Nitric Oxide Enhances Cytotoxicity of Lead by Modulating the Generation of Reactive Oxygen Species and Is Involved in the Regulation of Pb2+ and Ca2+ Fluxes in Tobacco BY-2 Cells

Lead is a heavy metal known to be toxic to both animals and plants. Nitric oxide (NO) was reported to participate in plant responses to different heavy metal stresses. In this study, we analyzed the function of exogenous and endogenous NO in Pb-induced toxicity in tobacco BY-2 cells, focusing on the role of NO in the generation of reactive oxygen species (ROS) as well as Pb2+ and Ca2+ fluxes using non-invasive micro-test technology (NMT). Pb treatment induced BY-2 cell death and rapid NO and ROS generation, while NO burst occurred earlier than ROS accumulation. The elimination of NO by 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) resulted in a decrease of ROS, and the supplementation of NO by sodium nitroprusside (SNP) caused an increased accumulation of ROS. Furthermore, the addition of exogenous NO stimulated Pb2+ influx, thus promoting Pb uptake in cells and aggravating Pb-induced toxicity in cells, whereas the removal of endogenous NO produced the opposite effect. Moreover, we also found that both exogenous and endogenous NO enhanced Pb-induced Ca2+ effluxes and calcium homeostasis disorder. These results suggest that exogenous and endogenous NO played a critical regulatory role in BY-2 cell death induced by Pb stress by promoting Pb2+ influx and accumulation and disturbing calcium homeostasis.


Introduction
Currently, heavy metal contamination is a major form of environmental pollution owing to emissions from industry, agricultural chemicals, vehicular traffic, and other human activities [1]. Lead is one of the most hazardous metals in the environment and is toxic to plants and other organisms. Like other heavy metals, Pb impairs plants by inhibiting seed germination and plant growth [2][3][4]; reducing nutrient uptake and biomass [5]; disrupting cell membrane permeability, photosynthesis, and cell division [4,6,7]; inhibiting fundamental enzymatic reactions; demolishing cell viability; and inducing cell death [8].
Heavy metals usually result in oxidative stress and a burst of reactive oxygen species (ROS) [9]. In addition to ROS, nitric oxide (NO) may also be rapidly induced in plant cells to regulate plant responses to abiotic stress, including heavy metal toxicity [10,11]. To date, there is a considerable amount of evidence addressing the relationship between ROS and NO signaling in plants. Some Next, we examined the internucleosomal fragmentation of DNA triggered by endonucleases using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. After 24 h of treatment with 100 and 250 μM Pb(NO3)2, the nuclei appeared TUNEL positive ( Figure S2, Supplementary Materials). Conversely, almost all of the nuclei in the control cells and 500 μM Pb(NO3)2-treated cells were TUNEL negative. Positive and negative controls were included and further confirmed the absence of artifacts.

Pb Triggered ROS and NO Bursts in Tobacco BY-2 Cells
Both ROS and NO are often produced in large amounts during plant response to various stresses and play key roles in plant PCD during development and defense [17,[27][28][29]. The influence of Pb on ROS and NO production in tobacco BY-2 cells was examined in this work. As shown in Figure 2, the ROS contents in the Pb-treated cells presented a time-dependent increase after Pb treatment and reached the highest peak at about 6 h after Pb exposure (Figure 2A,B). Measurements of NO released in the cells revealed an immediate increase right after Pb treatment in comparison with that in control cells. Following the treatment with 250 μM Pb(NO3)2, the release of NO increased rapidly. At 1.5 h, the NO reached the highest level, which was about 1.70-fold higher than that in control cells ( Figure 2C,D). These results suggest that the NO burst occurred earlier than that of ROS under Pb stress in tobacco BY-2 cells. Next, we examined the internucleosomal fragmentation of DNA triggered by endonucleases using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. After 24 h of treatment with 100 and 250 µM Pb(NO 3 ) 2 , the nuclei appeared TUNEL positive ( Figure  S2, Supplementary Materials). Conversely, almost all of the nuclei in the control cells and 500 µM Pb(NO 3 ) 2 -treated cells were TUNEL negative. Positive and negative controls were included and further confirmed the absence of artifacts.

Pb Triggered ROS and NO Bursts in Tobacco BY-2 Cells
Both ROS and NO are often produced in large amounts during plant response to various stresses and play key roles in plant PCD during development and defense [17,[27][28][29]. The influence of Pb on ROS and NO production in tobacco BY-2 cells was examined in this work. As shown in Figure 2, the ROS contents in the Pb-treated cells presented a time-dependent increase after Pb treatment and reached the highest peak at about 6 h after Pb exposure (Figure 2A,B). Measurements of NO released in the cells revealed an immediate increase right after Pb treatment in comparison with that in control cells. Following the treatment with 250 µM Pb(NO 3 ) 2 , the release of NO increased rapidly. At 1.5 h, the NO reached the highest level, which was about 1.70-fold higher than that in control cells ( Figure 2C,D). These results suggest that the NO burst occurred earlier than that of ROS under Pb stress in tobacco BY-2 cells.

NO Contributed to Pb-Induced ROS Production in Tobacco BY-2 Cells
The relationship of ROS and NO signaling in plants has been extensively studied, showing that NO may be an upstream signaling molecule for H 2 O 2 in the auxin signal transduction pathway during adventitious root development in marigold [30]. There are also reports that H 2 O 2 leads to quick NO production in guard cells of Phaseolus aureus [31]. In this work, Pb induced both ROS and NO production in tobacco BY-2 cells. Considering that the Pb-induced NO peak occurred in advance of the ROS peak, the NO-donor SNP and the NO-specific scavenger cPTIO were used to investigate the possible role of NO in ROS production. According to the emerging time of Pb-induced NO and ROS peaks, we treated tobacco BY-2 cells with 250 µM Pb(NO 3 ) 2 in combination with 0.5 µM SNP or 100 µM cPTIO for 6 h and 1.5 h, respectively, after which cells were detected for ROS and NO levels. As shown in Figure 3, compared with the control, Pb treatment resulted in obviously more NO and ROS released in tobacco BY-2 cells. When the exogenous NO-donor SNP was applied to Pb-treated cells, it resulted in a notable increase in the NO content, whereas the NO levels of the cells treated with cPTIO together with Pb were markedly decreased compared with those of the cells treated with Pb alone ( Figure 3A). Meanwhile, the presence of SNP markedly increased Pb-induced ROS production. In contrast, cPTIO could reverse, in part, the endogenous levels of ROS induced by Pb ( Figure 3B).

NO Contributed to Pb-Induced ROS Production in Tobacco BY-2 Cells
The relationship of ROS and NO signaling in plants has been extensively studied, showing that NO may be an upstream signaling molecule for H2O2 in the auxin signal transduction pathway during adventitious root development in marigold [30]. There are also reports that H2O2 leads to quick NO production in guard cells of Phaseolus aureus [31]. In this work, Pb induced both ROS and NO production in tobacco BY-2 cells. Considering that the Pb-induced NO peak occurred in  decreased compared with those of the cells treated with Pb alone ( Figure 3A). Meanwhile, the presence of SNP markedly increased Pb-induced ROS production. In contrast, cPTIO could reverse, in part, the endogenous levels of ROS induced by Pb ( Figure 3B). We further analyzed the effect of SNP and cPTIO on cell viability in tobacco BY-2 cells treated with 250 μM Pb(NO3)2 for 24 h. As shown in Figure 4, at 24 h, the portion of dead cells increased from 37.83% to 47.45% in the presence of 0.5 μM SNP, which was about 1.25-fold higher than that under Pb stress alone. Meanwhile, the number of dead cells was reduced by cPTIO from 37.83% to 16.21% after treatment with 250 μM Pb(NO3)2 for 24 h. The results suggest that NO played a key role in Pb-induced ROS production and, subsequently, cell death. We further analyzed the effect of SNP and cPTIO on cell viability in tobacco BY-2 cells treated with 250 µM Pb(NO 3 ) 2 for 24 h. As shown in Figure 4, at 24 h, the portion of dead cells increased from 37.83% to 47.45% in the presence of 0.5 µM SNP, which was about 1.25-fold higher than that under Pb stress alone. Meanwhile, the number of dead cells was reduced by cPTIO from 37.83% to 16.21% after treatment with 250 µM Pb(NO 3 ) 2 for 24 h. The results suggest that NO played a key role in Pb-induced ROS production and, subsequently, cell death. decreased compared with those of the cells treated with Pb alone ( Figure 3A). Meanwhile, the presence of SNP markedly increased Pb-induced ROS production. In contrast, cPTIO could reverse, in part, the endogenous levels of ROS induced by Pb ( Figure 3B). We further analyzed the effect of SNP and cPTIO on cell viability in tobacco BY-2 cells treated with 250 μM Pb(NO3)2 for 24 h. As shown in Figure 4, at 24 h, the portion of dead cells increased from 37.83% to 47.45% in the presence of 0.5 μM SNP, which was about 1.25-fold higher than that under Pb stress alone. Meanwhile, the number of dead cells was reduced by cPTIO from 37.83% to 16.21% after treatment with 250 μM Pb(NO3)2 for 24 h. The results suggest that NO played a key role in Pb-induced ROS production and, subsequently, cell death.

NO Increased Pb 2+ Influx in Tobacco BY-2 Cells
NMT is a promising technique for investigating the transfer of metal ions in certain regions of plants and organisms [32,33]. In this work, four-day-old tobacco BY-2 cells were incubated with 250 µM Pb(NO 3 ) 2 and Pb 2+ flux was immediately measured by NMT. A constant net Pb 2+ influx with a mean value of 70.40 ± 2.70 pmol cm −2 s −1 was detected after exposure to 250 µM Pb(NO 3 ) 2 ( Figure 5A). After the addition of 0.5 µM SNP, the Pb 2+ influx was significantly increased and reached a rate of 160.56 ± 32.83 pmol cm −2 s −1 , with a significant increase of 128.06%. In contrast, treatment with 100 µM cPTIO significantly inhibited the Pb 2+ influx in comparison with Pb treatment alone, and even a slight net Pb 2+ efflux of 6.75 ± 0.85 pmol cm −2 s −1 was observed ( Figure 5A,B). These results suggest that the Pb 2+ flux significantly changed in the presence of SNP or cPTIO within a short exposure time to Pb(NO 3 ) 2 .

NO Increased Pb 2+ Influx in Tobacco BY-2 Cells
NMT is a promising technique for investigating the transfer of metal ions in certain regions of plants and organisms [32,33]. In this work, four-day-old tobacco BY-2 cells were incubated with 250 μM Pb(NO3)2 and Pb 2+ flux was immediately measured by NMT. A constant net Pb 2+ influx with a mean value of 70.40 ± 2.70 pmol cm −2 s −1 was detected after exposure to 250 μM Pb(NO3)2 ( Figure 5A). After the addition of 0.5 μM SNP, the Pb 2+ influx was significantly increased and reached a rate of 160.56 ± 32.83 pmol cm −2 s −1 , with a significant increase of 128.06%. In contrast, treatment with 100 μM cPTIO significantly inhibited the Pb 2+ influx in comparison with Pb treatment alone, and even a slight net Pb 2+ efflux of 6.75 ± 0.85 pmol cm −2 s −1 was observed ( Figure 5A,B). These results suggest that the Pb 2+ flux significantly changed in the presence of SNP or cPTIO within a short exposure time to Pb(NO3)2.  We also measured the Pb 2+ flux in cells with different treatments for a long period of time. Here, 10 h treatment rather than 24 h was chosen, considering that the detection of ion fluxes across the membrane requires viable cells. As shown in Figure 6, a net Pb 2+ influx into the tobacco BY-2 cells, the mean value of which was 34.94 ± 2.98 pmol cm −2 s −1 , was found at 10 h under Pb treatment alone. Upon addition of SNP, the average Pb 2+ flux increased remarkably to 88.98 ± 10.38 pmol cm −2 s −1 . The mean value of Pb 2+ flux in the presence of SNP was about 2.55-fold higher than that in the control cells treated with Pb alone. However, cells exposed to cPTIO exhibited minimal Pb 2+ efflux, with a mean value of 0.37 ± 2.83 pmol cm −2 s −1 . The above results indicate that NO enhanced Pb 2+ influx into the cells at 10 h. We also measured the Pb 2+ flux in cells with different treatments for a long period of time. Here, 10 h treatment rather than 24 h was chosen, considering that the detection of ion fluxes across the membrane requires viable cells. As shown in Figure 6, a net Pb 2+ influx into the tobacco BY-2 cells, the mean value of which was 34.94 ± 2.98 pmol cm −2 s −1 , was found at 10 h under Pb treatment alone. Upon addition of SNP, the average Pb 2+ flux increased remarkably to 88.98 ± 10.38 pmol cm −2 s −1 . The mean value of Pb 2+ flux in the presence of SNP was about 2.55-fold higher than that in the control cells treated with Pb alone. However, cells exposed to cPTIO exhibited minimal Pb 2+ efflux, with a mean value of 0.37 ± 2.83 pmol cm −2 s −1 . The above results indicate that NO enhanced Pb 2+ influx into the cells at 10 h.

NO Promoted Pb Uptake to Aggravate Pb Toxicity
In order to determine the role of NO in Pb uptake, we investigated the effects of the NO-donor SNP and NO-specific scavenger cPTIO on the Pb content in tobacco BY-2 cells exposed to Pb. As shown in Figure 7, the Pb content of cells in the presence of 0.5 μM SNP significantly increased to 8.33 ± 0.55 mg g −1 DW, whereas it was 7.04 ± 0.13 mg g −1 DW in cells treated with Pb alone. It was about 18.3% higher with the addition of SNP. In contrast, the Pb content was remarkably reduced to 5.00 ± 0.22 mg g −1 DW when supplied with 100 μM cPTIO versus Pb-treated cells alone. Application of cPTIO reduced the Pb content in tobacco BY-2 cells by 28.9% as compared with Pb-stressed cells.

NO Promoted Pb Uptake to Aggravate Pb Toxicity
In order to determine the role of NO in Pb uptake, we investigated the effects of the NO-donor SNP and NO-specific scavenger cPTIO on the Pb content in tobacco BY-2 cells exposed to Pb. As shown in Figure 7, the Pb content of cells in the presence of 0.5 µM SNP significantly increased to 8.33 ± 0.55 mg g −1 DW, whereas it was 7.04 ± 0.13 mg g −1 DW in cells treated with Pb alone. It was about 18.3% higher with the addition of SNP. In contrast, the Pb content was remarkably reduced to 5.00 ± 0.22 mg g −1 DW when supplied with 100 µM cPTIO versus Pb-treated cells alone. Application of cPTIO reduced the Pb content in tobacco BY-2 cells by 28.9% as compared with Pb-stressed cells.

NO Enhanced Pb-Induced Calcium Homeostasis Disorder
Calcium, as nutrition and signal molecule, plays an important function in various life activities of plants. It is generally considered to alleviate heavy metal toxicities. Previous studies have found that Pb blocks calcium absorption in plants, thus producing toxic effects on plant growth [34]. In this work, NO promoted Pb 2+ influx and participated in Pb uptake by BY-2 suspension cells. We further detected the effect of NO on Pb-induced changes of Ca 2+ fluxes. As shown in Figure 8, a net Ca 2+ influx into tobacco BY-2 cells, the mean value of which was 13.54 ± 2.78 pmol cm −2 s −1 , was detected under control cells. Upon Pb stress, the average Ca 2+ influx was suppressed and the pattern of Ca 2+ influx changed to a Ca 2+ efflux, with a mean value of 17.88 ± 1.33 pmol cm −2 s −1 . Tobacco BY-2 cells exposed to 0.5 µM SNP exhibited a significantly elevated Ca 2+ efflux (29.42 ± 4.97 pmol cm −2 s −1 ) compared with that of the cells treated with Pb alone. The Ca 2+ efflux was decreased to a mean value of 13.18 ± 0.91 pmol cm −2 s −1 in the presence of 100 µM cPTIO in comparison with that with Pb treatment alone. However, this effect was not significant. These data suggest that NO enhanced Pb-induced calcium homeostasis disorder in tobacco BY-2 cells.

NO Enhanced Pb-Induced Calcium Homeostasis Disorder
Calcium, as nutrition and signal molecule, plays an important function in various life activities of plants. It is generally considered to alleviate heavy metal toxicities. Previous studies have found that Pb blocks calcium absorption in plants, thus producing toxic effects on plant growth [34]. In this work, NO promoted Pb 2+ influx and participated in Pb uptake by BY-2 suspension cells. We further detected the effect of NO on Pb-induced changes of Ca 2+ fluxes. As shown in Figure 8, a net Ca 2+ influx into tobacco BY-2 cells, the mean value of which was 13.54 ± 2.78 pmol cm −2 s −1 , was detected under control cells. Upon Pb stress, the average Ca 2+ influx was suppressed and the pattern of Ca 2+ influx changed to a Ca 2+ efflux, with a mean value of 17.88 ± 1.33 pmol cm −2 s −1 . Tobacco BY-2 cells exposed to 0.5 μM SNP exhibited a significantly elevated Ca 2+ efflux (29.42 ± 4.97 pmol cm −2 s −1 ) compared with that of the cells treated with Pb alone. The Ca 2+ efflux was decreased to a mean value of 13.18 ± 0.91 pmol cm −2 s −1 in the presence of 100 μM cPTIO in comparison with that with Pb treatment alone. However, this effect was not significant. These data suggest that NO enhanced Pb-induced calcium homeostasis disorder in tobacco BY-2 cells.

Discussion
Environmental pollution with toxic heavy metals poses a rising threat to both the ecosystem and human health [35]. Heavy metals also impose harmful effects on plant growth and metabolism. Among various heavy metals, lead is one of the most toxic and frequently faced contaminants owing to its toxic potential to plants and other organisms as well as its global-scale distribution [36,37]. Exposure to Pb stress causes damage to the chloroplast ultrastructure [38], disturbance of nutrient metabolism [39], inhibition of plant growth and photosynthesis [7,40], suppression of cell division [4], and, consequently, cell death [8]. In this study, different concentrations of Pb stress on tobacco BY-2 cells resulted in different degrees of cell death ( Figure 1). Furthermore, chromatin condensation and granular staining nuclei, which are considered the hallmark of PCD, were found in tobacco BY-2 suspension cells treated with 250 μM Pb(NO3)2 ( Figure S1, Supplementary Materials). In addition, DNA strand breaks were detected by the TUNEL assay in cells treated with 250 μM Pb(NO3)2 ( Figure S2, Supplementary Materials). Based on these morphological changes, these data confirmed that Pb leads to PCD in tobacco BY-2 cells, which is consistent with previous reports [41].

Discussion
Environmental pollution with toxic heavy metals poses a rising threat to both the ecosystem and human health [35]. Heavy metals also impose harmful effects on plant growth and metabolism. Among various heavy metals, lead is one of the most toxic and frequently faced contaminants owing to its toxic potential to plants and other organisms as well as its global-scale distribution [36,37]. Exposure to Pb stress causes damage to the chloroplast ultrastructure [38], disturbance of nutrient metabolism [39], inhibition of plant growth and photosynthesis [7,40], suppression of cell division [4], and, consequently, cell death [8]. In this study, different concentrations of Pb stress on tobacco BY-2 cells resulted in different degrees of cell death (Figure 1). Furthermore, chromatin condensation and granular staining nuclei, which are considered the hallmark of PCD, were found in tobacco BY-2 suspension cells treated with 250 µM Pb(NO 3 ) 2 ( Figure S1, Supplementary Materials). In addition, DNA strand breaks were detected by the TUNEL assay in cells treated with 250 µM Pb(NO 3 ) 2 ( Figure  S2, Supplementary Materials). Based on these morphological changes, these data confirmed that Pb leads to PCD in tobacco BY-2 cells, which is consistent with previous reports [41].
The toxicity of heavy metals quite often evokes the generation of ROS, which might react with many cellular organelles to cause cell damage [42]. It was reported that 0.5-1 mM Pb significantly induced cell death in rice root cells by triggering ROS production [43]. Besides ROS, NO (a bioactive molecule) has also been found to be a crucial messenger molecule in plant response to heavy metals [44][45][46]. The results presented in this work show that the production of ROS and NO increased dramatically in tobacco BY-2 cells treated with 250 µM Pb(NO 3 ) 2 . However, the peak of NO (at 1.5 h) appeared much earlier than that of ROS (at 6 h) (Figure 2). The levels of ROS and NO have been reported to be reciprocally controlled or affected by each other [47]. For example, it has been found that exogenous ROS such as H 2 O 2 induce NO generation in Hypericum perforatum cell cultures and Phaseolus aureus guard cells [13,31]. Meanwhile, some reports have demonstrated that NO provides protection as an antioxidant by scavenging active oxygen species generated by Cd 2+ stress in sunflower leaves [10] and wheat roots [46], while other reports have demonstrated that heavy-metal-induced NO production promotes ROS accumulation in the root of Solanum nigrum [48]. In the present study, Pb-induced NO reached peaks at about 1.5 h. However, ROS accumulation occurred at about 6 h. The time course suggests that NO might act upstream of ROS in tobacco BY-2 cell responses to Pb stress. We used the NO-donor SNP and the NO-specific scavenger cPTIO to investigate the role that NO plays in the Pb-induced generation of ROS. The NO-specific scavenger cPTIO not only diminished the NO content but also decreased the production of ROS. Accordingly, when exogenous NO was supplied by SNP, besides NO, ROS levels were also notably raised (Figure 3). Recently, it has been reported that stress-induced ROS generation in plants is modulated through NO crosstalk with ROS-scavenging enzymes, thereby modulating ROS status [49]. Kaur et al. reported that ROS generation decreased upon exogenous NO addition when wheat roots were treated with 50 and 250 µM Pb. It is attributed to the role of NO directly scavenging ROS as an antioxidant [22]. NO displays both antioxidant and pro-oxidant activity which is determined by the time and location of NO production, and the quantity of NO generated in cells. Here, under the same concentration of Pb stress, our study led to the opposite conclusion, that is, both exogenous and endogenous NO promote ROS generation in tobacco BY-2 cells upon Pb stress. The reason for the controversy may be attributed to the different plant species used as well as the different content of exogenous NO supplied.
It has been documented that NO and ROS could influence one another, and the interactions between them might be the real cause of cell death in plants [27]. In this study, we also analyzed the effect of exogenous and endogenous NO on the cell viability of BY-2 cells. We found that the addition of exogenous NO significantly enhanced Pb-induced cell death, whereas the removal of endogenous NO alleviated Pb-induced cell death compared with Pb treatment alone ( Figure 4). Hence, our study indicates that both exogenous and endogenous NO enhanced Pb toxicity in tobacco BY-2 cells. Most reports indicate that exogenous NO supplementation has a role in the protection of plants by alleviating heavy metal stress, including Cd [50], Cu [51], Pb [52], and so forth. There are a few reports showing that the application of exogenous NO in combination with heavy metals enhances metal toxicity [53]. The contribution of endogenous NO to plant metal stress was also reported to exert both cytotoxic and cytoprotective effects [14]. The reasons for this discrepancy can be probably due to the variety of the plant tissues used, the age of the plants, the concentrations and the duration of heavy metal exposure, and so forth.
NMT has been reported to be an effective approach to studying ion uptake and accumulation in plants and animals. The fluxes of ions, such as Cd 2+ , Ca 2+ , K + , Pb 2+ , and so forth, can be measured by NMT under normal physiological conditions [31,53,54]. Heavy metal transport is crucial for understanding metal uptake mechanisms in plants. In this study, we used NMT to show that a constant net Pb 2+ influx occurred in tobacco BY-2 cells under short-and long-term Pb treatment. Exogenous NO supplied with SNP increased the Pb 2+ influx, whereas the removal of NO by cPTIO resulted in a slight efflux of Pb 2+ (Figures 5 and 6). Moreover, our data on Pb content determination indicate that exogenous NO and Pb-induced endogenous NO promote Pb accumulation in tobacco BY-2 cells (Figure 7), which is consistent with our results of Pb 2+ fluxes determined by NMT. The results support a previous study showing that the Pb-induced production of NO plays a critical role in Pb uptake by Pogonatherum crinitum root cells [11]. The promotion of Pb uptake by exogenous and endogenous NO also implicates the enhancement of NO on Pb toxicity in BY-2 cells. Moreover, it was reported that Pb accumulated in plants reduced calcium uptake [55]. Using NMT to determine whether Pb alters the pattern of Ca 2+ flux across the membrane and to investigate the role of NO during this process, we also measured Ca 2+ flux upon Pb stress supplied with or without SNP and cPTIO. Our results indicate that Pb stress obviously induced Ca 2+ efflux from cells, and NO acted positively during this course ( Figure 8). Thus, NO induced Pb 2+ influx and enhanced Pb-induced calcium homeostasis disorder.

Cell Culture
Tobacco BY-2 cells were cultured in MS medium containing 30 g L −1 sucrose and 1 mg L −1 2,4-D (pH 5.8). The cells were grown in darkness at 25 ± 2 • C on a rotary shaker at 110 rpm and subcultured at a dilution of 1:10 per week.

Hoechst and PI Double Staining
Cell activity and nuclear morphology were detected using the Hoechst and PI double staining method [23]. Four-day-old tobacco BY-2 cells were treated under different conditions for 24 h. The cells were harvested by centrifugation at 1000× g for 2 min, washed twice with fresh medium, and then resuspended in 300 µL of assaying buffer containing the Hoechst 33342 and PI fluorescence (Beyotime, Jiangsu, China) dye for 30 min at room temperature. Next, the stained cells were washed twice with 0.1 M PBS (pH 7.4) and resuspended. The cells were observed with a fluorescence microscope (Olympus BX61, Tokyo, Japan) with an excitation filter of 330-385 nm. For each sample, five different nonoverlapping microscope fields, each containing at least 100 cells, were randomly chosen. Cell death was calculated as the percentage of dead cells to the total number of cells. All data are presented as the means ± SD of three replicates from three independent experiments.

Detection of NO and ROS Production
The generation of NO and ROS in tobacco BY-2 cells was investigated using the fluorescent dyes DAF-FM DA (3-amino,4-aminomethyl-2',7'-difluorescein diacetate) and DCFH-DA

Determination of Pb Content
Four-day-old tobacco BY-2 cells were treated with 250 µM Pb(NO 3 ) 2 in the presence or absence of 0.5 µM SNP or 100 µM cPTIO (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Collected cells were dried for 12 h at 70 • C and then digested with a mixture of HNO 3 /HClO 4 (5:1, v:v). The Pb content was determined by a flame atomic absorption spectrometer (Shimadzu AA-7000, Kyoto, Japan). All data are presented as the means ± SD of three replicates from three independent experiments.

Measurement of Pb 2+ and Ca 2+ Fluxes
Both Pb 2+ and Ca 2+ fluxes were investigated by using NMT (NMT100 Series, Younger USA LLC, Amherst, MA, USA) at Xuyue (Beijing) Sci. & Tech. Co., Ltd., Beijing, China. Four-day-old tobacco BY-2 cells were prepared according to the method described by Ma et al [23] and then transferred to a measuring chamber containing 3 mL of measuring solution for Pb 2+ (0.1 mM KCl, 0.05 mM CaCl 2 , 0.05 mM MgCl 2 , 0.5 mM NaCl, 0.25 mM Pb(NO 3 ) 2 , 0.3 mM Mes, and 3% sucrose; pH 5.8) in the presence of 250 µM Pb(NO 3 ) 2 . To measure the Pb 2+ flux at the initial start time, cells that showed stable fluctuations in the preliminary detection were chosen for the subsequent net Pb 2+ flux measurements with SNP or cPTIO. Briefly, the SNP or cPTIO stock solution was slowly added to the measuring solution until the final concentration reached 0.5 or 100 µM. Then, the recording of flux was restarted and continued for a further period of 5-10 min. Furthermore, the mean values for different treatments were determined from at least six cells to illustrate Pb 2+ flux variations upon different pharmacological applications. Cells incubated in standard medium with 250 µM Pb(NO 3 ) 2 , 250 µM Pb(NO 3 ) 2 , and 0.5 µM SNP, or 250 µM Pb(NO 3 ) 2 and 100 µM cPTIO for 10 h were also collected for Pb 2+ and Ca 2+ flux measurements. The measuring solution for Ca 2+ fluxes included 0.1 mM KCl, 0.05 mM CaCl 2 , 0.05 mM MgCl 2 , 0.5 mM NaCl, 0.3 mM Mes, and 3% sucrose (pH 5.8). The data obtained were converted into specific ion influx values (pmol cm −2 s −1 ) as described before [56]. At least six cells were used to measure the Pb 2+ or Ca 2+ fluxes in each treatment.

Statistical Analysis
The data were analyzed using a one-way analysis of variance (ANOVA) and significant differences among the experimental data were set to p = 0.05.

Conclusions
In conclusion, as shown in the schematic graphic (Figure 9), we showed that Pb stress induced Pb 2+ influx and the generation of ROS and NO. Exogenous and endogenous NO induced by Pb stress acted upstream of ROS and promoted the accumulation of ROS and subsequent cell death in tobacco BY-2 cells. Both exogenous and endogenous NO enhanced Pb toxicity in tobacco BY-2 cells, and the mechanism may attribute to the ability of NO to stimulate Pb 2+ influx and thus promote Pb uptake and aggravate Pb-induced Ca 2+ homeostasis disorder in BY-2 cells. These findings lead to a better understanding of the mechanism of NO underlying Pb cytotoxicity in plant cells.
measuring solution until the final concentration reached 0.5 or 100 μM. Then, the recording of flux was restarted and continued for a further period of 5-10 min. Furthermore, the mean values for different treatments were determined from at least six cells to illustrate Pb 2+ flux variations upon different pharmacological applications. Cells incubated in standard medium with 250 μM Pb(NO3)2, 250 μM Pb(NO3)2, and 0.5 μM SNP, or 250 μM Pb(NO3)2 and 100 μM cPTIO for 10 h were also collected for Pb 2+ and Ca 2+ flux measurements. The measuring solution for Ca 2+ fluxes included 0.1 mM KCl, 0.05 mM CaCl2, 0.05 mM MgCl2, 0.5 mM NaCl, 0.3 mM Mes, and 3% sucrose (pH 5.8). The data obtained were converted into specific ion influx values (pmol cm −2 s −1 ) as described before [56]. At least six cells were used to measure the Pb 2+ or Ca 2+ fluxes in each treatment.

Statistical Analysis
The data were analyzed using a one-way analysis of variance (ANOVA) and significant differences among the experimental data were set to p = 0.05.

Conclusions
In conclusion, as shown in the schematic graphic (Figure 9), we showed that Pb stress induced Pb 2+ influx and the generation of ROS and NO. Exogenous and endogenous NO induced by Pb stress acted upstream of ROS and promoted the accumulation of ROS and subsequent cell death in tobacco BY-2 cells. Both exogenous and endogenous NO enhanced Pb toxicity in tobacco BY-2 cells, and the mechanism may attribute to the ability of NO to stimulate Pb 2+ influx and thus promote Pb uptake and aggravate Pb-induced Ca 2+ homeostasis disorder in BY-2 cells. These findings lead to a better understanding of the mechanism of NO underlying Pb cytotoxicity in plant cells. Figure 9. A schematic graphic of NO function in enhancing cytotoxicity of Pb by modulating the generation of ROS, promoting Pb 2+ influx into the cells, and disturbing the ca 2+ homeostasis.

Supplementary Materials:
The following are available online at www.mdpi.com/link, Method: TUNEL assay, Figure S1: Typical cells with nuclei containing condensed or granular chromatin. Hoechst 33342 staining in cultured tobacco BY-2 cells treated with 250 μM Pb(NO3)2 for 24 h. Scale bar = 50 μm, Figure S2: Programmed cell death detection using the TUNEL assay. Tobacco BY-2 cells that received the same volume of distilled water