Exogenous application of melatonin to mitigate drought stress-induced oxidative damage in Phoebe sheareri seedlings

Plant material and experimental design

A pot experiment was performed from August 2020 to February 2021 under natural light at the west campus of Yangtze University, Jingzhou, China (111°15′∼114°05′E, 29°26′∼31°37′N). The seeds of P. sheareri were collected and stored in the laboratory yearly. The P. sheareri seeds were buried in sandy soil, growing naturally without special treatment to harvest the annual seedlings. One-year-old healthy P. sheareri seedlings were used in this experiment, and one P. sheareri seedling was planted per pot. The cultivating matrix consisted of nutritive soil, river sand, and perlite in 1:1:1. P. sheareri seedlings were grouped into four groups. Each group was randomly assigned 30 seedlings: control check (CK) group (n = 30), drought stress (D) group (n = 30), drought stress + melatonin treatment (MD) group (n = 30), and melatonin treatment (M) group (n = 30). After three months of routine management, the experimental treatment was started in November. Each group of plants underwent the following treatments:

For the CK group, the plants underwent normal water (Hoagland nutrient solution) treatment. For the D group, during drought stress, the watering of plants was stopped until the soil moisture content was reduced to 0%. For the MD group, during drought stress, the watering of plants was stopped until the soil moisture content was reduced to 0%. Then, 100 µmol/L of melatonin was sprayed on the foliar surface simultaneously. Once every four days, a total of five treatments were administered. For the M group, the plants underwent normal water (Hoagland nutrient solution) treatment and foliar sprayed with 100 µmol/L of melatonin once every four days. The leaves of 30 seedlings in each group were collected at the end of the experiment, mixed, and randomly divided into three parts for subsequent detection of physiological parameters.

Measurement of carotenoids (Car)

According to the product instructions, the Car content was evaluated with Plant Carotenoid Content Detection Kit (KLP1046, KangLang Bio. China) according to the product instructions. Briefly, Reagent I and Reagent II were mixed in 2:1 to obtain the extraction solution. The leaf samples were dried at 60 °C and crushed, then passed through a 100-mesh sieve. The sample (0.01 g) was dissolved in 2 mL extract solution and shaken at 37 °C for 60 min. Finally, the absorption values of blank and sample tubes were measured at 440 nm. Car content = 1.6×(OD_sample −OD_blank)/sample weight.

Activities of antioxidant enzymes

The SOD activity was measured by using Superoxide Dismutase (SOD) Test Kit (NBT method) (JN24888, Jining Shiye, China). Briefly, the leaf sample and extraction solution were mixed in 1:10 (g: mL) and shaken at 4 °C for 30 min. The samples were centrifuged at 4 °C for 10 min at 8,000 g, and the supernatant was tested. This was followed by the addition of 180 µL of supernatant, 45 µL of Reagent I, 2 µL of Reagent II, 35 µL of Reagent III, and 100 µL of Reagent IV into tubes, mixed and incubated for 30 min at 25 °C. Finally, the OD value was measured at 560 nm. SOD activity = 11.11× percentage inhibition/(1 − percentage inhibition)/Cpr. Cpr means the protein concentration of tissue homogenate.

The CAT activity was measured by the Catalase Test (CAT) Kit (ultraviolet absorption method) (JN24729, Jining Shiye, China). Briefly, the leaf sample and normal saline were mixed in 1:9 (g: mL) and shaken at 4 °C. The samples were centrifuged at 25 °C for 10 min at 2,500 g, and the supernatant was tested. This was followed by adding 0.05 mL of supernatant, 1 mL of Reagent I, and 0.1 mL of Reagent II, and placed in water maintained at 37 °C for 60 min. Afterward, 1 mL of Reagent III and 0.1 mL of Reagent IV were added. Finally, the OD values of samples were measured at 405 nm. CAT activity = (OD_blank –OD_sample) × 271 × [1 / (60 × 0.05)]/Cpr. Cpr means the protein concentration of tissue homogenate.

The POD activity was measured by the Peroxidase (POD) Test Kit (spectrophotometric method) (JN24754, Jining Shiye, China). Briefly, the leaf samples and extraction solution were mixed in 1:10 (g:mL) and shaken at 4 °C. The samples were centrifuged at 4 °C for 10 min at 8,000 g, and the supernatant was tested. The absorption value of POD was detected at 470 nm using a spectrophotometer. This was followed by adding 15 µL of supernatant, 270 µL of double distilled water, 520 µL of Reagent I, 130 µL of Reagent II, and 135 µL of Reagent III into 1 mL of the cuvette, quickly mixed, and then the OD values A1 and A2 at 30 s and 90 s were measured. ΔA = A1 − A2. POD activity = 7133  × ΔA/sample weight.

The APX activity was measured by the Ascorbate peroxidase (APX) Test Kit (A123-1-1, Nanjing Jiancheng Bioengineering Institute, China). Briefly, the leaf samples were dried at 60 °C and crushed, then passed through a 100-mesh sieve. The sample (0.1 g) was dissolved in 1 mL of Reagent I and shaken at 4 °C for 60 min. The samples were centrifuged at 4 °C for 20 min at 13,000 g, and the supernatant was tested. The absorption value of APX was detected at 290 nm using a spectrophotometer. This was followed by adding 100 µL of supernatant, 700 µL of preheated Reagent I, 100 µL of Reagent II, and 100 µL of Reagent III into 1 mL of quartz cuvette and quickly mixed. The OD values A1 and A2 at 10 s and 130 s were measured. ΔA = A1 − A2. APX activity = 1786  × ΔA/sample weight.

The PPO activity was measured by the Polyphenol Oxidase (PPO) Test Kit (Colorimetric method) (A136-1-1, Nanjing Jiancheng Bioengineering Institute, China). Briefly, the leaf samples and Reagent I extraction solution were mixed in 1:10 (g:mL) and shaken at 4 °C for 30 min. The samples were centrifuged at 25 °C for 10 min at 8,000 g, and the supernatant was tested. This was followed by adding 150 µL of supernatant, 600 µL of Reagent II, and 150 µL of Reagent III in 1 cm of the cuvette, mixed and incubated for 10 min at 37 °C, followed by 5 min in a boiling water bath. Finally, the OD values of the samples were measured at 420 nm. PPO activity = (OD_sample − OD_blank)/0.01× (V_ReagentI/sample weight) × (V_total/0.15 mL) × (1/V_total) × 0.1 min.

The GR activity was measured by the Glutathione Reductases (GR) assay kit (Ultraviolet colorimetric method) (A062-1-1, Nanjing Jiancheng Bioengineering Institute, China). Briefly, the leaf samples and normal saline were mixed in 1:9 (g:mL) and shaken at 4 °C for 30 min. The samples were centrifuged at 25 °C for 10 min at 2,500 g, and the supernatant was tested. This was followed by adding 20 µL of supernatant into 2.4 mL solution and mixed quickly. After 30 s of reaction, the OD value at 340 nm was measured in a 1 cm quartz cuvette and named A1. Next, the mixed solution was bathed at 37 °C for 2 min, followed by the measurement for 2.5 min of OD value, named A2. GR activity = [(A1 − A2)/6.22]/2 min/(V_sample × Cpr) × N. Cpr means the protein concentration of tissue homogenate, and N means the dilution ratio of the sample.

Oxidative stress indicators of malondialdehyde (MDA) and O₂^•−

The MDA content was measured with the Plant Malondialdehyde (MDA) assay kit (Colorimetric method) (A003-3-1, Nanjing Jiancheng Bioengineering Institute, China). Briefly, the leaf samples and Reagent V extraction solution were mixed in 1:9 (g:mL) and shaken at 4 °C for 10 min. The samples were centrifuged at 25 °C for 10 min at 4,000 g, and the supernatant was tested. This was followed by adding 50 µL of supernatant and 1 mL of working solution. The OD value at 530 nm was measured after boiling in a water bath for 20 min. The MDA content = [(OD_sample − OD_blank)/(OD_{standard sample} − OD_blank)] ×10 nmol/mL/(sample weight/V_total).

The O₂^•− content was measured by oxygen free radical test kit/colorimetric method (ml094977, Shanghai Enzyme-linked Biotechnology, China). Around 1 g of leaves were weighed, 2 mL of pre-cooled O2-LysisBuffer was added, and homogenized under the ice-bath condition. Next, the samples were centrifuged at 10,000 g at 4 °C for 1 min, and the supernatant was collected. After the NO₂-standard was restored to room temperature, it was diluted in different multiples to construct the standard curve. This was followed by adding 0.25 mL of supernatant, 0.25 mL of O₂-LysisBuffer, and 0.5 mL of hydroxylamine solution and placed in water maintained at 25 °C for 20 min. Afterward, 0.5 mL of aminobenzene sulfonic acid chromogenic solution and 0.5 mL of naphthylamine chromogenic solution were added. Finally, the OD values of the samples were measured at 530 nm after placing them in a water bath at 30 °C for 30 min. The O₂^•− content was calculated according to the standard curve.

Quantitation of soluble protein (SP), soluble sugar (SS), and proline (Pro) content

The SP content was tested by Bradford method according to a previous study (Abdel Latef & Tran, 2016). The SS content was detected by the plant soluble sugar content test kit (Colorimetric method) (A145-1-1, Nanjing Jiancheng Bioengineering Institute, China). Briefly, the leaf samples and double distilled water were mixed in 1:10 (g: mL), shaken, and placed in boiling water for 10 min. The samples were centrifuged at 25 °C for 10 min at 4,000 g, and the supernatant was tested. This was followed by adding 200 µL of supernatant, 100 µL of working solution, and 1 mL of concentrated sulfuric acid. The OD value at 620 nm was measured after placing the samples in a boiling water bath for 10 min. SP content = [(OD_sample − OD_blank)/(OD_{standard sample} − OD_blank)] ×100 µg/mL/(sample weight/V_total) × N. N means the dilution ratio of the sample.

The content of Pro was detected by Proline Assay Test Kit (Colorimetric method) (A107-1-1, Nanjing Jiancheng Bioengineering Institute, China), according to the product instructions. Briefly, the leaf samples and Reagent I extraction solution were mixed in 1:9 (g:mL) and shaken at 4 °C. The samples were centrifuged at 25 °C for 10 min at 3,500 g, and the supernatant was tested. This was followed by adding 0.5 mL of supernatant, 1 mL of Reagent II, and 1 mL of Reagent III. The OD value at 520 nm was measured after placing the samples in a boiling water bath for 30 min. Pro content = [(OD_sample − OD_blank)/(OD_{standard sample} –OD_blank)] ×5 µg/mL/(sample weight/V_total) × N. N means the dilution ratio of the sample.

Endogenous phytohormones detection

The extraction and content quantification of endogenous jasmonic acid methyl ester (JA-Me) and abscisic acid (ABA) were performed as described previously (Imran et al., 2021; Li et al., 2018). Endogenous phytohormones were detected by enzyme-linked immunosorbent assays kits that the Laboratory of Molecular Plant Breeding of Yangtze University supplied.

Drought resistance coefficient

The drought resistance coefficient (DRC) of 14 single indicators involved in this study was calculated using the following formula: DRC = I_d/ I_CK.

In the formula, I_d and I_M are the measured values of a certain index in the treatment and CK groups, respectively. The comprehensive coefficient of drought resistance was calculated using the following formula: Comprehensive coefficient of drought resistance = $\frac{1}{n}{\sum }_{i=1}^{n}DRC$.

Statistical analysis

After all the data passed the normality test of Kolmogorov–Smirnov, Brown-Forsythe test, and Bartlett’s tests were used to analyze the homogeneity of variance of the data. When the data satisfied the homogeneity of variance, one-way ANOVA following Tukey’s multiple comparisons test was conducted. Otherwise, Dunnett’s multiple comparisons test was used. The parameter contained a 95% confidence interval. The significance cutoff of the p-value was 0.05. The data were visualized by Graphpad Prism 9.0. Principal component analysis (PCA) and heatmap were analyzed by Sangerbox (http://www.sangerbox.com) and R v.4.1.0 for Windows (http://CRAN.R-project.org/).

Melatonin supported plant growth and carotenoid content

We first observed the appearance of P. sheareri seedlings in four groups in drought conditions (Fig. 1A). In the CK and M groups, the seedlings did not experience drought stress, and the plants grew upright with bright green leaves and sprouted new leaves. However, the phenotypic morphology of P. sheareri seedlings in the D group exhibited that all leaves were yellowish, wilted and drooped, curled, and dried with severe dehydration. The leaves withered, stem segments showed yellowish dehydration, and plant height was significantly lower than in the other three treatments. Additionally, we observed the effects of melatonin on fresh biomass. We found that the number of new leaves, the relative water content of leaves, and the length of shoots were significantly reduced in the D group compared to the CK group. In contrast, there was a significant rising in these three indicators in melatonin-treated seedlings under drought stress (Table 1). These results indicated that melatonin application improved plant growth under drought stress.

Consistent with the change in plant morphology, there were alterations in carotenoid content in the leaves (Fig. 1B). Drought stress (D group) significantly suppressed carotenoid content compared to the CK group, suggesting that photosynthesis in the leaves was impaired. Under drought conditions, melatonin spraying (MD group) slightly upregulated carotenoid content but not significantly, implicating that the protection of P. sheareri seedlings by melatonin is not strongly dependent on carotenoid. Taken together, melatonin can protect the photosynthesis of P. sheareri seedlings from drought stress to some extent.

Melatonin provoked activities of antioxidant enzymes

Melatonin protected plants from oxidative damage by acting as an antioxidant enzyme activator (Imran et al., 2021). In this study, the effects of melatonin on the activity of antioxidant enzymes in P. sheareri seedlings upon drought treatment, including SOD, POD, APX, PPO, CAT, and GR, were determined (Fig. 2). The results showed that compared to the CK group, the activities of the antioxidant enzymes were significantly decreased under drought stress (D group). However, melatonin (MD group) significantly restored their activity under drought stress conditions. Notably, melatonin treatment (M group) led to a slight increase in the activities of these antioxidant enzymes under routine water treatment, compared to the CK group. Therefore, these results suggested that melatonin application could protect P. sheareri seedlings from oxidative damage induced by drought stress by provoking antioxidant enzyme activity.

Melatonin restricted MDA and O₂^•− accumulation

Both MDA and O₂^•− accumulation are indicators of impaired ROS scavenging capacity in the plants; therefore, we examined the effects of melatonin under drought stress. The results showed that drought stress (D group) resulted in a significant increase in the MDA and O₂^•− accumulation in P. sheareri seedlings, compared to the CK group (Fig. 3). Expectedly, melatonin application (MD group) significantly ameliorated MDA and O₂^•− accumulation induced by drought stress in P. sheareri seedlings, indicating that melatonin could function as a highly effective ROS scavenger to protect P. sheareri seedlings from drought stress.

Melatonin affected SP, SS, and Pro contents

Pro, SP, and SS are all compatible solutes that increase cytoplasmic osmotic pressure, balancing water potential, and protect the membrane system, thus reducing water loss from the cells to boost the drought resistance of plants. As shown in Fig. 4, drought stress (D group) significantly increased the Pro and SP contents compared to the conventional water treatment in the CK group. It significantly reduced the SS content in P. sheareri seedlings, suggesting that its physiology was altered. In addition, under drought stress, melatonin treatment (MD group) significantly retracted SP content and increased SS content; however, melatonin treatment could not prevent the accumulation of Pro content. In conclusion, melatonin could coordinate plant resistance to drought by altering the SP and SS contents.

Melatonin increased phytohormones content

We investigated whether melatonin can respond to drought stress via phytohormone regulation, such as JA-Me and ABA. The results showed that drought stress significantly increased JA-Me and ABA contents, whereas melatonin treatment significantly counteracted this alteration due to drought conditions (Fig. 5). These results suggested that melatonin enhanced the drought tolerance of P. sheareri seedlings by correcting the content of endogenous hormones, JA-Me and ABA.

Correlation and dimensionality reduction analysis

To holistically evaluate the reliability of the results of this study, the samples were clustered using the PCA based on the results of all parameters. PCA reflected that the four groups have distinctly different clusters, with the CK and M groups nearby and the D group clustered away from the other three groups (Fig. 6A). The Pearson heatmap clustered a set of parameters together based on the similarity of patterns. For example, Car, SOD, POD, APX, PPO, CAT, GR, and SS were clustered into one group, and the remaining MDA, O₂^•−, SP, Pro, JA-Me, and ABA were clustered into one group (Fig. 6B). Moreover, the PCA of the responses of the P. sheareri seedlings to different treatments depicted 97.1% of variability in drought response (Fig. 6C). The PC1 component explained 78% of the variation and contributed positively via antioxidant enzymes (POD, APX, PPO, CAT, and GR), SS, and Car. The PC2 component explained 19.1% of the variation and contributed positively via SP, ABA, MDA, and JA-Me and negatively by O₂^•− and Pro. Similarly, the comparative heatmap revealed that the antioxidant enzymes, SS, and AR are clustered together and demonstrated high levels in the CK group (Fig. 6D). The remaining parameters in this study were grouped into another cluster, including SP, ABA, MDA, O₂^•−, JA-Me, and Pro. According to Figs. 6B–6D, we observed that the six tested antioxidants had highly similar response patterns under drought stress and upon melatonin treatment, suggesting that it may be sufficient to measure only two or three of them from a frugality perspective.

A comprehensive evaluation of drought resistance of P. sheareri seedlings under different treatment conditions

To comprehensively evaluate the effects of fourteen indicators on the drought resistance of P. sheareri seedlings, we introduced the DRC. The results showed that based on the DRC of fourteen single indicators, the comprehensive index of the MD group was the largest, followed by the D group, and the M group was the smallest (Table 2).