Seeds of apple (M. baccata) were sown in soppy vermiculite. Two weeks later, the seedlings were watered with half-strength Hoagland’s nutrient solution (Li M.Q. et al., 2016). When the seedlings developed to have four leaves, they were watered with complete nutrient solution. The plants were kept in green house with the temperature at a constant 22 ± 2°C and a 16/8 h light/dark cycle. The light intensity was approximately 100 μmol⋅m^-2⋅s^-1.

After the M. baccata seedlings developed to have four leaves, a total of 96 seedlings were transplanted into the glass container with sterilized matrix soil. They were divided into eight groups. Plants in group I were watered with the 200 mL normal nutrient solution. Totally 25 mL normal nutrient solution was added every 3 days as control. The waterlogging stress was conducted in the remaining seven groups by keeping the soil being covered with 300 mL the nutrient solution and added 25 mL normal nutrient solution every 3 days.

The seedlings from group II was waterlogging stressed without supply of exogenous melatonin. The waterlogging stressed seedlings from group III to VIII were treated with different concentration of melatonin by spraying or irrigation. Melatonin was dissolved in 100% ethanol at a concentration of 10 mM and stored at -20°C as a stock solution. When use, melatonin was then diluted into 50, 100, and 200 μM, respectively with deionized water. These different concentrations of melatonin were sprayed to the leaves of seedlings every other day in group III, IV, and V, respectively. Two pieces of hardboard were used to avoid sprayed melatonin dropping into the soil. In group VI, VII, and VIII, melatonin was directly supplemented to the nutrient solution at the concentrations of 200, 400, and 600 μM, respectively. Groups I and II was also applied with equal volume of ethanol. The experiments were independently repeated three times. Photos were taken before and after 9 days of waterlogging stress/melatonin treatments.

The leaves and roots were collected from the all groups of seedlings, respectively, before and after 9 days of waterlogging stress/melatonin treatment.

The leaves collected from the seedlings were immediately frozen at -80°C for future melatonin detection. Around 1 g of frozen leaves of M. baccata was ground to a fine powder in liquid nitrogen. The powder was mixed with 10 mL methanol and ultra-sonicated (80 Hz) for 35 min at 45°C. The sample preparation and HPLC detection of melatonin were performed as described by Zhao et al. (2013). Each experiment was independently repeated three times.

Total RNA was isolated from the leaves of seedlings of the eight groups, respectively, before and after 9 days of waterlogging stress/melatonin treatment, using the EASY spin Plant RNA Rapid Extraction Kit (Biomed, Beijing, China). The first-strand cDNA was synthesized following the protocol of Kit (Promega, Madison, WI, USA). The cDNAs were used as template for RT-PCR. The specific primers were designed according to the sequence of melatonin synthesized enzyme genes MbASMT9 (KJ156531), MbAANAT3 (KJ156532), and MbT5H1, respectively, by Primer 5 software and checked by BLAST search in the apple genome¹ (MbASMT9 Forward Primer 5′-TGATCTGCCCCATGTCGT-3′, Reverse Primer 5′-CTTTGTGGCGAGGGAAAC-3′; MbAANAT3 Forward Primer 5′-CGCTCCCTAACTACCAACCA-3′, Reverse Primer 5′-ACAAATCCCTTTCCCTACCAG-3′; MbT5H1 Forward Primer 5′-ATCCGTAAGATTTGTATACTTGAGCT-3′, Reverse Primer 5′-TCACCGACCAAGATAATAGCCT-3′).

RT-PCRs were performed use of 20 μL reaction mixtures containing 20 ng of first-strand cDNA, 2 × PCR Mix 5 μL (CWBIO, Beijing, China), 0.5 μM of each of the forward and reverse primers and appropriate amounts of ddH₂O. The Actin gene was used as the internal standard, and the PCR program for Actin was as following: 94°C for 5 min; 28 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and 72°C for 10 min, amplified with primers (Forward Primer 5′-CAATGCCTGCCATGTATG-3′, Reverse Primer 5′-CCAGCAGCTTCCATTCCAAT-3′). The PCR products were analyzed in 1% TAE-agarose gel stained by goodview. The quantification of amplified Actin, MbT5H1, MbAANAT3, and MbASMT9 fragment was done by the ImageJ software², the ratios of MbT5H1, MbAANAT3, MbASMT9 and Actin were calculated.

The coding frame of MbASMT9 was amplified and ligated into pMD 19-T Simple, which was then digested with BamHI/EcoRI and inserted into the pGEX-6p-1. The protein expression of MbASMT9 was analyzed according to Li et al. (2015). The purity of the GST-MbASMT9 protein was confirmed by SDS–PAGE.

The total protein was isolated from leaves of seedlings according to Wang et al. (2006). Western blot was applied with antibody of MbASMT9 (rabbit, 1:3000). The recombinant GST-MbASMT9 was used as an antigen to raise polyclonal GST-MbASMT9 antibodies in rabbit. The preparation of MbASMT9 antibody was carried out in accordance with relevant guidelines and regulations. The experimental protocols of the MbASMT9 antibody preparation were reviewed and approved by Beijing Municipal Science and Technology Commission. The chemiluminescent signals were detected using an ECL detection kit (Amersham-Pharmacia, USA). The loading control of the Western blot was stained by Coomassie Brilliant Blue.

A total of 0.3 g leaves or 0.3 g roots of M. baccata seedlings were ground with 8 mL chilled 50 mM phosphate buffer (pH 7.8), then they were transferred into 10 mL tubes and centrifuged at 4°C for 15 min at 10,000 g. The supernatants were diluted with phosphate buffer to 10 mL and used for enzyme activity detection. The enzyme activity of SOD was detected according to the method of Stewart and Bewley (1980). CAT activity was measured as the absorbance at 240 nm wave length according to the method of Ma et al. (2016). POD (Peroxidase) activity was measured by the changes in absorbance at 470 nm due to guaiacol oxidation according to the method of Shi et al. (2015b). Each experiment was independently repeated at least three times.

A total of 0.3 g roots for each group were collected to detect the enzyme activity of ADH and SDH before and after 9 days of waterlogging stress/melatonin treatment to identify the alterations of anaerobic and aerobic respiration. ADH activity was measured as reported by Yamashita et al. (2015). SDH activity was detected according to Landi et al. (2009). Each experiment was independently repeated at least three times.

The ROS level in roots was detected according to the method modified from Zuo et al. (2014). The intact roots were collected from each group and the fresh roots were immediately used for ROS detection before and after 9 days of waterlogging stress/melatonin treatment. Simply, the entire roots were incubated with the 5-(and 6)-chloromethyl-2′-7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) solution for 20 min and washed with distilled H₂O to remove excess CM-H₂DCFDA. The fluorescence images were obtained with a Leica stereoscope (Leica, Wetzlar, Germany) (× 10).

In addition to roots, the ROS level in leaves was also detected. The leaves were collected from the each group, respectively before and after 9 days of waterlogging stress/melatonin treatments and used for detection of O2•− and H₂O₂ immediately. For O2•− measurement, the leaf histochemical staining was vacuum infiltrated with 0.1 mgmL^-1 nitroblue tetrazolium in 25 mM K-HEPES buffer (pH 7.9) for 40 min. Then the samples were kept at 25°C in dark for an additional 4 h. For the H₂O₂ detection the leaves were vacuum infiltrated with 0.1 mg⋅mL^-1 DAB in 50 mM Tris-acetate (pH 3.8) and were incubated at 25°C in dark for 24 h. Then leaves for either O2•− or H₂O₂ detection were washed in 80% ethanol every 10 min at 80°C until the leaves lost green color completely (Gong et al., 2014). The MDA detection in the leaf samples were performed according to Zhao et al. (2013). Each experiment was independently repeated at least three times.

The extraction of Chl was conducted according to Porra et al. (1989). A total of 0.2 g leaves were homogenized in 2–3 mL 80% acetone. After filtration with four layers of gauze (1 mm × 1 mm), the homogenate was diluted with 80% acetone to 25 mL. The absorbance of the extract was measured at 645 and 663 nm. Chl concentration was calculated from the following equations: Chl a = 12.72 × OD663 -2.59 × OD645; Chl b = 22.88 × OD645 -4.67 × OD663; chl = chl a + chl b (Arnao and Hernández-Ruiz, 2009).

The photosynthetic rate was measured by LI-6400XT (LI-COR, Lincoln, NE, USA) according to the producer’s protocol. The light intensity was at 800 μmol⋅m^-2⋅s^-1. The humidity was about 50% and the temperature was 23°C. Each experiment was independently repeated three times.

The data are expressed as means ± SD. One-way ANOVA was used for the normality evaluation followed by a Tukey–Kramer multiple comparison test. The statistical significant difference was set up when P < 0.05. Statistical evaluations were carried out using SPSS software (IBM, Armonk, NY, USA).

The results showed that waterlogging stress significantly impaired the M. baccata seedling growth. Those plants which were under waterlogging stress for 9 days wilted severely compared to the normal controls (Figure 1A). When melatonin was sprayed to the seedlings at the concentrations of 50, 100, and 200 μM, respectively, it was apparent that melatonin spraying improved tolerance of seedlings against waterlogging stress. The protective effects of melatonin indicated a dose-responsive manner. The seedlings treated with 200 μM of melatonin, even under the waterlogging stress, showed a similar phenotype as the normal controls. The similar results were also observed in the seedlings those melatonin was irrigated (Figure 1B). When melatonin was irrigated to the seedlings at the concentrations of 200, 400, and 600 μM, respectively, it also increased waterlogging resistance of seedlings as a dose-dependent manner. The irrigation with 600 μM of melatonin, gained a similar phenotype as spraying with 200 μM melatonin. The percentage of leaf chlorosis was also analyzed. It was clearly to see that the percentage of leaf chlorosis was significantly increased after waterlogging treatment. After melatonin was applied to the seedlings under waterlogging, the percentage of leaf chlorosis were significantly decreased (Supplementary Figure 1).

To find the potential effects of waterlogging stress and melatonin application on the endogenous melatonin synthesis, the melatonin level and its synthetic gene expression were detected. The results showed that waterlogging stress significantly elevated plants melatonin levels in leaves (87 vs. 21 ng/g FW of control) and in roots (73 vs. 16 ng/g FW of control) (Figure 2A).

When exogenous melatonin was applied to the stressed seedlings, their endogenous melatonin was further increased compared to the plants under waterlogging stress alone (Figure 2A). For example, the endogenous leaf melatonin level in melatonin (200 μM) sprayed plants was 256 ng/g FW, which was 2.9 times higher than that in leaves of waterlogged seedling alone (87 ng/g FW). The similar results were observed in roots and also in melatonin irrigated seedlings. In accordance with the elevated melatonin production, the gene expressions of melatonin synthetic enzymes including ASMT (acetylserotonin O-methyltransferase), AANAT (aralkylamine N-acetyltransferase) and T5H (tryptamine 5-hydroxylase), were slightly upregulated under the waterlogging stress and melatonin treatment compared to the controls (Figure 2B). The upregulated gene expression of ASMT9, which is the supposed melatonin synthetic rate-limiting enzyme, failed to result in the increase in its protein level. In contrast, its protein level was declined when compared to the controls (Figure 2C). Obviously, the post-transcriptional regulation occurred for ASMT9.

The activities of the antioxidant enzymes including SOD, POD, CAT were measured in four groups of M. baccata seedlings. These groups included the normal control group, waterlogging stress alone, waterlogging stress treated with melatonin (200 μM spray and waterlogging stress treated with melatonin (600 μM) irrigation. Waterlogging stress significantly suppressed all antioxidant enzymes tested in both levels and roots of the seedlings. However, melatonin treatment could recover the activities of these antioxidant enzymes suppressed by waterlogging stress in a great degree. For example, melatonin (200 μM) spray recovered the activity of CAT in leaves to the level comparable to the control seedlings (Figure 3).

The activities of ADH and SDH indicate the anaerobic and aerobic respirations, respectively. It was found that the ADH activity (11.71 U/mg), the index of anaerobic respiration, was significantly higher in the roots of waterlogging stressed seedlings than that of normal control (7.01 U/mg). Both melatonin spray and irrigation reduced the anaerobic respiration indicated by decreased ADH activity (Figure 4A). In contrast, waterlogging stress suppressed the aerobic respiration indicated by the significantly reduced SDH activity compared to the control seedlings. This tendency was reversed by melatonin treatment in a great degree (Figure 4B).

Waterlogging stress significantly elevated the O2•− and H₂O₂ production both in leaves and in roots of the seedlings. These were indicated by the increased florescent staining intensities on them. Melatonin treatment significantly reduced O2•− and H₂O₂ productions, no matter melatonin was sprayed to the leaves or irrigated to the roots of the seedlings (Figures 5A,C). Accordantly, waterlogging stress caused oxidative damage in both leaves and roots of the seedlings. This was indicated by the increased content of MDA. It was expected that melatonin treatment significantly reduced the MDA levels in leaves and also in roots (Figures 5B,D).

Waterlogging stress dramatically reduced the Chl content in the leaves of seedlings. After 9 days of waterlogging stress, the Chl content of these plants (2.4 mg/g FW) was only roughly half of the value of control seedlings (4.1 mg/g FW). The photosynthetic rate of the waterlogging stressed plants was 1.67 μmol⋅m^-2⋅s^-1. The value was significantly lower than that of control seedlings (2.67 μmol⋅m^-2⋅s^-1). Melatonin treatments (spray or irrigation) improved both Chl content and photosynthetic rate which were suppressed by the waterlogging stress (Figures 6A,B).