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 mitochondrial-induced 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 broad-spectral 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.

Melatonin was discovered in the plant kingdom in Dubbels et al. (1995), Hattori et al. (1995) and significant progress has been made in defining its multiple roles in plants. Many researchers underline the fact that among its various roles, its antioxidant effectiveness and free radical scavenging ability, that protect plants against oxidative stress and alleviate or counteract cell damage, are crucial to plant physiology as in animals (Reiter et al., 1997; Tian et al., 2001). Melatonin is widely present in many higher plants (Arnao and Hernandez-Ruiz, 2015; Reiter et al., 2015). Elevated levels of melatonin protect plants against water and soil pollutants by acting as a direct free radical scavenger (Tan et al., 2007; Manchester et al., 2015) and/or as an indirect antioxidant stimulating antioxidant enzymes (Rodriguez et al., 2004; Bałabusta et al., 2016). Melatonin metabolites also posses antioxidant properties and they act in synergy with other antioxidants, such as ascorbic acid, glutathione, etc. (Gitto et al., 2001; Arnao and Hernández-Ruiz, 2009; Kołodziejczyk et al., 2015). Melatonin protects plant tissues and organs, particularly reproductive tissues, fruit and germ tissues of the seeds, from secondary oxidative stress caused by unfavorable environmental conditions, such as drought, salinity, cold, heat, ultraviolet light and ozone (Van Tassel et al., 2001; Dawood and El-Awadi, 2015; Reiter et al., 2015). The data indicate that exogenously applied melatonin also acts as plant biostimulator especially under suboptimal environmental conditions (Posmyk et al., 2008, 2009; Janas and Posmyk, 2013; Kołodziejczyk et al., 2016).

There is growing evidence that in plants melatonin action is associated with its antioxidant properties. Moreover melatonin as the antiapoptotic factor is well documented in various animal cells, but not in plants (Reiter et al., 1997, 2015). Thus, the aim of our investigation was to check melatonin influence on heavy metal-induced cell death in tobacco suspension cultures. There is rather little information on the mechanisms by which melatonin prevents plant cells from dying after Pb exposure.

Presented studies were preceded by analyses of influence the different melatonin concentrations as well as Pb, on BY-2 suspension cells (data not shown). The range of tested melatonin concentrations was 100–1000 nM, whereas Pb 0.5–50 μM. The concentration of Pb applied in presented experiments was chosen on the basis of measurement of LC50 on the 7th day of BY-2 cell cultivation. Our preliminary studies revealed that exogenous melatonin has ambiguous effects on BY-2 cells: it is an effective biostimulator when applied in concentration below 300 nM, but in excessive doses (above 300 nM) it significantly decreased both BY-2 cell proliferation and viability. This agrees with the previous publications of our team, where we also observed different effects of melatonin treatments dependent on the dosage used (Posmyk et al., 2008, 2009; Janas and Posmyk, 2013). This indicates, that melatonin, despite its potentially positive properties (e.g., antioxidative), can not be considered as being always protective since high concentration may have harmful side effects. The choice of dosage is crucial for the positive effects to be realized; in BY-2 cells melatonin was protective in the range 100–300 nM.

Our preliminary experiments led us to determine the optimal melatonin dose as 200 nM that stimulated proliferation of BY-2 cells and nearly completely reversed effects of Pb-stress. Thus, initially cell proliferation and viability during lag, log and stationary phases of growth both under optimal (C and MEL) as well as under heavy metal stress condition (Pb and MEL + Pb) were determined. The positive effects of preincubation of BY-2 cells with melatonin were visible during and after Pb-stress which suggests that melatonin at a concentration 200 nM fortifies cells against potentially stress conditions even before they appear. Proliferation of cells pretreated with melatonin but exposed to Pb (MEL + Pb) were only slightly worse than of the unstressed cells (C and MEL) whereas in the Pb-stressed cells the level of proliferation was more than 50% lower in comparison to the control. The protective role of melatonin against cell death was clearly visible in the cell mortality analyses during Pb-stress. The number of dead cells in the Pb-treated cells increased drastically while unexpectedly in the MEL + Pb variant BY-2 cell viability was about 80% higher than in the Pb samples (Figure 1B). The melatonin ability to prevent cell death was confirmed by the studies of fluorescence intensity of nuclear chromatin stained with AO/EB. In our experiments morphological changes in the nuclei of the Pb exposed cells, i.e., chromatin condensation, green–yellow and yellow color of nuclei, was shown (Figures 2, 3).

Plant cells that undergo PCD exhibit many of the same morphological characteristics as cells undergoing PCD in mammals and Caenorhabditis elegans including intensified formation of vesicles, cytoplasmic condensation, nuclear condensation, DNA fragmentation and chromatin condensation leading to DNA laddering as well as translocation of Cyt c (Balk et al., 2003; Lord and Gunawardena, 2012). Our results showed that in BY-2 cells DNA fragmentation appeared already at 4 h after Pb application and it persisted until the 2nd day after Pb application. In electrophoregrams of DNA samples isolated from the Pb-exposed cells extensive oligonucleosomal fragmentation caused by this heavy metal was observed, which was not noted in untreated cells (C and MEL). In the MEL + Pb treated cells melatonin completely blocked DNA laddering. Our results are in line with the studies of Lei et al. (2004) who showed that melatonin increased tolerance to cold in carrot suspension cells and protected their DNA against damage. Similar beneficial effects caused by exogenous melatonin were observed by Posmyk et al. (2008) in red cabbage seedlings subjected to copper stress and in cucumber seedlings subjected to chilling stress (Posmyk et al., 2009).

The data presented suggest that pre-incubation of BY-2 cells with melatonin limits the toxicity of Pb and protects cells against death. We used the plant model system to pinpoint the PCD phase when melatonin may act. It has been reported that release of Cyt c from mitochondrial intermembrane space to cytosol is a conserved pathway of PCD and it has been noted in many systems (Martínez-Fábregas et al., 2013). In plants, this issue has been poorly investigated and the mechanisms of Cyt c release, its role in determining cell death are still controversial. Vacca et al. (2006) reported that heat shock triggered Cyt c translocation but Cyt c was degraded en route to cell death. Moreover, Cyt c release is linked to ROS burst and strictly depends on ROS production, and it identifies the early phase of cell death. However, our Western blot analyses did not reveal translocation of Cyt c from mitochondria to cytosol 4 h after the heavy metal stress rather this occured much later (the 6th day of culture, the 2nd day after lead administration). Bolduc and Brisson (2002) as well as Kawai-Yamada et al. (2004) reported that oxidative metabolism leading to generation of ROS was one of the earliest events in PCD induced by biotic or abiotic stress in tobacco plants. Thus, this suggests that high levels of ROS mediate the signaling cascade for defenceive gene induction, e.g., hsp, lea, cor (Vincour and Altman, 2005). In our studies, ROS production and DNA laddering were the early features of PCD and translocation of Cyt c was independent of them. We observed DNA fragmentation before Cyt c translocation, which seems to support the hypothesis of Collazo et al. (2006) that NO/ROS ratio may induce a set of defense responses including cleavage of an inhibitor of caspase-activated DNAse (ICAD) (Krishnamurthy et al., 2000; Collazo et al., 2006). Although the presence of caspases in plants is debated, cysteine protease activity has been reported to be induced in plant systems undergoing cell death (Lam and del Pozo, 2000; Bozhkov et al., 2004). These proteases might function in a plant proteolytic network leading to disconnection of ICAD from DNAse (CAD) and fragmentation of DNA. Moreover, it would confirm our observations that in MEL + Pb cells, despite Pb application, DNA was not cleaved since this indoleamine is a highly effective antioxidant and it blocked caspase-like signaling leading to activation of CAD. Furthermore, our results revealed accumulation of superoxide radical in Pb exposed cells at the 4th hour after Pb treatment with only a slight detection in the control (C) and melatonin treated cells (MEL, MEL + Pb) (Figure 6). Thus, our finding are in line with the data of Vacca et al. (2006) who showed that ROS scavenging inhibited Cyt c release. Jou et al. (2004) documented that in rat brain astrocytes melatonin inhibited opening of mitochondrial permeability transition pore (MPT) and blocked MPT-dependent Cyt c release.

To test whether tobacco cells synthesize endogenous melatonin, and/or are capable of active absorption exogenous melatonin from the environment, the contents of this indoleamine in cell lysates were determined at the crucial points of the experiments, i.e., lag, log and stationary phase of growth.

Our results indicated that tobacco BY-2 cells are able to synthesize small amounts of this indoleamine depending on the phase of growth (its endogenous level increased slightly during experiment) as well as to absorb it actively from the medium (Table 1). It is known that biosynthesis and metabolism of this indoleamine are affected and modified by environmental conditions (i.e., stresses), and melatonin levels change during plant ontogenesis (Okazaki and Ezura, 2009). Elevated melatonin synthesis is often combined with the plant defense strategy because generally it was noticed that various plant species rich in melatonin had greater capacity for stress tolerance (Park et al., 2013; Bajwa et al., 2014; Zhang et al., 2015).

Availability of exogenous melatonin allowed BY-2 cells to take up its large quantities throughout the culture period (Table 1). Similar results were observed by Kołodziejczyk et al. (2015) in the case of cucumber and corn seeds which were primed with exogenous melatonin – they absorbed quantities of this indoleamine proportional to its concentration applied during priming (Kołodziejczyk et al., 2015). Interestingly, under unfavorable conditions, Pb-stressed cells (MEL + Pb) absorbed melatonin near 20% more intensively in comparison to the unstressed melatonin treated cells (MEL). Although, it is probably not a natural defense strategy of Nicotiana tabacum cells since endogenous melatonin content did not measurably increase in BY-2 cells under Pb stress conditions (Table 1). These results suggest that BY-2 cells readily absorb and use the accessed exogenous melatonin to counteract stress-induced damage. Survival of plants in polluted environments largely depends on their ability to sequester and/or detoxify toxic substances such as Pb. Tan et al. (2007) also found that melatonin was effective in preventing the death of pea plants grown in soil contaminated with copper.

We have shown that although tobacco is not a plant rich in endogenous melatonin, it is able to use it from an exogenous source as a potential effective factor for improving its stress defenses. This fact once again confirms the practical use of melatonin as a plant biostimulator (Janas and Posmyk, 2013; Kołodziejczyk and Posmyk, 2016; Nawaz et al., 2016). Moreover, we have presented novel findings concerning a decrease of mitochondrial Cyt c release together with limited DNA degradation, which suggest that the protection mechanism of melatonin is not only via limitation of secondary oxidative stress, but also via counteraction against PCD. In conclusion, melatonin has multiple actions as a factor fortifying cells against potential, harmful conditions.

AK: work conception, all experiments concerning Nicotiana tabacum BY-2 suspension cells realization, data acquisition and analysis, drafting of the manuscript. RR: research consultation/discussion, manuscript revision: language and editorial corrections. MP: methodological consultant, statistical calculations, data analysis and interpretation, manuscript revision.

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.