¹) improved the plant growth and photosynthetic efficiency of maize seedling under drought-stress conditions [28]. The 100 μM melatonin could enhance the boll distribution characteristics and control the boll shedding rate, which have been proven to be more helpful in stimulating cotton sympodial leaf physiological attributes including leaf gas exchange parameters, sugar metabolism, proline content, and antioxidant defense systems compared with less or no melatonin application during drought conditions [29]. In this study, the results showed that melatonin could promote the germination of rice seeds, the growth of roots and shoots, and the accumulation of dry matter. The best experimental effect appeared when the concentration of melatonin was 100 μM. The same study found that presoaking seeds with 100 μM melatonin had a positive effect on the number and opening of cotton seed coats, and it could alleviate drought stress by improving the germination rate, germination potential, germination index, and antioxidant enzymes of cotton seeds [30].

Drought stress can induce plants to accumulate a large amount of reactive oxygen species (ROS), induce oxidative stress, increase malondialdehyde (MDA) content, damage the structure and function of the plant cell membrane, and then inhibit the normal growth of plants [31]. The scavenging network of ROS, which includes enzymatic antioxidants such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and non-enzymatic antioxidants including glutathione, tocopherols, and ascorbate (non-enzymatic) antioxidants helps mediate the harmful effects of stressful factors in plants [32]. SOD could dismutase O₂⁻ into H₂O₂ to reduce cell damage, POD could use the different substrates as electron donors to restore H₂O₂ into water, and CAT could break H₂O₂ into water and oxygen [33]. Soluble protein is a key osmoregulatory substance that can enhance the osmoregulatory ability of plants [34]. Previous studies confirmed that improving the activity of plant antioxidant enzymes could improve the drought resistance of plants [35]. The 100 μM melatonin could enhance the total proline content and antioxidant activity, which caused a reduction in the total MDA content and hydrogen peroxide (H₂O₂) concentrations in cotton leaves during drought conditions [29]. The 100 μM melatonin application enhanced plant growth, alleviated ROS-induced oxidative damage by increasing the photosynthetic pigments, antioxidant enzyme activities, relative water content, and osmo-protectants of maize seedlings. In our experiment, the results showed that a certain concentration of melatonin could increase the activities of SOD, POD, and CAT in rice seed buds and roots under drought stress and reduce the content of MDA. In particular, when the melatonin concentration was 100 μM, the activities of SOD, POD, and CAT and the content of soluble protein in rice roots and shoots were significantly higher than those under drought treatment alone, and the content of MDA was significantly lower. This showed that melatonin can alleviate the damage to the antioxidant system of plants under drought stress by enhancing the activity of antioxidant enzymes and the content of osmoregulation substances in the roots and shoots of rice seeds and by reducing the degree of membrane lipid peroxidation. Under different drought stress conditions, different concentrations of melatonin have different regulation on the activities of various antioxidant enzymes. This finding may be due to the different characteristics of each antioxidant enzyme. Additionally, it has been confirmed that the increase in single antioxidant enzymes is not enough to increase the stress response of plants, which must be realized through the synergy between various protective enzymes [36]. Therefore, the interaction of SOD, POD, and CAT enzymes may play a role in balancing plant oxidative metabolism to reduce oxidative damage [37].

In the grey relational grade analysis, many factors (i.e., traits) are regarded as grey systems and integrated for a unified comparison. The importance of each factor is expressed according to the relational grade and order. The relative importance of factors also remains stable, which can then more accurately indicate the impact of each trait on the yield [38]. In this study, the grey correlation degree analysis was used to comprehensively evaluate the correlation between melatonin and various characteristics of rice seed germination. The results showed that germination rate (7 d), MDA content in the shoots, and MDA content in the roots accurately reflected the stress mitigation of melatonin, especially under drought conditions. This also shows that it is necessary to reduce the content of malondialdehyde in plants, in order to further promote the germination and growth of rice seeds under drought stress. Among these indexes, germination rate (7 d) was most closely related to the concentration of melatonin. Therefore, germination rate (7 d) is the first choice to evaluate the effect of melatonin on alleviating drought stress, and is the fundamental factor determining the drought tolerance of rice seeds. Therefore, in the evaluation of drought resistant germplasm and variety breeding of rice, the selection and utilization of germination rate (7 d) should be strengthened. The rice seed selection technology based on physiological and biochemical characters should be established to improve the efficiency and accuracy of seed selection.

When plants are subjected to environmental stress, the cell membrane is first damaged, resulting in membrane lipid peroxidation, extravasation of intracellular lysates, and accumulation of membrane lipid peroxidation product MDA [32]. MDA is an important product of membrane peroxidative decomposition, which can react with proteins. The accumulation of MDA can cause damage to the cells again, leading to a series of physiological and biochemical reactions [33]. To a certain extent, its content can indicate the level of membrane peroxidation and the degree of cell damage. Therefore, MDA is an important index to judge the degree of plant stress. The higher the content of MDA, the greater the degree of plant damage. In this study, the MDA content of shoots was significantly negatively correlated with root length and root dry weight (p < 0.01), while it was significantly negatively correlated with root fresh weight (p < 0.05). The MDA content of roots was negative correlated with root length and root dry weight (p < 0.01). This shows that the higher the content of MDA, the more unfavorable it is to the growth of rice seed roots. Previous studies have also shown that drought stress can cause membrane lipid peroxidation, destroy membrane structure and function, weaken tissue reduction ability, and inhibit rice seed germination [27].

The stress resistance characteristics of plants such as drought resistance, cold resistance, and salt resistance are essentially a cross and complex physiological change process. In the evaluation of the drought tolerance of rice seeds, the drought tolerance threshold based on germination rate is often used to directly compare the degree of drought tolerance during germination, but using only the germination rate may not fully and effectively reflect the drought tolerance during germination. The membership function method converts each index into the value of 0–1 through dimensionless to put different indexes to the same order of magnitude, enhance the comparability between different indexes, and can better quantitatively evaluate each treatment [39]. To determine the critical point in the response of rice seeds to drought stress, the membership function comprehensive analysis method was used to comprehensively evaluate various indices. The study found that the critical gradient of single drought stress was 35%, and the critical gradient of interactive treatment was 35% + 100 μM. Compared with the single drought stress treatment, the maximum value of the membership function of the interactive treatment was the best, which further verified that melatonin could improve the drought resistance of rice seeds. This was the first study exploring the mechanism by which melatonin mitigates the inhibitory effects of rice seed germination under drought stress. It may provide a new idea for melatonin modulation in the regulation of plant drought defenses. Moreover, these results provide a theoretical basis for melatonin to alleviate drought stress in rice. In the future, we would like to explore the molecular mechanism of melatonin regulation with respect to rice resistance to drought stress.

5. Conclusions

In conclusion, melatonin pretreatment (100 μM) can promote rice seed germination and seedling growth, improve agronomic traits such as shoot length, root length, and biomass, improve the antioxidant enzyme system activity and the soluble protein content, and reduce the malondialdehyde content to alleviate the inhibitory effect of drought stress on rice growth. A schematic diagram showing the physiological and biochemical mechanisms in response to 100 μM melatonin to relieve drought stress is presented in Figure 10.