Twelve-day-old seedlings were washed with tap water, and dried briefly with absorbent paper to remove surface water. There were three different ways for application of melatonin. The first method (I) was soaking seeds with 20 or 100 µM melatonin and watered with the Hoagland's nutrient solution, and soaking seeds with water as a control. The second method (II) was that the rice seedlings were watered with the Hoagland's solution containing 20 or 100 µM melatonin for 2 days. The seedlings were grown in the Hoagland's solution as a control. The third method (III) was that the rice seedling leaves were sprayed every 4 h with 20 or 100 µM melatonin for 2 days. The control seedlings were sprayed with a similar solution without melatonin. After melatonin pretreatment, the 14-day-old seedlings were transferred to 12◦C, and the control seedlings remain in day/night temperatures of 28/22◦C. After 6-day treatment, the second leaves were used for the measurements, and all experiments were repeated at least three times.

## Extraction and Quantification of Melatonin by HPLC-MS/MS

Extraction of melatonin from rice was performed as described by Stürtz et al. (2011). The endogenous melatonin quantification was performed using liquid a chromatography (HPLC) system (1290 LC, Agilent, USA) couple to a mass spectrum (MS) system (6470 LC-MS/MS, Agilent, USA) according to Stürtz et al. (2011) with some modifications. MS–MS parameters were as follows: positive ion mode; turbo 1 speed, 100%; turbo 2 speed, 100%; sheath gas temperature, 300◦C; sheath gas flow, 11.0 L/min; capillary current, 59 nA; capillary, 3368 V; MS 1 heater, 100◦C; MS 2 heater, 100◦C; rough vac, 9.91E-1 Torr; high vac, 3.60E-5 Torr; and m/z, 159.0. Separations were carried out on a 150 × 2.1 mm, 1.8 µm, Eldath RS-C18 column. Two different solvents were used in the mobile phase: solvent A (methanol) and solvent B (methanol with 0.1% formic acid, v/v), at a flow rate of 0.3 mL/min, injection volume of 1 µL and methanol was from 20 to 80%.

## Determination of Shoot and Root Dry Weight

After 6 days cold stress, shoots and roots were separated, then washed with tap water and rinsed twice with distilled water, gently

wiped dry with a paper towel, and then were oven dried at 80◦C to constant weight for DW determination, respectively.

### Determination of Leaf Relative Water Content (RWC) and Root Activity

The RWC was measured according to Cao et al. (2015). The second leaves were excised and their fresh mass (FM) was determined immediately, after floated on deionized water for 5 h, the saturated mass (SM) was recorded. Then the leaves were dried at 80◦C to constant weight to measure their dry mass (DM). The RWC was calculated from the following equation:

RWC [%] = [(FM – DM)/(SM – DM)] × 100.

Root activity was assessed by TTC (triphenyl tetrazolium chloride) method (Chen, 2003), and was expressed as the deoxidization ability (µg g−1 h −1 ).

### Determination of Chlorophyll and Carotenoids Contents

Chlorophyll (Chl) content was measured as previously described Lichtenthaler and Wellburn (1983). Fresh leaves (0.1 g) were cut and homogenized with 20 mL of 80% (v/v) acetone in a pre-chilled mortar and pestle, then centrifuged at 8000 g for 10 min. The supernatant was separated and the absorbance was measured with a spectrophotometer (UV-1750, Shimadzu, Japan) at 663, 646 and 470 nm. The content of chlorophyll and carotenoids were calculated using the Lichtenthaler and Wellburn formula (Lichtenthaler and Wellburn, 1983) and expressed in mg g−1 FW.

### Determination of Electrolyte Leakage (EL) and Malondialdehyde (MDA)

Electrolyte leakage was determined using a electrical conductivity meter (DDS-309+, Chengdu, China) following Ning et al. (2015). The second leaves were taken and cut into 5 mm segments, weighted and placed in 50 mL capped polypropylene centrifuge tubes containing 25 mL distilled water. The initial electrical conductivity (EC1) of the bathing solutions was measured after the samples were incubated at 25◦C for 24 h. Then, the samples and bathing solutions boiled in water for 30 min and measured electrical conductivity (EC2) again after cooling the solution to room temperature. EL was calculated using the formula: EL = EC1/ EC2 × 100.

The degree of membrane lipid peroxidation in leaves was estimated by MDA content. MDA was evaluated using thiobarbituric acid assay (Nahar et al., 2015). Fresh leaves (0.5 g) was homogenized in 5 mL of trichloroacetic acid (TCA) (5%, m/v) and centrifuged at 8000 g and 4◦C for 10 min. 2 mL of the supernatant was mixed with the equal volume 0.5% thiobarbituric acid. The mixture was bathed at 95◦C for 30 min and then instantly cooled on ice and centrifuged at 5000 g and 4◦C for 10 min. The absorbance of the supernatant was recorded at 450, 532, and 600 nm, respectively. MDA content was calculated according to Nahar et al. (2015) and expressed as µmol g −1 FW.

#### Determination of H2O2 and O− 2

O − 2 and H2O2 were visually detected in leaves with nitro blue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB), respectively, as described previously method (Yang et al., 2004). The leaves were excised at the base and immersed into NBT (1 mg/mL) solution for 2 h or into DAB (0.5 mg/mL) solution for 12 h in the dark, then boiling in ethanol (90%, v/v) for 0.5–2 h to decolorize the leaves. At least five leaves were used for each staining.

The content of H2O2 was determined as described by Velikova et al. (2000). 0.5 g fresh leaves were homogenized with 5 mL 0.1% (m/v) TCA in an ice bath and centrifuged for 15 min at 12 000 × g and 4◦C. 0.5 mL of the supernatant was added to 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI. The absorbance of the mixture was recorded at 390 nm. Finally, a standard curve of H2O2 reagent was used to calculated the H2O2 content. The content of H2O2 was expressed as µmol g−1 FW.

Production of O− 2 was estimated following Nahar et al. (2015) with some modification. 0.2 g fresh leaves was homogenized with 2 mL 65 mM phosphate buffer (pH 7.8), and centrifuged at 10000 g and 4◦C for 10 min. Then, 0.5 mL supernatant was mixed with 0.5 mL 65 mM phosphate buffer (pH 7.8) and 0.1 mL 10 mM hydroxylamine hydrochloride. After 20 min incubated at 25◦C, the solution was added to 1 mL 17 mM sulfanilamide and 1 mL 7 mM naphthylamine and incubated at 25◦C for 20 min. Absorbance was recorded at 530 nm. The formation O− 2 was calculated using a standard curve of a NaNO2 reagent.

### Trypan-Blue Staining

The dead cells were visually detected using a trypan-blue staining method as described by Liang et al. (2015) with some modifications. Leaves were detached and stained with lactophenol-trypan blue solution (10 mL of lactic acid, 10 mL of glycerol, 10 g of phenol, 10 mg of trypan blue, dissolved in 10 mL of distilled water) at 70◦C for 1 h and then boiled for approximately 5 min and left staining overnight. After destaining in chloral hydrate solution (2.5 g of chloral hydrate dissolved in 1 mL of distilled water) for 3 days to reduce background staining, samples were equilibrated with 70% glycerol for scanning.

### Enzyme Extraction and Assay

Fresh leaves (0.2 g) were homogenized with 2 mL of ice-cold 50 mM phosphate buffer (pH 7.8) containing 0.2 mM Na2EDTA, 2 mM ascorbate and 2% (m/v) polyvinylpyrrolidone using a chilled mortar and pestle. The homogenate was centrifuged at 12000 g for 15 min at 4◦C. The supernatant was used for assays of specific enzymatic activities. The activity of SOD was assessed according to Giannopolitis and Ries (1977) by assay its ability to inhibit the photochemical reduction of NBT. One unit of SOD activity was defined as the amount of enzyme that caused 50% inhibition of NBT reduction. The analysis of POD activity was based on the oxidation of guaiacol using H2O2 according to the method as described by Zhang and Kirham (1994). The CAT activity was measured as decline in absorbance at 240 nm due to the consumption of H2O2 according to the method of Hamurcu et al. (2013).

### Determination of Proline, Reduced and Oxidized Glutathione (GSH and GSSG) and Soluble Sugars Contents

Proline was extracted and determined by the method described by Mahdavian et al. (2016) with a minor modification. 0.5 g of fresh leaves were homogenized 5 mL 3% (m/v) sulphosalicylic acid and the homogenate was centrifuged at 5000 g for 10 min. 2 mL supernatant was added to 2 mL glacial acetic acid and 2 mL acid ninhydrin. The mixture was incubated in a boiling water bath for 1 h and terminated in an ice bath. The reaction mixture was then extracted with 4 mL of toluene with vigorous stirring. The upper toluene phase was separated and an absorbance was recorded at 520 nm. The proline content was calculated using a standard curve and expressed as µmol g −1 FW.

Reduced glutathione (GSH) content was determined using the method of Kraj (2016) with some modifications. 0.5 g samples were extracted in an ice bath with 5 mL 100 mM potassium phosphate buffer (pH 7.5) containing 5 mM EDTA, and then centrifuged at 12000 g for 20 min. Two milliliter supernatant was mixed with 1 mL 100 mM phosphate buffer (pH 7.5) and 0.5 mL 4 mM DTNB (5,50 -dithio-bisnitrobenzoic acid). The reaction mixture was incubated at 25◦C for 10 min, and the absorbance was measured at 412 nm. For GSSG assay, the GSH in supernatant was cleared first, and GSSG content was quantified as described by Knörzer et al. (1996). The GSH and GSSG content was calculated according to their standard curves and expressed as µmol g−1 (FW).

Soluble sugar content was measured based on the anthrone method (Azarmi et al., 2016). Fresh leaf samples (0.5 g) were homogenized with 5 mL of 80% ethanol, and the homogenate was centrifuged at 5000 g for 15 min. Then, 0.1 mL of the supermatant was mixed with 3 mL of anthrone. The reaction mixture was placed in a boiling water bath for 30 min and then cooled on ice. Absorbance was recorded at 625 nm. The concentration of soluble sugars was calculated using a glucose standard curve and exhibited as mg g−1 FW.

### Measurement of Photosynthetic Parameters and Chlorophyll Fluorescence

The photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 (Ci) and water use efficiency (WUE) of leaves were measured using a potable photosynthesis system (GSF-3000, Heinz-Walz Instruments, Effeltrich, Germany). Intact leaves were measured at a temperature of 25◦C, a photosynthetically active radiation (PAR) of 750 µmol m−2 s −1 , a CO2 concentration of 400 µmol mol−1 and relative humidity between 40 and 60%.

A modulated imaging chlorophyll fluorometer (IMAGING-PAM M-Series, Heinz-Walz Instruments, Effeltrich, Germany) was used to measure chlorophyll fluorescence parameters according to the instructions provided by the manufacturer. After dark adaptation for 30 min, the fluorescence (F0), maximum fluorescence (Fm) and the non-photochemical quenching (NPQ) were determined (Zhou et al., 2010). The maximal quantum yield of PSII photochemistry in the dark-adapted state was calculated as Fv/Fm.

### Protein Analyses

Thylakoid membrane proteins were isolated as described by Fristedt et al. (2011). Denaturing 12% (w/v) polyacrylamide containing SDS was prepared by the method of Aro et al. (1993). Western blotting analysis was performed according to Chen et al. (2009). Antibodies used (all raised in rabbits) were anti-Arabidopsis D1, D2, CP43, LHCb1, LHCb2, LHCb3 and ACTIN (against 100 amino acids of recombinant actin conserved peptide). These antibodies were purchased from Agrisera Comp. (Umea, Sweden).

### Statistical Analysis

All experiments were repeated at least three times, and mean values were presented with standard deviations (SDs; n = 3). Duncan's multiplication range test was used for comparison among different treatments. The difference was considered to be statistically significant when P < 0.05.

## RESULTS

Effects of cold stress and exogenous melatonin on growth of rice seedlings are shown in **Figure 1**. The low temperature stress inhibited the growth of rice seedlings, while melatonin pretreatment alleviated this inhibition to a certain extent (**Figure 1A**). And 100 µM melatonin pretreated plants with higher endogenous melatonin levels grew better than that of 20 µM melatonin pretreated plants (**Figures 1A,B**). Plant DM, RWC and root activity were significantly decreased by cold treatment compared with the control (**Figures 1C–E**). Consistently, melatonin pretreated rice seedlings exhibited significantly greater dry weight and higher RWC than control (**Figures 1C,D**). Seed soaking and root immersing methods showed better protection from cold stress than leaf spraying methods (**Figures 1C,D**). However, only Seed soaking methods in the 100 µM melatonin showed a significant increase in root activity (**Figure 1E**). These results indicate that exogenous melatonin application improved cold stress resistance in rice seedlings.

Chlorophyll and carotenoid contents significantly decreased after 6-day cold stress (**Figures 2A,B**), and the decline of pigments caused by the cold stress was mitigated by melatonin application. The chlorophyll and carotenoid contents in melatonin-pretreated seedlings were higher than plants without melatonin pretreatment. The chlorophyll content of 100 µM melatonin pretreated plants was higher than the plants that treated with 20 µM melatonin with root immersing methods (**Figure 2A**). For carotenoid contents, there was no significant difference among the three methods.

The accumulations of MDA and the EL of leaf tissues are markers of lipid peroxidation and damage of cellular membranes. The content of MDA and EL increased significantly in response to cold treatment (**Figures 2C,D**). While the melatonin pretreatment significantly lowered MDA concentration and EL.

according to Duncan's multiple range test at P < 0.05. I – seed soaking, II – root immersion, III – leaf spraying.

And the 100 µM melatonin pretreatment resulted in lower MDA content and EL than the 20 µM melatonin pretreatment. These results indicate that melatonin effectively protected the membrane system, and seed soaking and root immersing methods provided better protective effect on membrane system against cold stress (**Figures 2C,D**).

H2O2 and O− 2 are two major ROS induced by environmental stresses (Chen et al., 2016). Slight H2O2 and O− 2 staining was observed in the control leaves and intensive staining in the cold-treated plants was observed (**Figures 3A,B**). Histochemical staining showed that the H2O2 and O− 2 accumulations were alleviated by melatonin application during cold stress. To validate the results of histochemical staining, we measured H2O2 and O− 2 contents at the same time. H2O2 and O− 2 were extensively accumulated under cold treatment, but they were lower in melatonin-pretreated plants than the control seedlings (**Figures 3C,D**). By seed soaking and root immersing methods of melatonin application, the 100 µM melatonin pretreatment resulted in lower H2O2 content than 20 µM melatonin pretreatment. These results also suggested that the methods of seed soaking and root immersion could prevent the accumulation of ROS in leaf tissue more effectively than the method of leaf spraying (**Figure 3**).

We also detected cell death under cold stress with or without melatonin application by trypan-blue staining (**Figure 4**). After cold stress for 6 days, obvious cell death occurred in rice seedlings

cold stress (12◦C). Others are the same as in Figure 1.

in comparison with non-stressed plants. While, the cell dead was significantly alleviated in melatonin pretreated seedlings. The protective effect of 100 µM melatonin pretreatment was more significant than 20 µM melatonin pretreatment.

The effects of cold stress and melatonin applications on specific activities of antioxidant enzymes and on non-enzymatic antioxidants in rice seedlings are showed in **Figure 5**. The activities of antioxidant enzyme (SOD, CAT, and POD) were greatly induced by the cold stress (**Figures 5A–C**). Additionally, melatonin pretreated plants showed more higher activities of antioxidant enzymes (SOD, CAT, and POD), especially when the concentrations of melatonin were 100 µM (**Figures 5A–C**). The three different methods of melatonin application did not show significant differences in SOD and POD activities (**Figures 5A,B**), while the seed soaking and root immersion caused more increasement of CAT activity (**Figures 5C**).

We also measured the contents of proline, GSH/GSSG and soluble sugar, because they are important non-enzymatic antioxidants. After cold stress for 6 days, compared with the control, the contents of proline, GSH, GSSG and soluble sugar were significantly increased (**Figures 5D–F**). Moreover, the melatonin application further increased their accumulation under cold stress conditions. Meanwhile, compared to the cold treated seedlings alone, the 100 µM melatonin pretreatment increased the proline, GSH and soluble sugar more than the 20 µM melatonin pretreatment. Besides, the effects of three melatonin-application methods showed no significant difference (**Figures 5D–F**).

The effects of cold stress and melatonin application on photosynthesis were evaluated by monitoring gas exchange parameters [net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 (Ci) and WUE]. Cold stress significantly decreased all of these parameters, as shown in **Figures 6**. These parameters were higher in melatonin pretreated rice seedlings than the control plants. Melatonin caused significant increases in net photosynthetic rate, stomatal conductance and intercellular CO2 (**Figures 6A–C**), especially in the group of 100 µM melatonin pretreatment by root immersing method. While, only the root immersing method caused a significant increase in water use efficiency (**Figure 6D**).

After cold treatment, the maximum photochemical efficiency of PSII (Fv/Fm) was significantly decreased, but it was higher in melatonin pretreated plants (**Figures 7A,C**). And there were no significant differences among the three melatoninapplication methods. In contrast to the change of Fv/Fm, the

FIGURE 3 | Effects of exogenous melatonin on H2O2 (A,C), and O− 2 (B,D) after 6-day cold stress (12◦C). Histochemical detection (A,B) and content (C,D). Others are the same as in Figure 1.

non-photochemical quenching (NPQ) was significantly increased after 6-day cold stress (**Figures 7B,D**). This increase was partly reversed by melatonin application (**Figures 7B,D**). The chlorophyll fluorescence data suggested that the seed soaking and root immersing applications were more effective than the leaf spraying (**Figure 7**).

PSII proteins were further detected by Western blotting. The results showed that the contents of D1, CP43, Lhcb1, and Lhcb2 were significantly reduced after exposure to cold conditions, while the contents of D2 and Lhcb3 proteins showed no significant change (**Figure 8**). However, higher D1, CP43, Lhcb1, and Lhcb2 contents were found for the melatonin pretreated plants, compared with the seedlings without melatonin treatments. For these PSII proteins, seedlings of 100 µM melatonin pretreatment showed better protective effects than those of the 20 µM melatonin pretreatment. While the three methods did not show any significant difference.

### DISCUSSION

Low temperature stress critically inhibits plant growth. In the present study, we found that the growth of rice seedlings was significantly inhibited under low temperature conditions. However, the application of exogenous melatonin significantly alleviated the cold-stress-induced growth inhibition. These results are in agreement with the previous reports that melatonin application can enhance cold tolerance of Arabidopsis (Bajwa et al., 2014), wheat (Hulya et al., 2014), and bermudagrass

(Hu et al., 2016). In the present study, we also investigated the effects of three different methods of melatonin application and our results showed the seed soaking and root immersion was more effective than leaf spraying. However, quantitative analysis of melatonin showed that the melatonin content of the leafspraying method was higher than the other two methods, especially in the plants treated with100 µM melatonin. We presume that large amounts of melatonin may stay on the leaf surface and only a few was absorbed into the leaf tissue when melatonin was sprayed to the leaves.

Photosynthetic pigments are susceptible to environmental stresses (Ashraf and Harris, 2013). Chlorophylls, the most abundant and essential pigments in higher plants, responsible for capturing light energy and driving electrons to the reaction center during photosynthesis. Under biotic or abiotic stresses, seedlings have decreased chlorophyll contents due to impaired chlorophyll biosynthesis or accelerated chlorophyll degradation (Perveen et al., 2010). In the present study, we showed that chlorophylls and carotenoids were significantly decreased when the seedlings were exposed to cold stresses. However, this

efficiency (WUE) (D) after 6-day cold stress (12◦C). Others are the same as in Figure 1.

inhibitory effect of cold stress on pigments was alleviated by melatonin pretreatments, and they showed some dose-dependent response. A high concentration of 100 µM melatonin provided better protective effects than the 20 µM treatment. These results indicate that exogenous melatonin pre-treatment improved cold stress tolerance in rice seedlings.

Lipid peroxidation and the subsequent increase in MDA content are reported in previous studies during cold stress (Hulya et al., 2014; Hu et al., 2016). EL is a good indicator of the permeability of the plasma membrane and increased under stress conditions (Liu et al., 2015; Zeng et al., 2015). Compare with the control seedlings, the cold-stressed seedlings showed a 180% increase in MDA content, and a 49% increase in EL (**Figures 2C,D**). These findings are consistent with prior researchers that abiotic stress markedly enhanced MDA content and EL in plants (Hulya et al., 2014; Liu et al., 2015).

Reactive oxygen species are the byproducts of photosynthesis, respiration and other normal metabolic processes, and they play an important role in stress tolerance (Chen et al., 2016). ROS are also involved in programmed cell death, abiotic stress response and virus defense (Shi et al., 2015a,b,c). However, excessive accumulation of ROS can stimulate membrane lipid peroxidation, and then lead to the damage of cell membrane, loss of cellular integrity, and cell death (Chen, 2003; Zeng et al., 2015). In the present study, O− 2 and H2O2 increased significantly under cold stress conditions. However, pretreatments with melatonin significantly decreased ROS levels in leaf tissues, and the alleviatory effects were more obvious for the seed soaking and root immersion methods than the leaf spraying method.

Plants have evolved enzymatic antioxidant system or the nonenzymatic antioxidant system to maintain the redox balance. Among enzymatic antioxidant defense systems, SOD is a key enzyme that dismutates O− 2 to O2 and H2O2, while CAT and POD can break down H2O2 to H2O through different pathway in plant cells (Mittler, 2002; Hu et al., 2016). While the non-enzymatic antioxidant system plays an important role in maintaining normal cell metabolism and increasing plant tolerance to stresses (Liu et al., 2015). Melatonin is a broad spectrum antioxidant and a crucial radical scavenger and could stimulate the activities of antioxidant enzymes (Hulya et al., 2014; Hu et al., 2016), and exogenous melatonin application might activate enzymatic antioxidant system or the nonenzymatic antioxidant system to maintain the redox balance. Shi et al. (2015a) found that exogenous melatonin alleviated ROS accumulation and cold-induced oxidative damages by directly scavenging ROS and enhancing antioxidative enzymes in bermudagrass. In present study, melatonin pretreatments led to increasing in SOD, POD and CAT activities of rice seedlings. GSH

is the main component of non-enzymatic antioxidant system in plants which is involved in direct and indirect regulations to ROS levels (Ning et al., 2015). GSH regulates the redox homeostasis of cell together with its oxidized form (GSSG) and based on the reduction potential of the GSH/GSSG couple (Szalai et al., 2009). Both of GSH and GSSG contents increased under the cold stress in this study, which could be further promoted by melatonin applications (**Figures 5E**). These results indicated that melatonin applications might promote GSH accumulation in plants suffered from cold stress.

Photosynthesis is an important physiological process occurring in green plants, which is the basis of the biological world, and is also an important medium for the earth's carbon and oxygen cycle. It is well known that photosynthesis is very sensitive to low temperatures (Dalal and Tripathy, 2012), which could induced the decrease of photosynthetic pigments, destruction of chloroplasts structure, and stomatal closure, and so forth (Cai et al., 2016). In the present study, rice seedlings experienced a pronounced depression in Pn under low temperature (**Figure 6A**), and this phenomenon has been also reported in other stress conditions (Wang et al., 2013a; Cai et al., 2016; Li et al., 2017). We observed that the decreased Pn was accompanied by significant declines in Gs and Ci (**Figures 6B,C**). Therefore, we presume that decrease of Pn under low temperature conditions may be attributed to stomatal limitation. In addition, both stomatal and non-stomatal inhibitions could reduce Pn. The decrease in stomatal conductance could result in a declined Pn and reduced assimilation products, thus causing an inhibited growth and a lower yield (Easwar Rao and Chaitanya, 2016). Melatonin applications can alleviate cold-stress-induced inhibitions to photosynthetic activities, and the seed soaking and root immersion applications were more effective than leaf spraying.

Chlorophyll fluorescence analysis was one of the most powerful and widely used means for obtaining the functional status of PSII (Wang et al., 2013b), and the Fv/Fm ratio represents the maximal photochemical efficiency of PSII (Zhou et al., 2010). Fv/Fm decreased because of photoinhibition or PSII damages. While the thermal dissipation, represented by NPQ, increased (Wang et al., 2013a; Zhang et al., 2013). Under the cold stress, the value of Fv/Fm decreased significantly, but it was still higher in melatonin pretreated plants, and NPQ was also kept at a high value. Melatonin treated rice seedlings maintained lower NPQ, also suggesting that melatonin could protect photosynthetic system from cold-stress damages. Similar changes were also observed in other abiotic stresses (Wang et al., 2013a; Zhang

et al., 2013). For chlorophyll fluorescence, we did not find any significant difference among the three methods of melatonin application, while the decrease of NPQ was more obvious in the seed soaking method than the other two methods.

PSII is a multi-subunit pigment-protein complex that catalyzes the light-driven electron transfer from water to plastoquinone (PQ) in the thylakoid membrane of high plants, algae, and cyanobacteria (Baena-González et al., 2013). PSII plays an especially important role in the responses of photosynthesis in the higher plants to environment stresses (Yuan et al., 2005). Previous studies have shown that D1 protein, one of the core proteins of PSII, is the most vulnerable component in PSII reaction center under stress conditions (Luo et al., 2009; Li et al., 2017). It has been shown that the D1 protein can be significantly damaged by environmental stresses (Luo et al., 2009). In the present study, the lighter decreases in PSII proteins indicated the protective role of melatonin to PSII under cold stress.

In summary, the applications of exogenous melatonin alleviated the cold-stress-induced inhibition to plant growth by enhancing the activities of antioxidative enzymes and the levels of non-enzyme antioxidants. Melatonin could protect photosynthetic system from oxidative damages. The melatonin application by the seed soaking and root immersion was more effective than the leaf spraying method. All the effects of melatonin depended on its concentrations and the application methods. Our results imply that melatonin could be considered as an utilizable substance to improve plant cold tolerance.

### AUTHOR CONTRIBUTIONS

fpls-08-00785 May 10, 2017 Time: 16:37 # 12

SY and MY designed the experiments; Q-HH, BH, C-BD, Z-WZ, Y-EC, CH, L-JZ, YH, and J-QL performed the experiments and data analysis; and Q-HH, SY, and MY wrote the manuscript.

### REFERENCES

### ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (31300207), the Sichuan Natural Science Foundation (014z1700) and the Preeminent Youth Fund of Sichuan Province (2015JQO045).

reduction of oxidative stress in Bermuda grass (Cynodon dactylon (L). Pers.). J. Pineal Res. 59, 120–131. doi: 10.1111/jpi.12246

fpls-08-00785 May 10, 2017 Time: 16:37 # 13

Zhou, S. B., Liu, K., Zhang, D., Li, Q. F., and Zhu, G. P. (2010). Photosynthetic performance of Lycoris radiata var. radiate to shade treatments. Photosynthetica 48, 241–248. doi: 10.1007/s11099-010-0030-7

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Han, Huang, Ding, Zhang, Chen, Hu, Zhou, Huang, Liao, Yuan and Yuan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Comparative Physiological and Transcriptomic Analyses Reveal the Actions of Melatonin in the Delay of Postharvest Physiological Deterioration of Cassava

Wei Hu1† , Hua Kong1† , Yunling Guo1† , Yuliang Zhang1 , Zehong Ding1 , Weiwei Tie1 , Yan Yan1 , Qixing Huang1 , Ming Peng1 , Haitao Shi2 \* and Anping Guo1 \*

1 Key Laboratory of Biology and Genetic Resources of Tropical Crops, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China, 2 Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, College of Agriculture, Hainan University, Haikou, China

### Edited by:

Basil J. Nikolau, Iowa State University, USA

#### Reviewed by:

Thomas J. Bach, Université de Strasbourg, France Jie Zhou, Zhejiang University, China

#### \*Correspondence:

Haitao Shi<EMAIL_ADDRESS>Anping Guo<EMAIL_ADDRESS>
†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Plant Metabolism and Chemodiversity, a section of the journal Frontiers in Plant Science

Received: 26 March 2016 Accepted: 12 May 2016 Published: 27 May 2016

#### Citation:

Hu W, Kong H, Guo Y, Zhang Y, Ding Z, Tie W, Yan Y, Huang Q, Peng M, Shi H and Guo A (2016) Comparative Physiological and Transcriptomic Analyses Reveal the Actions of Melatonin in the Delay of Postharvest Physiological Deterioration of Cassava. Front. Plant Sci. 7:736. doi: 10.3389/fpls.2016.00736 Melatonin plays important roles in various aspects of biological processes. However, it is less known on the effects and mechanism of melatonin on the postharvest physiological deterioration (PPD) process of cassava, which largely restricts the potential of cassava as a food and industrial crop. In this study, we found that exogenous application of melatonin significantly delayed PPD of cassava tuberous roots by reducing H2O2 content and improving activities of catalase and peroxidase. Moreover, 3425 differentially expressed genes by melatonin during the PPD process were identified by transcriptomic analysis. Several pathways were markedly affected by melatonin treatments, including metabolic-, ion homeostasis-, and enzyme activity-related processes. Further detailed analysis revealed that melatonin acted through activation of ROS-scavenging and ROS signal transduction pathways, including antioxidant enzymes, calcium signaling, MAPK cascades, and transcription factors at early stages. Notably, the starch degradation pathway was also activated at early stages, whereas it was repressed by melatonin at middle and late stages, thereby indicating its regulatory role in starch metabolism during PPD. Taken together, this study yields new insights into the effect and underlying mechanism of melatonin on the delay of PPD and provides a good strategy for extending shelf life and improvement of cassava tuberous roots.

Keywords: cassava, melatonin, postharvest physiological deterioration, reactive oxygen species, starch metabolism

## INTRODUCTION

In the late 1950s, melatonin was first identified from the borine pineal gland (Lerner et al., 1958, 1959).  In addition, melatonin was reported to be a direct scavenger of reactive oxygen species (ROS) and of other free radicals, but also through activating antioxidant enzymes and augmenting the efficiency of other

antioxidants (Tan et al., 1993, 2003; Barlow-Walden et al., 1995; Pablos et al., 1998; Rodriguez et al., 2004; Hardeland, 2005; Koláˇr and Macháckova, 2005 ˇ ; Reiter et al., 2009; Galano et al., 2013; Zhang and Zhang, 2014). Dubbels et al. (1995), and Hattori et al. (1995) melatonin was discovered in plants. Further, endogenous concentrations of melatonin were found to be different in plants as function of growth state, growth location, plant tissue, and harvest time (Hattori et al., 1995; Burkhardt et al., 2001; Pape and Lüning, 2006; Hernández-Ruiz and Arnao, 2008; Arnao and Hernández-Ruiz, 2009, 2013; Murch et al., 2009; Okazaki et al., 2010; Ramakrishna et al., 2012; Wang L. et al., 2014).  Because melatonin can act as an important antioxidant in animals, many studies emphasized the importance of melatonin in directly or indirectly scavenging ROS in plants (Arnao et al., 2001; Cano et al., 2006).

Cassava is the third most important crop besides rice and maize in Africa, Asia, and Latin America (Oliveira et al., 2014; Hu et al., 2015a,b). Due to its high starch content and limited input needs, cassava can provide a source of dietary carbohydrate for over 600 million people worldwide, and is also considered as a producer of industrial starch and bioethanol (Zidenga et al., 2012). The potential of cassava as a food and industrial crop is largely restricted by the rapid postharvest physiological deterioration (PPD) of the tuberous root that starts within 72 h after harvest (Zidenga et al., 2012; Vanderschuren et al., 2014). PPD is induced by mechanical injury, handling operations, and storage conditions (Vanderschuren et al., 2014). To this end, more attention has been paid to the physiology and biochemistry of PPD (Reilly et al., 2001, 2004; Iyer et al., 2010; Zidenga et al., 2012; Xu et al., 2013; Vanderschuren et al., 2014). These studies have revealed that ROS production is one of the earliest events in the PPD process, and reduction of ROS accumulation by overexpression of scavenging enzymes could lead to the delay of PPD. However, less is known on the effect and mechanism of melatonin on the PPD process of cassava tuberous roots.

In this study, to gain insight into the roles and regulatory mechanism of melatonin in PPD of cassava, we investigated the effect of melatonin on the phenotype, physiology, and gene transcription rate during the postharvest period of cassava tuberous roots, and found that melatonin plays a role in the delay of PPD by reducing ROS and improving ROS signaling network. We also found that exogenous application of melatonin alters the expression of genes involved in the starch metabolism pathway during the PPD process. This study provides insights into the roles and molecular mechanisms of the actions of melatonin in the delay of PPD in cassava.

## MATERIALS AND METHODS

### Plant Material and Treatment

Cassava tuberous roots were harvested from 10-month-old cassava varieties SC124 (Manihot esculenta cv SC124) grown in a greenhouse (60% humidity, 16 h light, 35/20◦C). The tuberous roots were separated into two sample groups, water-treated PPD and melatonin-treated PPD. For water-treated PPD, whole tuberous roots were soaked in water for 2 h; for melatonin-treated PPD, whole tuberous roots were soaked in 100 µM melatonin for 2 h. Tuberous roots were cut into slices approximately 5 mm thick, then were moved into petri dishes containing a wet filter paper, and then were incubated at 28◦C and 60% relative humidity in the dark (Vanderschuren et al., 2014). After incubation for 0, 6, 12, 24, 48, and 72 h, the slices were sampled and frozen in liquid nitrogen until extraction of total RNA and transcriptomic analysis. Each sample contains four replicates from independent experiments.

### Determination of H2O2 Content and Antioxidant Enzyme Activities

The activities of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) and H2O2 content were measured spectrophotometrically. Approximately 0.5 g of roots slices was ground and homogenized in 5 mL of extraction buffer with 0.05 M phosphate buffer (pH7.8). The homogenate was centrifuged at 10000 g for 10 min at 4◦C and the resulting supernatant was collected for analysis of H2O2 content and enzyme activities. Total POD activity was examined according to the changes in absorbance at 470 nm (Polle et al., 1994). H2O2 content and activities of CAT and SOD were detected using H2O2, CAT and SOD Detection Kits (A064, A007 and A001, Nanjing Jiancheng, Nanjing city, China) according to manufacturer's instructions. Each sample contains four replicates consisting of three root slices each.

### Transcriptomic Analysis

Total RNA was extracted from tuberous roots of SC124 variety after 6, 12, and 48 h of incubation with or without melatonin according to the manufacturer's instructions for its plant RNA extraction kit (DP432, TIANGEN, Beijing city, China). Each sample contains three replicates from independent experiments. Three micrograms of total RNA from each sample were converted into cDNA with an AMV Reverse Transcriptase (Promega, Madison, WI, USA). Eighteen cDNA libraries were generated and sequenced by the use of Illumina GAII following the Illumina RNA-seq protocol. A total of 1667.1 million 51-bp raw reads was produced from the 18 samples. Using FASTXtoolkit, adapter sequences in the raw sequence reads were removed. After examining the sequence quality and removing low quality sequences by FastQC, 1589.5 million clean reads were generated. Using Tophat v.2.0.10, 84.2% clean reads were mapped to the cassava reference genome (version 4.1) (Trapnell et al., 2009). The transcriptome assemblies were performed by Cufflinks with alignment files (Trapnell et al., 2012). Gene expression levels were calculated as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Differentially expressed genes (DEGs) were identified by DEGseq according to the read count for each gene (Wang et al., 2010).

### Statistical Analysis

fpls-07-00736 May 25, 2016 Time: 16:1 # 3

Statistical analyses were carried out with SPSS (SPSS Inc., Chicago, IL, USA). Physiological data was analyzed by Duncan's multiple range test. Means denoted by the same letter do not significantly differ at P < 0.05 (lower case letters) or P < 0.01 (upper case letters) (n = 4). Transcriptomic data was analyzed using Student's t-tests at a significance level of ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (n = 3).

### RESULTS

### Exogenous Application of Melatonin Delayed PPD of Cassava Tuberous Roots

To investigate the effect of melatonin on PPD of cassava tuberous roots, SC124 variety widely planted in China were treated with water (control) or melatonin for 2 h, then the PPD symptoms were observed at 0, 6, 12, 24, 48, and 72 h after treatment (**Figure 1**). After 6 h of water treatment, PPD symptoms could be observed in the tuberous roots with slight 'vascular streaking.' Further, 'vascular discoloration' symptom with brown color becoming spread on the whole surface of cassava tuber slices became visible after 12 h of water treatment. In comparison to water treatment, melatonin-treated cassava tuberous roots showed 'vascular streaking' after 12 h of treatment and 'vascular discoloration' after 48 h of treatment. These results indicate that exogenous application of melatonin delays PPD occurrence in cassava tuberous roots.

### Exogenous Application of Melatonin Decreased H2O2 Content and Improved Activities of CAT and POD during the PPD Process

To test whether melatonin-induced delay in PPD is related to ROS scavenging in tuberous roots during the postharvest period, H2O2 content was examined at different time points. Generally, H2O2 content increased in the tuberous roots of SC124 during 0–72 h postharvest with or without melatonin treatment, indicating the increasing of oxidative damage during postharvest. Compared with water-treated samples, melatonin-treated cassava tuberous roots showed lower H2O2 accumulation during the 6 to72 h postharvest period (**Figure 2**).

To detect whether melatonin-induced ROS scavenging is related to the activities of antioxidative enzymes during the postharvest period, CAT, POD, and SOD activities were tested at different time points (**Figure 2**). During the PPD process, CAT activity was significantly higher in melatonin-treated cassava tuberous roots than that in control samples at stages of 6, 12, and 24 h after treatment. Additionally, POD activity significantly increased in melatonin-treated samples compared with controls at stages of 6, 24, and 48 h after treatment. There were no significant differences for SOD activity between controls and melatonin-treated samples. These results indicate that increased activities of CAT and POD may be involved in melatonininduced ROS scavenging during the PPD process.

### Differential Expression Profiling between Water-Treated and Melatonin-Treated Cassava Tuberous Roots

To study the transcriptionally regulatory mechanism underlying the melatonin-mediated delay of PPD in cassava, we performed comparative transcriptomic analysis between water-treated and melatonin-treated SC124 cassava tuberous roots during PPD. A total of 3425 differentially expressed genes (DEGs) (fold change ≥2; P-value ≤0.05) were identified by exogenous melatonin treatment, including 1439 (1079 up- and 360 down-regulated) genes from M6/CK6, 1211 genes from M12/CK12 (531up- and 680 down-regulated), and 1844 genes from M48/CK48 (682 upand 1162 down-regulated) (**Figure 3A**; Supplementary Tables S1–S4). Gene ontology (GO) enrichment analysis showed that many metabolic-, ion homeostasis-, and enzyme activity-related pathways were extensively changed after melatonin treatment during PPD (**Supplementary Figure S1**; Supplementary Table S5). Notably, some genes associated with response to oxidative stress, catalytic activity, antioxidant activity, and oxidationreduction were enriched, indicating that melatonin-induced antioxidant response was activated during PPD process.

Additionally, venn diagram analysis showed the amounts of 168 DEGs (fold change ≥ 2; P-value ≤ 0.05) present in all three stages, indicating that these genes were involved in the melatonin-mediated delay of PPD during 6–48 h (**Figure 3B**). Among these, some genes related to ROS scavenging were found, suggesting their crucial roles in the melatonin-mediated delay of PPD in cassava tuberous roots (Supplementary Table S6). GO enrichment analysis indicated that several pathways were significantly affected by melatonin treatments, including carbohydrate metabolic process, polysaccharide catabolic/metabolic process, biological process, glucosidase activity, hydrolase activity, and catabolic activity (Conesa et al., 2005) (**Supplementary Figure S2**; Supplementary Table S7).

### Melatonin-Mediated ROS Scavenging Activity by Antioxidant Enzymes during PPD Process

To better understand the action of melatonin on ROS scavenging during the PPD process, 34 genes encoding antioxidant enzymes were identified from the DEGs (fold change ≥2; P-value ≤0.05 at least in one stage of PPD). Heat map analysis indicated that 30, 10, and 8 genes were up-regulated after 6, 12, and 48 h melatonin treatment, respectively; whereas 4, 24, and 25 genes were down-regulated after 6, 12, and 48 h of melatonin treatment, respectively (**Figure 4**; Supplementary Table S8). From these data, it can be concluded that many genes were transcriptionally induced at an early storage stage (6 h), whereas they were repressed following middle (12 h) and late stages (48 h), indicating the rapid activation the expression of genes encoding antioxidant enzymes by melatonin.

### Melatonin-Mediated ROS Signaling Network during the PPD Process

To gain insight into the melatonin-mediated ROS signaling network during the PPD process, 161 genes related to ROS response were identified from the DEGs (fold change ≥2; P-value ≤0.05 at least in one stage of PPD), including calcium signaling-, phospholipase signaling-, MAPK cascades-, NADPH oxidase-, and transcription factor-related genes (**Figure 5**; Supplementary Table S9). Expression profiles showed that 114, 98, and 83 genes showed up-regulation after 6, 12, and 48 h melatonin

treatment, respectively; whereas 47, 61, and 76 genes showed down-regulation after 6, 12, and 48 h melatonin treatment, respectively. This indicates that more ROS-responsive genes were activated at early stages relative to middle and late stages by melatonin.

### Melatonin-Mediated Starch Metabolism during the PPD Process

To get some clues on the roles of melatonin in regulating the starch synthesis and degradation processes, 17 starch metabolic associated genes were identified from the DEGs (fold change ≥ 2; P-value ≤ 0.05 at least in one stage of PPD). Transcriptomic data indicated that 9, 3, and 3 genes were up-regulated after 6, 12, and 48 h melatonin treatment, respectively; whereas 8, 14, and 14 genes were down-regulated after 6, 12, and 48 h melatonin treatment, respectively (**Figure 6**; Supplementary Table S10). Genes related to starch synthesis, including sucrose synthase, glucose phosphomutase, and ADP-glucose pyrophosphorylase were mainly repressed, indicating the repression of starch synthesis process by melatonin during PPD. Most of genes involved in starch degradation, such as starch phosphorylase, alpha-amylase, and beta-amylase were induced at the early stage (6 h); however, repressed at the middle and late stages (12 and 48 h). These results suggest that melatonin may be a crucial regulator of starch metabolism during PPD process of cassava tuberous roots.

### DISCUSSION

Cassava is one of the most important crop in Africa, Asia, and Latin America. Nevertheless, the rapid PPD of its tuberous roots, a unique phenomenon compared with other root crops, renders the roots unpalatable and unmarketable, thereby adversely impacting farmers, processors, and consumers alike (Xu et al., 2013). Previous studies demonstrated that ROS production is one of the earliest events in the deterioration process and reduction of ROS accumulation could lead to a delayed onset of PPD (Reilly et al., 2001, 2004; Iyer et al., 2010; Zidenga et al., 2012; Xu et al., 2013; Vanderschuren et al., 2014). Melatonin was reported to function in the reduction of ROS through scavenging ROS directly and enhancing the levels of antioxidants and the activities of antioxidative enzymes (Tan et al., 1993, 2003; Barlow-Walden et al., 1995; Pablos et al., 1998; Rodriguez et al., 2004; Hardeland, 2005; Koláˇr and Macháckova, 2005 ˇ ; Reiter et al., 2009; Galano et al., 2013; Zhang and Zhang, 2014; Zhang et al., 2014a; Shi et al., 2015d). Therefore, we hypothesized that exogenous application of melatonin might delay PPD of cassava tuberous roots by scavenging ROS. To confirm this, cassava tuberous roots were treated with melatonin. Compared with controls, melatonin-treated cassava tuberous roots showed delayed PPD both for 'vascular streaking' and 'vascular discoloration' symptoms, indicating that melatonin has a clear effect on PPD (**Figure 1**). Physiological analyses also support that melatonin-mediated delay of PPD is related to reduced H2O2 and increased activities of CAT and POD (**Figure 2**).

To provide the possible molecular evidence, transcriptomic analysis was carried out between water-treated and melatonintreated cassava tuberous roots during the PPD process. Among the DEGs, 30 out of 34 genes encoding enzymes associated with ROS scavenging, including SOD, POD, CAT, APX, PrxR, were transcriptionally induced by melatonin at early storage stage (6 h) during PPD (**Figure 4**; Supplementary Table S8). These genes provide cells with highly efficient machinery for detoxifying O2− and H2O2 in plants (Mittler et al., 2004). Accordingly, the decreased H2O2 content, increased activities of POD and CAT, and lightened PPD symptoms after melatonin treatment were also observed at early storage stage (6 h) during PPD (**Figures 1** and **2**). These results further support that the melatonin mediated ROS-scavenging system may contribute to reducing oxidative injury of cassava tuberous roots.

Previous studies demonstrated that an oxidative burst occurred at the early stage of PPD (Reilly et al., 2001, 2004; Iyer

et al., 2010; Zidenga et al., 2012; Xu et al., 2013; Vanderschuren et al., 2014). Coincidently, melatonin acted through scavenging ROS by antioxidant enzymes, thus assisting in reduction of cellular ROS levels at early stage. After oxidative burst, cells still need to maintain a homeostasis of ROS. Thus, the downregulation of genes encoding ROS-scavenging enzymes at

mitogen-activated protein kinase cascades; WRKY, WRKY transcription factor; ZAT, zinc finger protein; HSF, heat stress transcription factor; MYB, MYB transcription factor; NAC, NAC transcription factor; AP2/ERF and B3 domain-containing transcription factor; NADPH, NADPH oxidase.

middle (12 h) and late stages (48 h) by melatonin implied that melatonin may be a flexible regulator of ROS during the PPD process (**Figure 4**; Supplementary Table S8). In addition, although melatonin repressed the expression of genes encoding ROS-scavenging enzymes at middle and late stages, melatonintreated samples still maintained lower H2O2 levels and higher activities of CAT and POD relative to control samples (**Figures 1** and **2**). This implies that melatonin may extend the effects of early transcriptional activation to enzyme activity levels at middle and late stages through a series of post-transcriptionally regulatory mechanisms.

Plant cells sense ROS through at least three different mechanisms, including unidentified receptor proteins, redox-sensitive transcription factors, and direct inhibition of phosphatases. Downstream signaling events include calcium and phospholipid signaling pathways, and hence activate oxidative signal-inducible protein (OXI1), MAPK cascades, NADPH oxidase, and transcription factors (Mittler et al.,

2004; Kim et al., 2011). To address the question how does melatonin regulate cells to perceive and transduct ROS signaling, those genes related to the ROS network were identified in the melatonin-treated cassava tuberous roots. Notably, many calcium signaling pathway connected genes, including calcium-sensing, -transport, -transduction genes, and downstream transcription factors, were significantly induced, whereas almost all of the phospholipase C (PLC) and phospholipase D (PLD) genes in phospholipid signaling pathway were repressed by melatonin during the PPD process (**Figure 5**; Supplementary Table S9). Accordingly, phosphoinositide-dependent kinase (PDK1), a downstream component of phospholipid signaling pathway, did not show significant difference at transcriptional levels with melatonin treatment. This suggests that melatonin-induced ROS signaling is positively transducted through calcium signaling components. In response to melatonin, 14 genes related to calcium-dependent signaling were induced in Arabidopsis by RNA-seq analysis (Weeda et al., 2014). Calcium-related protein kinase (CRK), calcium-dependent protein kinase (CDPK), and calcineurin B-like (CBL)-interacting protein kinase (CIPK) were reported to be commonly regulated by melatonin treatment in Bermuda grass (Shi et al., 2015a). Many transcription factors, including WRKY, NAC, ZAT, and HSF were up-regulated after melatonin treatment and some of them were confirmed to play a melatonin-mediated protective role in abiotic stress response and leaf senescence by functional analyses (Shi and Chan, 2014;

Zhang et al., 2014b; Shi et al., 2015a,c). Also, the crosstalk between melatonin and calcium signaling has been demonstrated to modulate various calcium-dependent cellular functions in animal cells (Posmyk and Janas, 2009). Additionally, calmodulin proteins were also reported to be up-regulated during early PPD of cassava (Owiti et al., 2011). Therefore, the extensive activation of calcium signaling and related transcription factors may be an important event in melatonin-mediated ROS pathway during PPD.

Besides, we also noted that the number of genes related to ROS network induced by melatonin was greater at early stages than that at middle and late stages (**Figure 5**; Supplementary Table S9). This is consistent with the expression patterns of genes encoding ROS-scavenging enzymes after melatonin treatment in general (**Figure 4**; Supplementary Table S8). Based on this consistent trend, it is concluded that coincidently with an oxidative burst melatonin acted by inducing cells to sense and transduct ROS signaling through calcium signaling and related transcription factors, thus resulting in the induction of ROS-scavenging genes to relieve ROS injury.

Interestingly, we observed that numerous starch metabolismrelated genes showed transcriptional changes after melatonin treatment according to GO analyses (**Supplementary Figures S1** and **S2**). Most of genes related to starch synthesis were repressed during the PPD process (**Figure 6**; Supplementary Table S10). After harvest, the primary carbon source supplied by photosynthesis was cut off, which limited the function of starch synthesis-related genes. The repression of starch synthesis pathway by melatonin may represent an action of melatoninmediated saving of cell resources in tuberous roots after harvest.

Additionally, exogenous application of melatonin resulted in upregulation of starch degradation related genes at early stage of PPD (6 h) (**Figure 6**; Supplementary Table S10), which is consistent with the expression profiles of genes in ROS-scavenging and ROS signal transduction pathways. Starch degradation produces glucose that supplies energy for various biological process. It is possible that melatonin-mediated starch degradation may provide energy for the active ROS-scavenging process, such as ascorbate-glutathione cycle, at early stage of PPD.

As reported by Uarrota et al. (2016), degradation of starch occurred during the PPD process, which resulted in reducing starch content, thus decreasing the commercial value of cassava. Exogenous application of melatonin decreased the transcripts of starch degradation associated genes at the middle and late stages (12 and 48 h) of postharvest (**Figure 6**; Supplementary Table S10). This indicates that melatonin cannot only alleviate PPD symptoms, but also can synchronously inhibit starch

FIGURE 7 | Overview of melatonin action on the delay of PPD of cassava tuberous roots. At early stage of the PPD process, coincidently with oxidative burst, melatonin acted on inducing cells to sense and transduct ROS signaling through calcium signaling and related transcription factors, thus resulting in the induction of ROS-scavenging genes to relieve ROS injury and delay of PPD. Melatonin-mediated starch degradation may provide energy for the active ROS-scavenging process at early stage of PPD. At middle and late stages, melatonin function on repressing starch degradation, benefit for improving food characteristics and quality of cassava. During all three stages, the repression of starch synthesis pathway by melatonin may represent an action of melatonin-mediated saving resources of cells in tuberous roots.

degradation, which is of great importance for improving food characteristics and quality of cassava. ROS have been implicated in the oxidative-reductive depolymerization of carbohydrates (Uarrota et al., 2016). The early activation of ROS-scavenging events by melatonin may contribute to the subsequent repression of starch degradation during PPD process.

Recent studies suggested that low doses of melatonin accelerated starch catabolism at night, whereas high doses of melatonin significantly decreased this process and led to starch accumulation in photosynthetic tissues of maize (Zhao et al., 2015). Additionally, melatonin could alter the metabolic status and increase the levels of starch in leaf tissue of Malus hupehensis (Wang et al., 2013a). These studies indicate the physiolgical effect of melatonin on starch metabolism in photosynthetic tissues. The evidences presented here expand the role of and transcriptionally regulatory mechanism of melatonin on starch metabolism in nonphotosynthetic tissue.

### CONCLUSION

This study demonstrated the effect of melatonin on the delay of PPD in cassava tuberous roots is related to ROS-scavenging pathway, ROS-signaling transduction, and starch metabolism (**Figure 7**). These findings would contribute to extend shelf life and improve quality of casssava tuberous roots.

### REFERENCES

### AUTHOR CONTRIBUTIONS

HS, HK, MP, and AG conceived the study. WH, YG, YZ, and YY performed the experiments. ZD, QH, and WT carried out the analysis. WH and HS designed the experiments and wrote the manuscript.

### ACKNOWLEDGMENTS

This study was funded by the National Natural Science Foundation of China (31171610), the Natural Science Foundation of Hainan Province (314122, 20153048), and the startup funding of Hainan University (kyqd1531). We thank Professor Kaimian Li and Songbi Chen for providing plant materials.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016.00736

FIGURE S1 | Differentially expressed genes at 6, 12, and 48 h storage after melatonin treatment by GO enrichment analysis. Asterisks indicate a significant difference (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001).

FIGURE S2 | Common genes differentially expressed at 6, 12, and 48 h storage after melatonin treatment by GO enrichment analysis. Asterisks indicate a significant difference (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001).

analysis in functional genomics research. Bioinformatics 21, 3674–3676. doi: 10.1093/bioinformatics/bti610

expression profiling of the ROS scavenging and signal transduction pathways. J. Plant Physiol. 168, 1960–1971. doi: 10.1016/j.jplph.2011.05.008

Reiter, R. J., Tan, D. X., and Galano, A. (2014). Melatonin: exceeding expectations. Physiology 29, 325–333. doi: 10.1152/physiol.00011.2014

dactylon (L). Pers.] by exogenous melatonin. J. Exp. Bot. 66, 681–694. doi: 10.1093/jxb/eru373

fpls-07-00736 May 25, 2016 Time: 16:1 # 11

Zidenga, T., Leyva-Guerrero, E., Moon, H., Siritunga, D., and Sayre, R. (2012). Extending cassava root shelf life via reduction of reactive oxygen species production. Plant Physiol. 159, 1396–1407. doi: 10.1104/pp.112. 200345

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Hu, Kong, Guo, Zhang, Ding, Tie, Yan, Huang, Peng, Shi and Guo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Melatonin-Producing Endophytic Bacteria from Grapevine Roots Promote the Abiotic Stress-Induced Production of Endogenous Melatonin in Their Hosts

Jian Jiao1 , Yaner Ma2 , Sha Chen3 , Chonghuai Liu2 , Yuyang Song1 , Yi Qin1 , Chunlong Yuan1 \* and Yanlin Liu1 \*

1 College of Enology, Northwest A&F University, Yangling, China, 2 Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou, China, 3 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

### Edited by:

Haitao Shi, Hainan University, China

### Reviewed by:

Yuan Huang, Huazhong Agricultural University, China Shan Yuan, China Agricultural University, China

#### \*Correspondence:

Yanlin Liu<EMAIL_ADDRESS>Chunlong Yuan<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Metabolism and Chemodiversity, a section of the journal Frontiers in Plant Science

Received: 02 August 2016 Accepted: 31 August 2016 Published: 21 September 2016

#### Citation:

Jiao J, Ma Y, Chen S, Liu C, Song Y, Qin Y, Yuan C, and Liu Y (2016) Melatonin-Producing Endophytic Bacteria from Grapevine Roots Promote the Abiotic Stress-Induced Production of Endogenous Melatonin in Their Hosts. Front. Plant Sci. 7:1387. doi: 10.3389/fpls.2016.01387 Endophytes form symbiotic relationships with plants and constitute an important source of phytohormones and bioactive secondary metabolites for their hosts. To date, most studies of endophytes have focused on the influence of these microorganisms on plant growth and physiology and their role in plant defenses against biotic and abiotic stressors; however, to the best of our knowledge, the ability of endophytes to produce melatonin has not been reported. In the present study, we isolated and identified root-dwelling bacteria from three grapevine varieties and found that, when cultured under laboratory conditions, some of the bacteria strains secreted melatonin and tryptophan-ethyl ester. The endophytic bacterium Bacillus amyloliquefaciens SB-9 exhibited the highest level of in vitro melatonin secretion and also produced three intermediates of the melatonin biosynthesis pathway: 5-hydroxytryptophan, serotonin, and N-acetylserotonin. After B. amyloliquefaciens SB-9 colonization, the plantlets exhibited increased plant growth. Additionally, we found that, in grapevine plantlets exposed to salt or drought stress, colonization by B. amyloliquefaciens SB-9 increased the upregulation of melatonin synthesis, as well as that of its intermediates, but reduced the upregulation of grapevine tryptophan decarboxylase genes (VvTDCs) and a serotonin N-acetyltransferase gene (VvSNAT) transcription, when compared to the un-inoculated control. Colonization by B. amyloliquefaciens SB-9 was also able to counteract the adverse effects of salt- and drought-induced stress by reducing the production of malondialdehyde and reactive oxygen species (H2O2 and O2 −) in roots. Therefore, our findings demonstrate the occurrence of melatonin biosynthesis in endophytic bacteria and provide evidence for a novel form of communication between beneficial endophytes and host plants via melatonin.

#### Keywords: melatonin, endophytic bacteria, UPLC-MS/MS, grapevine, abiotic stress

**Abbreviations:** IAA, indole-3-acetic acid; MDA, malondialdehyde; ROS, reactive oxygen species; TEE, tryptophan-ethyl ester.

### INTRODUCTION

fpls-07-01387 September 17, 2016 Time: 16:0 # 2

Melatonin (N-acetyl-5-methoxytryptamine) was first isolated from the bovine pineal gland (Lerner et al., 1958) and is now recognized as ubiquitous among living organisms, including humans, animals, plants, bacteria, fungi, and macroalgae (Tilden et al., 1997; Rodriguez-Naranjo et al., 2012; Tan et al., 2012). In vertebrates, the molecule functions as a biological modulator of mood, sleep, sexual behavior, seasonal reproductive physiology, and circadian rhythms (Reiter, 1993; Hardeland, 2008; Reiter et al., 2010); as a potent antioxidant, with free radical-scavenging activities; and as a stimulator of antioxidant enzyme activities (Reiter et al., 2005). Meanwhile, the occurrence of melatonin in higher plants wasn't documented until almost 50 years later, when it was reported by both Dubbels et al. (1995) and Hattori et al. (1995). Since then, melatonin has been recognized to function as an abiotic antistressor, by protecting plants against ROS that are produced as a result of harmful abiotic stresses (Posmyk et al., 2008, 2009; Arnao and Hernández-Ruiz, 2009a; Nawaz et al., 2016). In addition, melatonin also functions as a plant regulator, with growth-promoting effects similar to those of IAA, which is a plant hormone in the auxin class (Hernández-Ruiz and Arnao, 2008).

Since the discovery of melatonin in higher plants, the factors that influence the endogenous melatonin levels in plants have remained relatively unexplored. Recently, reports have demonstrated that different abiotic stressors are able to elevate the endogenous melatonin levels of plants (Arnao and Hernández-Ruiz, 2013a,b; Shi et al., 2015; Hernández-Ruiz and Arnao, 2016) and that stress-induced ROS bursts may be the common factor that triggers the accumulation of melatonin (Arnao and Hernández-Ruiz, 2015). Under natural conditions, the internal organs of plants are frequently colonized by a vast number of diverse microbes that are able to interact with their hosts and, thereby, modulate plant growth and development (Sturz et al., 2000). Soil microbes, for example, have been shown to enter and proliferate within plant roots (Hardoim et al., 2008), and in grapevines, naturally occurring endophytes have been isolated from roots, stems, leaves, and various reproductive tissues (e.g., inflorescences, seeds, and fruits; Compant et al., 2011). These symbiotic organisms are important in defending their hosts against phytopathogens (Lindow and Brandl, 2003; West et al., 2010) and may also promote the growth of their host plants via nitrogen fixation (Elbeltagy et al., 2001), phosphorus solubilization (Richardson et al., 2009), and the enhancement of plant hormones levels (Ali et al., 2009). Therefore, endophytes are generally recognized as important and beneficial components of plant micro-ecosystems. In fact, since endophytes often supply their hosts with plant hormones, we speculate that endophytes are capable of producing melatonin and that they provide melatonin to their plant hosts. This conjecture is based on observations that (i) melatonin has been identified in microorganisms, such as aerobic photosynthetic bacteria (Tilden et al., 1997), recombinant E. coli (Byeon and Back, 2016) and some fungi (Manchester et al., 1995; Hardeland and Poeggeler, 2003); (ii) melatonin biosynthesis is likely to be evolutionarily conserved (Tan et al., 2014); (iii) the cellular machinery for melatonin synthesis in eukaryotes may have been inherited from bacteria, as a result of endosymbiosis (Tan et al., 2013); and (iv) bioinformatic analyses has revealed that enzymes involving in melatonin synthesis occur in bacterial genomes (Pavlicek et al., 2010; Falcón et al., 2014). However, to the best of our knowledge, the ability of endophytic bacteria to produce melatonin has not been reported, and the synthetic pathway of melatonin in heterotrophic bacteria remains to be elucidated.

Therefore, in the present study, we used a culture-dependent method to isolate endophytic bacteria from the roots of three grapevine varieties and screened the resulting cultures for their in vitro capacity to produce melatonin. We subsequently used a promising endophytic strain that produced high levels of melatonin to investigate the intermediates of the melatonin biosynthesis pathway, and in addition, we also performed root colonization experiments, in order to evaluate the effect of the strain on the endogenous melatonin production of host plants under abiotic stress. Finally, to examine whether stress-induced changes in melatonin levels were associated with the induction of melatonin synthesis, we performed qRT-PCR analysis of VvSNAT and several VvTDCs genes, both of which play a pivotal role in regulating melatonin biosynthesis in plants (Byeon et al., 2013; Zhao et al., 2013).

### MATERIALS AND METHODS

### Isolation of Endophytic Bacteria from Grapevine Roots

We selected three grapevine varieties for our experiments, including the Chinese wild grapevine Vitis amurensis 'Changbai 9,' V. vinifera 'Cabernet Sauvignon,' and V. labruscana 'Summer Black,' and collected root samples from plants that were cultivated at the National Grape Germplasm Repository (113◦ 700 E; 34◦ 720 N), Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou, Henan, China. The root samples were collected from one vine of each variety, when the vines were in flower, and a total of 5 g roots were collected from five randomly chosen root sections (∼20 cm below ground) each plant and pooled in sterile 15-mL tubes. The pooled root samples were then kept in refrigerated boxes and transported to the laboratory within 1 h of collection for subsequent processing.

The roots were surface-sterilized in 70% ethanol for 3 min, followed by soaking in sodium hypochlorite (3% available chlorine) for 2 min, and were then rinsed three times with sterile water. Next, each sample was ground and homogenized in 1.5 mL of PBS using sterile quartz sand in individual mortars. The resulting homogenate was serially diluted (10−3 , 10−4 , and 10−5 ) and plated on nutrient agar (3 g/L beef extract, 10 g/L tryptone, 5 g/L NaCl, and 20 g/L agar, pH 7.4) with 100 mg/L actidione to inhibit the growth of fungi, and each dilution was prepared in triplicate. In parallel, we also checked the efficiency of our surface sterilization procedure by plating 100 µL of the last washing solution (i.e., sterile water used for third rinse) onto nutrient agar. Then, after incubation at 28◦C for 2–3 days, the number of colony-forming units was counted, and four to six

representative isolates of each morphology were collected and purified by streaking onto fresh nutrient agar plates. Each purified isolate was maintained at −80◦C in 1 mL sterile nutrient broth that contained 20% glycerol.

### Genomic DNA Extraction and Species Assessment

DNA extraction was performed using the TIANamp bacteria DNA Kit (Tiangen, Beijing, China), according to the manufacturer's instructions, and in order to identify each of the isolates, we amplified the 16S rRNA sequence, using the primers 8F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1063R (50 -ACGGG CGGTGTGTRC-30 ) (Wang and Qian, 2009), as well as the gyrB sequence, which encodes the B subunit of the type II topoisomerase DNA gyrase, using the degenerate primers UP-1 (50 - GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTY GA-30 ) and UP-2r (50 -AGCAGGATACGGATGTGCGAGCCRTC NACRTCNGCRTCNGTCAT-30 ) reported by Yamamoto and Harayama (1995). The resulting PCR products were purified and bi-directionally sequenced, using the same primers that were used for PCR amplification. The sequences were then compared with reference sequences in GenBank, using the online Blastn software1 , and identification was considered valid when the identity of a contiguous sequence was ≥99%.

### Screening for Melatonin-Producing Endophytic Bacteria

After determining the identities of the individual isolates to the lowest possible taxonomic level (i.e., species or genus), we randomly selected individual strains to represent each taxon. The selected strains were inoculated into 10 mL lysogeny broth medium, and incubated for 24–36 h, until they reached an OD600 of ∼1.0–1.5. Once this concentration was achieved, the liquid medium was removed by centrifugation at 8000 × g and 4◦C for 10 min, and the resulting pellets were washed once with 10 ml sterile PBS and re-suspended in 10 mL minimal medium (Voigt et al., 2007). The cultures were then incubated at 28◦C for 8 h to allow for the depletion of amino acids, after which the cell concentration of the cultures was determined using a Petroff–Hausser counting chamber and the cultures were standardized to 108 cells/mL. Next, 1 mL of each of the standardized bacterial cultures was inoculated into individual brown bottles (250 mL) that contained 100 mL of nutrient broth with 200 mg/L tryptophan, and the cultures were incubated in a rotary shaker at 28◦C with a rotational speed of 150 rpm in the dark. Cell viability was quantified using the plate counting method with appropriate dilutions. After 36 h, the bacterial cultures were centrifuged and the supernatants were diluted 1:1 with methanol, and the resulting mixtures were passed through a 0.22-µm filter and used for preliminary screening of melatonin and TEE production.

To investigate the potential conversion of L-tryptophan to melatonin in the bacterial strain that produced the highest concentration of melatonin, we measured the concentrations of several intermediate molecules of melatonin biosynthesis in its culture medium. The strain was cultivated using the procedure described above. In addition, every 6 h, we sampled the bacterial cultures, centrifuged the samples at 8000 × g for 10 min, and diluted the supernatant 1:1 with methanol. Then, after being passed through a 0.22-µm filter, the resulting mixtures were analyzed for tryptamine, 5-hydroxytryptophan, serotonin, N-acetylserotonin, and melatonin contents using UPLC-MS/MS.

### Colonization Assay

To determine whether the melatonin-producing endophytic bacteria could influence the endogenous melatonin level in roots, grapevine plantlets were inoculated with a bacterial isolate that produced the highest amount of melatonin. The bacterial inoculum was prepared by inoculating the strain into 100 mL nutrient broth, incubating the culture for 24–36 h at 28◦C, centrifuging the culture at 6000 × g for 10 min, and re-suspending and standardizing the inoculum to 107–108 cells/mL with 0.9% sterilized saline solution. The standardized inoculum was then used to inoculate 6-week-old tissue-cultured V. labruscana 'Summer Black' plantlets. The roots of the experimental plantlets were immersed in the bacterial inoculum for 1 min, whereas the roots of the control plantlets were treated with 0.9% sterile saline solution, and afterward, all the plantlets were transferred to 500 mL culture bottles that contained 150 g sterile nutrient soil (Pindstrup, Ryomgaard, Denmark) and 40 mL nutrient-rich water that was prepared with the MS (Murashige and Skoog) basic nutrient medium (Cat# M519; Phytotechnology2 ). The plants were randomly distributed in a greenhouse with a 16-h light/8-h dark cycle at 26◦C and irrigated with distilled water (5 mL) every 2 days.

After 20 days of endophyte colonization, we randomly selected 12 plantlets from each of the inoculated and control plantlets, in order to compare their growth, which we assessed by measuring root length, root fresh weight (FW), plant height, and chlorophyll content. The chlorophyll content of fully expanded leaves was analyzed using a chlorophyll ELISA Kit, according to the manufacturer's instructions (Lvyuan, Beijing, China). In addition, the roots were also sampled to determine counts of viable bacteria in the roots of inoculated and control plants, as described in the "Isolation of endophytic bacteria from grapevine roots" section above, and the plate counts of viable bacteria were considered an indicator of bacterial invasion capacity.

Meanwhile, the rest of plantlets were assigned to one of four experimental treatment groups: (i) inoculated plantlets subject to salt stress; (ii) control plantlets subject to salt stress; (iii) inoculated plantlets subject to drought stress; and (iv) control plantlets subject to drought stress. Briefly, 20 mL NaCl solutions (60 or 120 mM) were applied to a series of inoculated and control plantlets in order to simulate salt stress, and 20 mL 10% PEG-6000 solution was added to another series of inoculated and control plantlets in order to simulate drought stress, with 12 plantlets per treatment. After 4 days, the roots were sampled

1http://www.ncbi.nlm.nih.gov/BLAST

2http://www.phytotechlab.com/

from each plantlet at 9–10:00 AM and ground into powder using liquid nitrogen in individual mortars, and 0.5 g of each root powder was extracted with 2 mL of methanol, as described previously (Boccalandro et al., 2011). The resulting extracts were mixed with 2 mL ultrapure water, centrifuged, passed through a 0.22-µm filter, and stored in amber vials for analysis of 5 hydroxytryptophan, tryptamine, serotonin, N-acetylserotonin, and melatonin via UPLC-MS/MS.

The H2O2 levels of the roots were measured according to Patterson et al. (1984), and superoxide production was estimated using the method of Elstner and Heupel (1976). In addition, we assessed the extent of lipid peroxidation in the roots by measuring the MDA content as described by Zhao et al. (2013) with little modification. Briefly, 0.1 g of each root powder was extracted with 1 mL of 10% (w/v) trichloroacetic acid (TCA), vortexed, and centrifuged at 8000 × g for 10 min. Then, 0.2 ml of each supernatant was mixed with 0.2 ml 0.5% (w/v) thiobarbituric acid (TBA) in 20% (w/v) TCA. The mixtures were heated at 100◦C for 20 min, cooled, and centrifuged at 8000 × g for 10 min. Absorbances were read at 440, 532, and 600 nm, and the MDA concentration (nmol/g FW) was calculated according to the formula: [6.45 × (A532 – A600) – 0.56 × A450] × V/W, where V (mL) is the volume of the tissue extract, and W (g) is the FW.

### RNA Extraction and Quantitative Real-Time PCR Analysis

Total RNA was isolated from the root tissue of stress-induced plantlets, following the CTAB method (Reid et al., 2006), and the resulting RNA was treated with DNase I and converted to cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (Takara, Dalian, China).

Three TDC homologs, which shared conserved functional domains and >30% homology with the amino acid sequence of rice tryptophan decarboxylase (GenBank No. XP\_015648768), were identified by searching the non-redundant protein and nucleotide sequence data of grapevine (Vitis Vinifera L.) at the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MD, USA), using the tBLASTn, BLASTp, and PSI-BLAST programs (Altschul et al., 1997); and sequence of the grapevine serotonin N-acetyltransferase gene (VvSNAT; GenBank No. XM\_002266325) was previously predicted by Byeon et al. (2014a). The expression levels of all four genes in the roots of stress-induced plantlets were determined using quantitative real-time PCR (qRT-PCR) analysis with a Roche 480 light cycler System and SYBR Fast qPCR Mix (TaKaRa, Dalian, China). All primers were designed using the NCBI Primer-BLAST service3 (**Table 1**), and the qRT-PCR amplification was performed with the following conditions: 95◦C for 5 min, followed by 40 cycles of 95◦C for 5 s, 60◦C for 10 s, and 72◦C for 15 s; and melting curve analysis was performed using 95◦C for 5 s, 60◦C for 1 min, 97◦C continuously, and then 40◦C for 30 s. All the qRT-PCR reactions were performed in triplicate, and the expression levels of the target genes were normalized using the EF1-α gene as an internal reference.

TABLE 1 | Primers used for quantitative real-time PCR.

### UPLC-MS/MS Analysis of Metabolites

Tryptophan-ethyl ester, tryptamine, 5-hydroxytryptophan, serotonin, N-acetylserotonin, and melatonin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was produced using a Millipore Milli-Q ultrapure water purification unit (Millipore, Bedford, MA, USA). Other solvents, including methanol and formic acid (HPLC grade), were purchased from Merck (Darmstadt, Germany). Stock solutions were prepared by dissolving 10 mg of each standard in 1 mL methanol under low light conditions; the solutions were then stored at −80◦C to prevent degradation. Fresh working solutions were prepared in a methanol:water solution (50:50, v/v).

Quantitative detection was conducted using a UHPLC-ESI-QQQ-MS (Agilent 1290 and 6460 triple quadrupole mass spectrometry series; Agilent Corporation, Santa Clara, CA, USA). In the solvent system, Milli-Q water that contained 0.1% (v/v) formic acid was used as eluent A, and methanol was used as eluent B. The analytes were separated using an Agilent ZORBAX Eclipse XDB-C18, Rapid Resolution HT column (1.8 µm, 3.0 mm × 50 mm) at 42◦C with linear elution gradient protocols of 0–6 min, 5–55% B, 6–15 min, 55–100% B, at 0.2 mL/min flow rate. Next, 100% B was kept constant for 2 min and the column was re-equilibrated for 5 min using the initial solvent composition. The injection volume was 1 µL. The metabolites were quantitatively detected using the multiple reactions monitoring mode under unit mass-resolution conditions (tryptamine m/z 161→144, 5 hydroxytryptophan m/z 221→204, serotonin m/z 177→160, N-acetylserotonin m/z 219→160, and melatonin and TEE m/z 233→174) in positive ion mode. To quantify the analytes, we constructed eight-point calibration curves, using diluted working solutions of external standards. All points on the curves represented the average of three independent determinations. The linearity of the calibration graphs was determined using regression analysis. The limits of detection (LOD) were calculated based on the S/N ratio of 3:1. The limits of quantitation (LOQ) were defined as the lowest level that had an S/N ratio of 10:1. All the investigated analytes displayed excellent linearity, with correlation coefficients (R 2 ) ranging from 0.9975 to 0.9988 (**Table 2**).

3http://www.ncbi.nlm.nih.gov/tools/primer-blast/

Melatonin 0.12–1.2 0.9984 0.04 0.12 Tryptophan-ethyl ester 0.1–10 0.9979 0.03 0.1

TABLE 2 | UPLC-MS/MS quantitation data for six analytes.

fpls-07-01387 September 17, 2016 Time: 16:0 # 5

LOD, limit of detection; LOQ, limit of quantification.

### Statistical Analysis

For each experiment, the results were expressed as the mean ± standard deviation of data from 3–12 replicates. Statistical evaluation was performed using one-way ANOVA, followed by Tukey's test for the data of preliminary screening of melatonin production and Student's t-test for the colonization assay. All the statistical analyses were performed using SPSS (version 19.0; IBM, Armonk, NY, USA), and a P-value of <0.05 was considered statistically significant.

### RESULTS

### Melatonin and TEE Production by Endophytic Bacteria from Grapevine Roots

No colonies grew on the plates that were inoculated with water from the final washing step of the root sterilization procedure, suggesting that the surface sterilization procedure was effective. For the remaining plates, the highest bacterial count (5.75 ± 0.26 log10 CFU/g FW) was detected on those inoculated with the homogenized roots of V. labruscana 'Summer Black,' followed by those inoculated using V. vinifera 'Cabernet Sauvignon' roots (5.23 ± 0.18 log10 CFU/g FW) and V. amurensis 'Changbai 9' roots (4.89 ± 0.22 log10 CFU/g FW). A total of 98 endophytic bacteria strains were isolated from the surface-sterilized roots, and 16S rRNA sequences were amplified from each strain, whereas gyrB sequences were only amplified from 64. Based on comparison with related sequences deposited in the GenBank DNA database, we identified seven different bacterial genera, which included Agrobacterium, Bacillus, Variovorax, Pseudomonas, Streptomyces, Sphingomonas, and Ensifer.

The Streptomyces strains were excluded from the screening of melatonin- and TEE-producing abilities, owing to their abnormal growth and low biomass in nutrient broth. We randomly screened eight endophytic bacterial strains that represented eight species (**Table 3**). With the exception of B. cereus CS-17 and Sphingomonas sp. VA-16, all the investigated strains secreted tryptophan derivatives into the medium and exhibited species-specific levels of production (**Figure 1A**). Five of the strains secreted melatonin in vitro, and the highest level (7.75 ng/1012 cells; 0.87 ng/mL; cell count, 11.15 log10 CFU/mL) was produced by B. amyloliquefaciens SB-9, followed by B. thuringiensis CS-9 (3.33 ng/1012 cells; 0.53 ng/mL; cell count, 11.20 log10 CFU/mL) and Agrobacterium tumefaciens CS-30 (2.90 ng/1010 cells; 0.22 ng/mL; cell count, 10.88 log10 CFU/mL). TEE has previously been considered as one of the melatonin isomers, and this compound produced the same fragmentation pattern of melatonin using the triple quadrupole mass spectrometry (**Figure 1B**). We found that six strains, including all the melatonin-producing isolates, were able to produce TEE, with amounts ranging from 0.24 to 19.83 ng/1012 cells.

### Characterization of Melatonin Formation in B. amyloliquefaciens SB-9

The melatonin synthetic pathways of animals and plants have been reported previously (**Figure 2A**); however, the mechanism of melatonin synthesis in bacteria is currently unknown. We detected 5-hydroxytryptophan, serotonin, N-acetylserotonin, and melatonin after 6 h of incubation; however, we did not find tryptamine during the incubation. The concentrations of 5-hydroxytryptophan, serotonin, and N-acetylserotonin increased throughout the incubation period, and reached maximum values of 8.82 ± 1.08, 3.81 ± 0.46, and 8.41 ± 0.82 ng/mL, respectively, after 30 h (**Figure 2**), whereas the concentration of melatonin reached a maximum value of 1.19 ± 0.12 ng/mL after 24 h of incubation and declined slightly thereafter. When the results were expressed as ng/1012 viable cells, the production capacity for all the investigated metabolites peaked at 6 h (cell number, 9.87 log10 CFU/mL) and, thereafter, declined with increasing cell density (11.76 log10 CFU/mL at 30 h).

### Growth Responses of V. labruscana 'Summer Black' Plantlets Treated with B. amyloliquefaciens SB-9

Based on its high melatonin-producing capacity, we selected B. amyloliquefaciens SB-9 for the inoculation of grapevine plantlets. We observed no symptoms of pathogenicity in the inoculated V. labruscana 'Summer Black' plantlets. At 20 days after inoculation, B. amyloliquefaciens SB-9 was successfully re-isolated from inoculated roots, and its identity was verified via sequencing of the 16S rRNA region. In addition, the colonies recovered from the inoculated seedling roots exhibited a single morphotype (**Figure 3A**), and the population density was 5.74 ± 0.22 log10 CFU/g FW (**Figure 3B**), which indicated a high level of colonization, whereas no colonies were isolated from the control plant roots. Furthermore, we also observed that inoculation with B. amyloliquefaciens SB-9 significantly promoted the growth of the grapevine plantlets. In fact, the root length, plant height, FW, and leaf chlorophyll content of the inoculated plantlets were 48.58, 19.46, 41.82, and 41.76% greater, respectively, than those of the control plantlets (**Figures 3C,D**), which indicates that the strain can be regarded as a valuable plant growth-promoting rhizobacteria.

—, no PCR product.

### Effect of B. amyloliquefaciens SB-9 on Levels of Endogenous Melatonin and Its Intermediates in Roots under Stress

When the roots of inoculated and control plantlets were subjected to salt or drought stress, they responded by synthesizing melatonin and its intermediates (tryptamine, 5-hydroxytryptophan, serotonin, and N-acetylserotonin), albeit to different extents. We found that melatonin synthesis was similar in the inoculated and control plants under normal growth conditions; however, after exposure to salt or drought stress, the melatonin levels in the roots of inoculated plantlets were 52.61, 37.90, and 53.07% higher, respectively, than those in the roots of control plantlets (**Figure 4E**). Similarly, in the absence of abiotic stresses, 5-hydroxytryptophan levels in inoculated plants and control plants were similar, but after NaCl or 10% PEG 6000 treatment, the synthesis of 5-hydroxytryptophan in the roots of inoculated plantlets was significantly higher than that in the roots of control plants (**Figure 4B**). A similar trend was also observed for N-acetylserotonin, and in salt-stress plantlets (120 mM only), its level was significantly upregulated in inoculated plantlets, when compared to control plants (**Figure 4C**); however, the tryptamine levels in the roots of inoculated plantlets were significantly lower than those in the roots of control plants after exposure to abiotic stresses (**Figure 4A**), and the serotonin levels in inoculated and control plantlets were statistically similar (P > 0.05; **Figure 4D**). Therefore, it is likely that the synthesis of melatonin and its intermediates in the roots of plants exposed to abiotic stress is influenced by B. amyloliquefaciens SB-9 colonization.

### Effect of B. amyloliquefaciens SB-9 on Stress-Induced Oxidative Damage

The MDA level in plant tissue is an indicator of lipid peroxidation status, and it is accompanied by ROS production (H2O2 and O2 −). In the absence of abiotic stresses, the MDA level was markedly lower in the roots of inoculated plantlets than

FIGURE 2 | Evolution of metabolite production in melatonin biosynthesis pathway in Bacillus amyloliquefaciens SB-9. (A) Melatonin biosynthesis pathway in animals and plants. Red arrows indicate the animal pathway, and green arrows indicate the plant pathway. TPH, tryptophan hydroxylase; AAAD, aromatic amino acid decarboxylase; TDC, tryptophan decarboxylase; T5H, tryptamine 5-hydroxylase; AN-SNAT, animal serotonin N-acetyltransferase; PL-SNAT, plant serotonin N-acetyltransferase; AN-ASMT, animal N-acetylserotonin O-methyltransferase; PL-ASMT, plant N-acetylserotonin O-methyltransferase; CAMT, caffeic acid O-methyltransferase. (B) Chemical structures and UPLC-MS/MS chromatograms of five standards (40–60 ng/mL). (C) Levels of metabolites involved in the melatonin biosynthesis in a B. amyloliquefaciens SB-9 culture grown in medium containing 200 mg/L tryptophan. The levels are expressed as ng/mL (solid line) and ng/1012 cells (dotted line), and the data points represent the means ± SD (n = 3).

in the roots of un-inoculated plantlets (3.8 nmol/g FW vs. 5.2 nmol/g FW). Additionally, inoculated and un-inoculated plantlets exposed to salt and drought stress had increased MDA levels; however, the MDA levels were significantly lower in the roots of endophyte-associated plantlets than in the roots of un-inoculated plantlets (**Figure 5A**). We observed similar trends in ROS production between un-inoculated and inoculated plantlets (**Figures 5B,C**). Our findings indicate that colonization with Bacillus amyloliquefaciens SB-9 counteracted the adverse effects of abiotic stress by reducing the production of MDA and ROS.

### Relative Expression of Melatonin Synthesis Genes

The VvTDC1, VvTDC2, and VvTDC3 genes are located on grapevine chromosomes 7, 10, and 4, respectively, and their

predicted amino acid sequences possessed 48.50, 47.87, and 47.49 % homology with rice tryptophan decarboxylase (TDC1). Similar to the pattern observed for endogenous melatonin levels, we observed that the relative expression levels of VvTDC1, VvTDC2, VvTDC3, and VvSNAT in the roots of control plantlets were significantly upregulated by both salt and drought stress (**Figure 6**), again suggesting that melatonin synthesis is stressinducible. Interestingly, the transcript levels of the VvTDCs and VvSNAT in the roots of SB-9-inoculated plants were also increased by both salt and drought stress; however, the extent of upregulation for these genes was significantly lower (P < 0.05) than that of control plants when they were subject to identical stressors, with the exception of VvTDC1 under 60 mM salt stress.

### DISCUSSION

Melatonin was previously identified in the primitive photosynthetic bacterium Erythrobacter longus (Tilden et al., 1997) and in the gram-negative bacterium Escherichia coli (Hardeland and Poeggeler, 2003); however, few other bacteria are known to produce melatonin. In the present study, we demonstrated that endophytic bacteria, such as A. tumefaciens and B. amyloliquefaciens, are capable of secreting melatonin into extracellular media (**Figure 1A**). In addition, we also found that the level of melatonin in seedling roots was greater when the roots were colonized by B. amyloliquefaciens SB-9 and subjected to salt or drought stress (**Figure 2A**). These findings are in accordance with those of Arnao and Hernández-Ruiz (2015), who proposed an association between beneficial endophytes and the enhanced melatonin levels in their host plants. However, we cannot be sure that the enhanced levels of endogenous melatonin were derived from the endophytic bacteria. Alternatively, root cells might utilize intermediate metabolites of melatonin that are produced by endophytic bacteria. In fact, in the present study, B. amyloliquefaciens SB-9 secreted serotonin and N-acetylserotonin.

The melatonin biosynthesis pathway of plants was recently described (Byeon et al., 2014b) and was shown to differ markedly from that of vertebrates (Tan et al., 2014). One difference is that plants initially decarboxylate tryptophan to form tryptamine and subsequently hydroxylate tryptamine to form serotonin, whereas vertebrates initially hydroxylate tryptophan to form 5-hydroxytryptophan. In the present study, we failed to detect tryptamine in the B. amyloliquefaciens SB-9 culture; however, the concentration of 5-hydroxytryptophan increased throughout the incubation period (**Figure 2C**). This suggests that the first step of melatonin biosynthesis in the endophytic bacterium B. amyloliquefaciens SB-9 may be similar to that in vertebrates. However, the entire melatonin biosynthesis pathway remains to be elucidated, and further studies of the genes involved in the melatonin biosynthesis pathway of B. amyloliquefaciens SB-9 are required.

Endophytic bacteria play an important role in promoting plant growth; however, their influence might be the result of cumulative effects from the various bioactivities of individual endophytes. Indeed, N2-fixing bacteria are also capable of producing IAA (Pedraza et al., 2004), solubilizing phosphate, and releasing amino acids (Liba et al., 2006). Furthermore, it has also been reported that IAA and gibberellins frequently occur simultaneously in culture broth (Piccoli et al., 2011), which indicates that individual endophytic bacterium may be capable of synthesizing multiple phytohormones. All of the above-mentioned factors are beneficial to plant growth. We found that endophytic bacteria produced an additional growth regulator, melatonin, which was previously reported to stimulate

lateral root and shoot growth in several plants, even at low concentrations (Chen et al., 2009; Park and Back, 2012; Bajwa et al., 2014; Wei et al., 2014; Hernández-Ruiz and Arnao, 2016). In the present study, plant height, FW, leaf chlorophyll content, root length, and number of lateral roots (data not shown) were all enhanced by SB-9 colonization. Although we are not sure whether the other B. amyloliquefaciens strains could enhance the endogenous melatonin level in roots, this growthpromoting activity has also been confirmed by other researchers (Idriss et al., 2002; Zhang et al., 2015), Until now, however, we have no direct evidence that the SB-9-derived melatonin enhancement played a role in the growth attributes we measured. This is especially true since the growth promotion, including main/lateral roots development, were likely derived from the combined effects of plant growth-promoting rhizobacteria (PGPR), such as nitrogen fixation, phosphorus solubilization, the production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase or other PGPR-induced physical and chemical (gibberellin, auxin, cytokinin, and unknown metabolites) changes in plants. Further studies, using a mutant that is unable to increase the endogenous melatonin level of roots, are needed to determine the correlation between the enhanced level of melatonin in endophytic bacteria and the promotion of growth in their host plants.

Recent studies have suggested that some plants accumulate melatonin as a defense against a variety of environmental abiotic stressors, such as salt (Mukherjee et al., 2014), chemical agents (Arnao and Hernández-Ruiz, 2009b; Byeon et al., 2015),

low temperature (Shi et al., 2015), and drought (Arnao and Hernández-Ruiz, 2013a,b). The results of the present study concur with those of previous reports, and we also found that stress-induced melatonin synthesis was accompanied by the upregulation of several VvTDCs and VvSNAT, as well as the increased production of melatonin intermediates, such as tryptamine, 5-hydroxytryptophan, serotonin, and N-acetylserotonin (**Figure 4**).

Endophytes have mostly been reported to counteract the adverse effects of stress by reducing the production of MDA and ROS in plants (Jungwook et al., 2009; Khan et al., 2012). This behavior is likely derived from the combined effects of endophytes, such as the enhancement of plant antioxidant enzyme (POD, CAT, POD and APX) activities and the production of phenolic compounds or other antioxidant compounds (Torres et al., 2012). We found that, in grapevine plantlets exposed to salt or drought stress, the production of MDA and ROS, as well as the transcript levels of the grapevine VvTDCs and VvSNAT in inoculated roots were relatively lower than those in un-inoculated controls. Therefore, it seems logical that the endogenous levels of melatonin and its intermediates in inoculated roots could be also lower than those in the un-inoculated controls. However, only tryptamine levels exhibited this trend, whereas levels of the other intermediates in the roots of inoculated plants were similar or higher than those in the un-inoculated controls. We speculate that the lower expression of VvTDCs and VvSNAT could be associated with lower oxidative damage in the roots of inoculated plantlets (**Figures 5** and **6**) because the transcript levels of melatonin synthesis genes were reported to show a positive correlation with ROS levels caused by abiotic stress (Li et al., 2014); however, the higher endogenous 5-hydroxytryptophan, N-acetylserotonin and melatonin levels, as well as the statistically similar serotonin level, in the roots of these plantlets was due to SB-9 colonization, which may promote the production of these compounds via supplementary bacterial melatonin biosynthesis, a possible exchange of metabolites between plants and the strain,

or additional promoting factors produced by the strain. In fact, in the present study, B. amyloliquefaciens SB-9 was able to secrete these compounds in vivo, except tryptamine. In primitive bacteria, melatonin is thought to function as an antioxidant and free radical scavenger that reduces the harmful effects of ROS (Tan et al., 2014). This is the primary role that was reported in both animals and plants (Tan et al., 2002, 2003; Reiter et al., 2005). Therefore, based on these observations, if abiotic stress induces a burst of ROS in the internal tissues of host plants, the living conditions for root-inhabiting microbes presumably become toxic, and as a result, endophytes upregulate their melatonin biosynthesis accordingly, in order to alleviate ROS-induced cell damage.

In summary, the findings of the present study demonstrate that the melatonin-producing ability of endophytic bacteria and the potential application of these bacteria in promoting endogenous melatonin level in plants. However, it remains unclear (i) whether the enhanced levels of endogenous melatonin in roots were derived from production by endophytic bacteria; (ii) whether endophytic fungi produce melatonin; (iii) whether fruit-colonizing endophytes enhance the melatonin levels of fruit tissue, especially in grapevines; (iv) whether any endophytic bacteria, even those with low melatoninproducing abilities, are able to enhance the endogenous melatonin levels in their host plants by providing intermediate metabolites; or (v) which specific internal and external elements influence melatonin production in endophytes and their host plants? Therefore, more comprehensive and detailed investigations are needed to characterize the role

### REFERENCES

of melatonin biosynthesis in naturally occurring symbiotic relationships.

## AUTHOR CONTRIBUTIONS

YL, CY, JJ, YS, and YQ conceived the study; JJ and YM performed the experiments, analyzed the data, and wrote the manuscript; SC analyzed the UPLC-MS/MS data; CL provided suggestions and revised the manuscript. All authors approved the final manuscript and agreed to be accountable for all aspects of the work, i.e., ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

### FUNDING

This work was supported by a project grant from the National Natural Science Fund Program of China (No. 31571812; http: //www.nsfc.gov.cn).

### ACKNOWLEDGMENTS

We thank Zhang Hongming for valuable advice and are grateful to Dr. Dun-Xian Tan (Department of Cellular and Structural Biology, The University of Texas, Health Science Center at San Antonio) for critical comments.

isomers during evolution: relation to their biological functions. Int. J. Mol. Sci. 15, 15858–15890. doi: 10.3390/ijms150915858

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Jiao, Ma, Chen, Liu, Song, Qin, Yuan and Liu. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Melatonin Protects Cultured Tobacco Cells against Lead-Induced Cell Death via Inhibition of Cytochrome c Translocation

#### Agnieszka Kobylinska ´ 1 , Russel J. Reiter2 and Malgorzata M. Posmyk1 \*

1 Laboratory of Plant Ecophysiology, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland, 2 Department of Cellular and Structural Biology, UT Health Science Center, San Antonio, TX, United States

### Edited by:

John Love, University of Exeter, United Kingdom

### Reviewed by:

Vinay Kumar, Central University of Punjab, India Renata Bogatek, Warsaw University of Life Sciences, Poland Roberto Luis Benech-Arnold, University of Buenos Aires, Argentina

> \*Correspondence: Malgorzata M. Posmyk<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Metabolism and Chemodiversity, a section of the journal Frontiers in Plant Science

Received: 22 September 2016 Accepted: 28 August 2017 Published: 14 September 2017

### Citation:

Kobylinska A, Reiter RJ and ´ Posmyk MM (2017) Melatonin Protects Cultured Tobacco Cells against Lead-Induced Cell Death via Inhibition of Cytochrome c Translocation. Front. Plant Sci. 8:1560. doi: 10.3389/fpls.2017.01560 Melatonin was discovered in plants more than two decades ago and, especially in the last decade, it has captured the interests of plant biologists. Beyond its possible participation in photoperiod processes and its role as a direct free radical scavenger as well as an indirect antioxidant, melatonin is also involved in plant defense strategies/reactions. However, the mechanisms that this indoleamine activates to improve plant stress tolerance still require identification and clarification. In the present report, the ability of exogenous melatonin to protect Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells against the toxic exposure to lead was examined. Studies related to cell proliferation and viability, DNA fragmentation, possible translocation of cytochrome c from mitochondria to cytosol, cell morphology after fluorescence staining and also the in situ accumulation of superoxide radicals measured via the nitro blue tetrazolium reducing test, were conducted. This work establishes a novel finding by correcting the inhibition of release of mitochondrial ctytocrome c in to the cytoplasm with the high accumulation of superoxide radicals. The results show that pretreatment with 200 nm of melatonin protected tobacco cells from DNA damage caused by lead. Melatonin, as an efficacious antioxidant, limited superoxide radical accumulation as well as cytochrome c release thereby, it likely prevents the activation of the cascade of processes leading to cell death. Fluorescence staining with acridine orange and ethidium bromide documented that lead-stressed cells additionally treated with melatonin displayed intact nuclei. The results revealed that melatonin at proper dosage could significantly increase BY-2 cell proliferation and protected them against death. It was proved that melatonin could function as an effective priming agent to promote survival of tobacco cells under harmful lead-induced stress conditions.

Keywords: BY-2 tobacco cells, cytochrome c, DNA fragmentation, melatonin, programmed cell death

## INTRODUCTION

In their natural environment, plants are exposed to many different biotic and abiotic stresses. Among various stressors, heavy metals, especially lead (Pb), are major environmental pollutants, particularly in areas with high anthropogenic pressure (Gill, 2014) and its accumulation has adverse effects on plant growth and crop productivity. Pb is phytotoxic and found in dust, fumes, mists,

vapors and in soil as minerals (PbCO, PbS, PbSO4) (Nicholls and Mal, 2003). Although the level of heavy metals in agricultural soil is normally very low, the repeated use of phosphate fertilizers over long periods may cause dangerously high concentrations of some of these toxins (Gill, 2014). Pb is taken up via roots along with water, or it can be absorbed from the air via shoots and foliage (Fahr et al., 2013). Unfortunately, plant roots are not selective and absorb Pb with other minerals where accumulates. In a number of species, high Pb levels cause abnormal plant morphology, reduced plant growth and finally it induces cell death (Pourrut et al., 2012). Toxic Pb concentrations inhibit the activity of key enzymes, e.g., acid phosphatase, esterases, peroxidases, malic dehydrogenase, by reacting with their sulfhydryl groups. Moreover, Pb contributes to water imbalance, alterations in cell membrane permeability and it limits mineral nutrition. Pb excess also induces oxidative stress in tissues by increased reactive oxygen species (ROS) generation. Simultaneously, Pb provokes DNA damage, gene mutations, protein oxidation, lipid peroxidation and finally it promotes signal transduction cascades that promote cell death (Wierzbicka, 1999; Gill, 2014).

Programmed cell death (PCD) is an indispensable process for animals and plant development. In plant systems, PCD falls within two broad categories, environmentally induced and developmentally regulated cell death. Environmentally induced PCD is usually a consequence of external factors including heat shock (Vacca et al., 2006; Lord and Gunawardena, 2012), cold (Lei et al., 2004), pathogen infection leading to a hypersensitivity response (HR) (Mur et al., 2008; Pietrowska et al., 2015) and death caused by heavy metals (Iakimova et al., 2007; Iwase et al., 2014). PCD is an event displayed by many different organisms throughout evolution; however, despite the enormous evolutionary distance across organisms there are some common features including: increased formation of vesicles, cytoplasmic condensation, nuclear condensation, DNA laddering and translocation of cytochrome c (Cyt c) from mitochondria to the cytosol (Isbat et al., 2009; Martínez-Fábregas et al., 2014). In plant cells, Cyt c release occurs during PCD and is a result of many stimuli such as menadione, D-mannose, heat or ROS (Sun et al., 1999; Stein and Hansen, 1999; Tiwari et al., 2002; Vacca et al., 2004).

Petrosillo et al. (2003) documented that mitochondrialinduced ROS production promotes Cyt c release from mitochondria by a two-step process, including dissociation of Cyt c from cardiolipin, followed by permeabilization of the outer membrane, probably by interaction with voltage dependent anion channels. However, the function of cytoplasmic Cyt c is still controversial since Vacca et al. (2006) found that Cyt c release depended on ROS production, but it may not trigger PCD. Furthermore, after Cyt c translocation, caspase-like proteases inactivate it, leading to Cyt c degradation en route to PCD (Vacca et al., 2006). However, data of Martínez-Fábregas et al. (2014) indicated that extra-mitochondrial Cyt c had a double role in causing living cells to die, by triggering the pro-apoptotic routes, e.g., cysteine protease response to dehydration 21 - RD21, hydroxyacylglutathione

hydrolase 2 (GLY2) as well as by inhibiting the pro-survival factors including SET protein (which acts as an inhibitor of p53 acetylation and blocks both p53-mediated cell cycle arrest and apoptosis after stress) or luminal binding protein 1 and 2 (BiP1 and BiP2) whose overexpression increased cell tolerance to endoplasmic reticulum stress as shown in tobacco protoplast (Leborgne-Castel et al., 1999; Martínez-Fábregas et al., 2014).

To reduce the negative impact of various stresses, including Pb pollution, the best solution may be biostimulators, which improve plant tolerance and protect them against harmful factors. Among many different protective substances naturally occurring in plants, melatonin (N-acetyl-5-methoxytryptamine) seems to have great biostimulatory potential (Janas and Posmyk, 2013). Melatonin has been detected in numerous plant species (Reiter et al., 2015). This indoleamine is a broadspectral antioxidant. It stimulates antioxidant enzymes and synthesis of glutathione, and activates other antioxidants (Bałabusta et al., 2016). It also increases the efficiency of mitochondrial electron transport chain thereby decreasing electron leakage thus limiting free radical generation (Kładna et al., 2003; Rodriguez et al., 2004; Leon et al., 2005; Tan et al., 2007; Reiter et al., 2015; Bałabusta et al., 2016). Moreover, the work of Galano et al. (2013), Tan et al. (2014) and Kołodziejczyk et al. (2015) indicated that the melatonin metabolites, e.g., cyclic-3-hydroxymelatonin, 2-hydroxylmelatonin and especially N1-acetyl-N2-formyl-5-methoxykynu-ramine (AFMK) also possessed antioxidant activity. These facts, together with melatonin small size makes it particularly capable of translocating easily between cell compartments and of protecting cell structures against excessive ROS.

Melatonin is also useful to protect plants against heavy metal-induced stresses (Tan et al., 2007). Presowing melatonin treated seeds eliminated the toxic effects of copper ions in Brassica oleracea rubrum during germination (Posmyk et al., 2008) and zinc sulfate in Hordeum vulgare L. roots (Arnao and Hernández-Ruiz, 2009). Relatively little is known about the specific mechanisms of melatonin action at the subcellular level in plants. Lei et al. (2004) showed that pretreatment with melatonin of carrot suspension cells attenuated cell damage caused by cold exposure.

Studying the molecular pathways of PCD in whole plants introduces many difficulties, because it often occurs in a small number of directly stress-affected cells (McCabe and Leaver, 2000). Thus, for analysis of cytotoxic effects, cell lines are of particular suitable. Nicotiana tabacum L. cv Bright Yellow 2 (BY-2) suspension cells are fast growing higher plant cells, which provide an excellent model for examining plant physiology, biochemistry and molecular biology (Nagata et al., 1992). They allow research both at the level of a single cell and in its compartments. The objective of the present study was to determine if pretreatment of a suspension Nicotiana tabacum BY-2 cells with melatonin inhibits Pb-induced PCD. The findings show that melatonin significantly limited the negative effects of this heavy metal and acted as a biostimulating, pro-survival factor.

### MATERIALS AND METHODS

fpls-08-01560 September 12, 2017 Time: 15:58 # 3

### Plant Material

Sterile suspensions of in vitro cell cultures of Nicotiana tabacum, L. cv Bright Yellow 2 (BY 2) were used. The cells were cultivated in Linsmaier and Skoog (1965) basal medium (LS) supplemented with 30 g l−1 sucrose, 0.2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D; synthetic auxin), 1 mg l−1 thiamine, 0.1 g l−1 myo-inositol and 10−2 M KH2PO4. The initial pH of the medium was established as 5.3.

### Cell Culture and Growth Conditions

BY-2 suspended cells were routinely propagated and cultured at 25◦C. From the stationary growth phase (day 7th) of the base culture, 2 ml of cell suspension were passaged into the fresh LS medium as a control (C) and LS with 200 nM melatonin (MEL). The optimal dose of melatonin was chosen experimentally. In the middle of the logarithmic phase of growth (day 4th) Pb(NO3)2 was added to LS (Pb) and LS with melatonin (MEL + Pb) media to the final Pb2+concentration 15 µM. Thus, the experiments were performed in four variants: (i) C: BY-2 cells cultured under optimal conditions on LS medium, (ii) MEL: BY-2 cells cultured on LS medium supplemented with melatonin from the start of new culture; (iii) Pb: BY-2 cells cultured on LS medium with Pb2+ added on the 4th day of culture and (iv) MEL + Pb: BY-2 cells cultured on LS medium with melatonin added from the start of culture and stressed with Pb2+ added on the 4th day of culture. The cultures were maintained to the 7th day (stationary phase of the control cell growth). The applied concentration of lead was chosen after measurement of LC50 on the 7th day.

### Determination of Cell Growth and Viability

The cell number was determined with the use of a Fuchs-Rosenthal haemocytometer under a light microscope Olimpus CX-31 equipped with MicroScan v.15. digital system of image analysis; additionally the number of dead cells was assessed after selective staining with methylene blue. Living cells do not take up the stain and retain their natural color whereas damaged cells are stained blue as they are unable to keep the methylene blue from penetrating their membranes. The number of cells and their viability were analyzed every experimental day.

### Melatonin Determination

Melatonin was extracted according to the modified methods of Guerrero et al. (2001) and Hernandez-Ruiz et al. (2004). Its concentration was measured during lag, log and the stationary phases of growth. After filtration and separation of the cells from the medium concentrations of melatonin in the extracts were determined using high-performance liquid chromatography (HPLC-MS/MS). For extraction, 5 g of fresh weight of the cells was homogenized with 5 mL of 50 mM sodium phosphate buffer (pH 8.0) containing 1 mM EDTA and 5 µM butylated hydroxytoluene (BHT) as an antioxidant. The homogenate was

The homogenate was centrifuged at 15000 g for 10 min at 5◦C. Initial purification consisted in two steps by solventpartitioning using ethyl acetate and 50 mM sodium phosphate buffer (first at pH 8.0 and second at pH 3.0). The two organic phases were evaporated together under vacuum. Dry residue was re-dissolved in 1 mL of mobile phase, filtered through Supelco ISO-Disc filters (PTEF-4 – 2.4 mm × 0.2 m; Supelko, Bellefonte, PA, United States), and frozen at −70◦C until HPLC-MS analysis. The purified extract was subjected to HPLC-MS/MS analysis using an Agilent 1200 LC System coupled with AB Sciex 3200 QTRAP mass detector equipped with TurboSpray Ion Source (ESI). Each sample was injected onto Agilent SB-C18 column.

### Assay of Cell Death by Fluorescent Microscopy

Detection and verification of cell death in the suspension of cells were carried out according to Byczkowska et al. (2013) procedure: (1) 0.5 mL of the culture medium with 0.5 mL of the appropriate cell suspension was supplemented with 0.5 mL of 0.02 M phosphate buffer pH 7.4 (PHB). (2) The cells were stained with the AO/EB mixture containing 50 µg cm−3of acridine orange and 50 µg cm−3 of ethidium bromide in PHB. (3) Drops of cell suspension were immediately put on glass slides and analyzed for 5 min using fluorescent microscopy with a blue light excitation filter of the Optiphot-2 epi-fluorescence microscope (Nikon) equipped with a camera and Act-1 software (Precoptic, Poland) for fluorescent microscopy and preparation of microphotographs according to Byczkowska et al. (2013). AO/EB staining included the use of acridine orange which penetrates whole cells and stains the nuclei green and with ethidium bromide which dyes nuclei red and it is only absorbed by damaged cells with impaired cellular and nuclear membrane integrity. From the above data, a curve of the fluorescence intensity of nuclear chromatin after AO/EB staining was prepared, as described by Byczkowska et al. (2013). This scale allows the recognition of living, dying and death cells. Living cells have intact nuclei stained green, while dying cells have green–yellow, yellow, yellow–orange, or bright orange nuclei with slightly condensed or fragmented chromatin at the early stage of death whereas with condensed and fragmented chromatin at the late stage. Necrotic cells have structurally normal orange nuclei.

When the color is changed from green to red, values of fluorescence intensity of acridine orange and ethidium bromide increase (Byczkowska et al., 2013).

### Cell Fractionation

Fractionation of cells was performed using the digitonin method according to Ganju and Eastman (2003) with modification of Kobylinska et al. (2006) ´ . In all experimental variants the cells were washed twice with PBS and next permeabilized for 30 min in a buffer containing: 1 mM NaH2PO4, 8 mM Na2HPO4, 75 mM NaCl, 250 mM sucrose, digitonin (0.05% of cells weight), 20 µl/g cells 1 mM phenylmethylsulfonyl fluoride (proteases inhibitor), and cocktail of enzymes for cell wall lysis (CellLytic Sigma). Cell homogenate was obtained by centrifugation at 3000 g for 1 min. at 4◦C to remove cell debris. The cleaned homogenate after centrifugation at 12000 × g was divided into two fractions: the supernatant was removed as the cytosolic fraction and the pellet (mitochondrial fraction) was resuspended in the above buffer (without digitonin). To both fractions sufficient volumes of Laemmli sample buffer supplemented with 10% β-mercaptoethanol were added (Laemmli, 1970) and the mixtures were boiled for 5 min.

### Western Blot Analysis

fpls-08-01560 September 12, 2017 Time: 15:58 # 4

Fractionated BY-2 cell lysates (50 µg of proteins) were electrophoretically separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 15% gel (Laemmli, 1970) and transferred to Immobilon PSQ at the voltage of 20 V overnight, at 4◦C according to Towbin et al. (1979). After blocking in 3% non-fat dry milk in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 60 min, the membranes were incubated with primary antibodies specific to Cyt c in TBST in a cold room overnight. Subsequently, the membranes were washed several times in TBST and incubated with appropriate secondary antibodies conjugated with alkaline phosphatase (Sigma Chemical Co.) in TBS for 2 h at room temperature. Next the membranes were washed several times with TBST, and the proteins were visualized by incubation with the substrate solution (0.33 mg/ml of nitro blue tetrazolium, 0.17 mg/ml of 5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl and 5 mM MgCl2), prepared according to Leary et al. (1983).

### DNA Isolation

DNA digestion was performed using the cetyl-trimethylamonnium bromide (CTAB) method previously described by Murray and Thompson (1980). BY-2 cells from log phase of growth (4th day, 4 h after Pb2+ addition) and from the beginning of the stationary phase (6th day, 48 h after Pb2+ addition) were frozen in liquid nitrogen and ground in a mortar to a fine powder. Then, CTAB buffer (100 mM Tris-HCl pH 8.0, 1,4 M NaCl, 20 mM EDTA and 2% CTAB) was added and extraction was performed for 30 min at 65◦C. After immediate cooling on ice, DNA preparation was continued in the extraction mixture of chloroform/isoamyl alcohol (24:1) until a fine emulsion was created. The organic phase was separated from the aqueous phase by centrifugation at 12000 × g for 15 min at 4◦C. DNA was precipitated with isopropanol at −20◦C, 20 min. The DNA precipitates were spun at 12000 × g for 10 min at 4 ◦C, washed two times in 70% ethanol and air dried. DNA pellets were dissolved in 100 µl TE buffer (10 mM Tris-HCl pH 7.5, and 1 mM EDTA) containing 10 µl 1% RNase A. RNA digestion was conducted 2 h at 37◦C. Purity of the obtained DNA preparations was determined spectrophotometrically by analysis of the absorbance spectra in the range of 230–320 nm. The value of A260/280 within the limits of 1.8 – 2.0 was the criterion of DNA purity. Then, 5 µl of a loading buffer was added to each tube, and the DNA preparations were electrophoresed in 2% agarose gels and run at 5 V/cm. The gels were stained with ethidium bromide and visualized under ultraviolet (UV) light.

### Statistical Analysis

The data represent the means ± standard deviation (±SD). Each variant of culture was replicated three times and at least three independent samples were used for measurement. The data were analyzed using STATISTICA v.10.0\_MR1\_PL [StatSoft] software. One-way or two-way analysis of variance (ANOVA) and then the post hoc Duncan multiple range test was carried out to find the significant differences at p < 0.001 in each experiment.

## RESULTS

### Cell Growth and Viability

Preincubation with melatonin prior to Pb treatment protected tobacco suspension cells from death and improved cell proliferation. Cell growth intensity in C and MEL variants was similar during culture time. After Pb addition on the 4th day, a significant inhibition of tobacco cell proliferation was observed (**Figure 1A** – see the variants MEL + Pb and especially Pb). From the first day after heavy metal stress induction, proliferation of the MEL + Pb cells was about 40% higher in comparison to those treated with Pb but not primed with melatonin (Pb) (**Figure 1A**) this tendency was maintained throughout the duration of the Pb-stress.

The effects of melatonin pretreatment on viability of Pb-stressed tobacco suspension cells were verified in all experimental samples. Methylene blue staining documented the protective effect of melatonin against cell death induced by Pb. The mortality of cells exposed to Pb but preincubated with melatonin (MEL + Pb) was slightly higher than in C and MEL variants. Culture medium supplementation with melatonin did not result in cell death acceleration. The number of dead cells in the Pb exposed cells increased significantly and it was 24.2, 37.3, and 49.2% for the 1st, 2nd, and 3rd day after Pb application, respectively (**Figure 1B**). In contrast, mortality of MEL + Pb cells was about 80% lower than in the Pb cells (**Figure 1B**).

### Detection of Cell Death

Fluorescence analyses after successive addition of AO/EB fluorochromes showed that after Pb treatment BY-2 cells died via PCD. Yellow and green-yellow nuclei with slightly condensed chromatin dominated among dying cells (**Figure 2**), but some yellow-stained nuclei with condensed chromatin were also observed. This observation indicates that already 4 h after Pb treatment, BY-2 cells underwent the initial stages of cell death. At the end of cell culture (the 7th day; the 3rd day after Pb stress) nuclei with dark orange chromatin were not found, indicating that necrotic type of cell death after Pb exposure in BY-2 tobacco cells was not detected.

Unexpected effects were obtained for cells exposed to Pb but pre-incubated with melatonin (MEL + Pb). The fluorescence intensity of randomly selected nuclei for this treatment was estimated at 16% and it was similar to the Pb-untreated samples:

14 and 17% for control and melatonin treated cells respectively. In contrast fluorescence intensity in Pb variant increased to 53%, and was expressed as yellow/yellow–orange nuclei color (**Figure 3**).

### Profile of DNA Fragmentation

To gain insight into the mechanism of plant PCD induced by Pb, DNA fragmentation and release of Cyt c from mitochondria into cytosol was checked. We found an inhibitory effect of melatonin on DNA laddering, one of the hallmarks of PCD. **Figure 4** shows that DNA isolated from control and melatonin treated cells remained intact, whereas DNA from Pb samples exhibits significant fragmentation; this was more intensive 4 h after Pb stress then 2 days later (the 6th culture day, the 2nd day after lead treatment). The analyses showed that melatonin completely blocked/reversed the cytotoxic Pb influence and protected tobacco cells against DNA damage caused by the heavy metal.

### Cytochrome c Translocation