Zinc-Dependent Protection of Tobacco and Rice Cells From Aluminum-Induced Superoxide-Mediated Cytotoxicity

Al³⁺ toxicity in growing plants is considered as one of the major factors limiting the production of crops on acidic soils worldwide. In the last 15 years, it has been proposed that Al³⁺ toxicity are mediated with distortion of the cellular signaling mechanisms such as calcium signaling pathways, and production of cytotoxic reactive oxygen species (ROS) causing oxidative damages. On the other hand, zinc is normally present in plants at high concentrations and its deficiency is one of the most widespread micronutrient deficiencies in plants. Earlier studies suggested that lack of zinc often results in ROS-mediated oxidative damage to plant cells. Previously, inhibitory action of Zn²⁺ against lanthanide-induced superoxide generation in tobacco cells have been reported, suggesting that Zn²⁺ interferes with the cation-induced ROS production via stimulation of NADPH oxidase. In the present study, the effect of Zn²⁺ on Al³⁺-induced superoxide generation in the cell suspension cultures of tobacco (Nicotiana tabacum L., cell-line, BY-2) and rice (Oryza sativa L., cv. Nipponbare), was examined. The Zn²⁺-dependent inhibition of the Al³⁺-induced oxidative burst was observed in both model cells selected from the monocots and dicots (rice and tobacco), suggesting that this phenomenon (Al³⁺/Zn²⁺ interaction) can be preserved in higher plants. Subsequently induced cell death in tobacco cells was analyzed by lethal cell staining with Evans blue. Obtained results indicated that presence of Zn²⁺ at physiological concentrations can protect the cells by preventing the Al³⁺-induced superoxide generation and cell death. Furthermore, the regulation of the Ca²⁺ signaling, i.e., change in the cytosolic Ca²⁺ ion concentration, and the cross-talks among the elements which participate in the pathway were further explored.

Introduction

Aluminum is the most abundant metal and the third most abundant chemical element in the Earth’s crust. The increase in free aluminum ions (chiefly, Al³⁺) accompanying soil acidification is considered to be toxic to plants (Poschenrieder et al., 2008) and animals (Markich et al., 2002). Al³⁺ toxicity in growing plants is considered as one of the major factors limiting the production of crops on acidic soils worldwide (Poschenrieder et al., 2008; Panda et al., 2009).

A number of studies documented the toxic impact of Al³⁺ especially on roots (Le Van et al., 1994; Lukaszewski and Blevins, 1996; Sanzonowicz et al., 1998). It has been proposed that early effects of Al³⁺ toxicity at growing root apex, such as those on cell division, cell extension or nutrient transport, involve the binding to (Ma et al., 1999) or uptake of Al³⁺ by plants (Lazof et al., 1994; Babourina and Rengel, 2009). Accordingly, actin cytoskeleton and vesicle trafficking are primary targets for Al³⁺ toxicity in the root tips of the sensitive variety (Amenós et al., 2009).

In the last 15 years, it has been proposed that Al³⁺ toxicity are mediated with distortion of the cellular signaling mechanisms such as calcium signaling pathways (Kawano et al., 2003a, 2004; Rengel and Zhang, 2003; Lin et al., 2005, 2006a), and production of cytotoxic reactive oxygen species (ROS) causing oxidative damages (Yamamoto et al., 2002; Kawano et al., 2003a). Recently, Al³⁺-induced DNA damages in the root cells of Allium cepa was shown to be blocked by calcium channel blockers suggesting that Al³⁺-stimulated influx of extracellular Ca²⁺ into cytosol causes the programmed cell death-like decomposition of DNA (Achary et al., 2013).

To date, two independent groups have proposed the likely modes of ROS production in Al³⁺-treated plant cells. While Yamamoto et al. (2002), propounded the role of mitochondria challenged by Al³⁺ using the cultured cells of tobacco (Nicotiana tabacum L., cell line SL) and the roots of pea (Pisum sativum L.); our group (Kawano et al., 2003a) emphasized the involvement of NADPH oxidase, thus sensitive to an inhibitor of NADPH oxidase, diphenylene iodonium (DPI) in tobacco BY-2 cells. While ROS is slowly produced through mitochondrial dysfunction (ca. 12 h after Al³⁺ treatment; Yamamoto et al., 2002), the NADPH oxidase-mediated production of superoxide anion radical (O₂^•–) takes place immediately after Al³⁺ treatment (Kawano et al., 2003a).

The action of Al³⁺ for induction of O₂^•– generation which is sensitive to DPI was recently confirmed in the cells of Arabidopsis thaliana (Kunihiro et al., 2011). Furthermore, the Al³⁺-induced oxidative burst showed biphasic signature consisted with an acute transient spike and a slow but long-lasting wave of O₂^•– generation. In addition, among six respiratory burst oxidase homologs (Atrbohs) coding for plant NADPH oxidase, solely AtrbohD was shown to be responsive to Al³⁺ in biphasic manner by showing rapid (1 min) and long-lasting (24 h) expression profiles (Kunihiro et al., 2011).

Interestingly, the mechanism of Al³⁺-induced oxidative burst (production of O₂^•–) is highly analogous to the response of tobacco cell suspension culture to other metal cations, chiefly trivalent cations of lanthanides such as La³⁺ and Gd³⁺ (Kawano et al., 2001). Therefore, we assume that some known chemical factors reportedly interfere with the lanthanide-induced plant oxidative burst might be active for protection of plant cells from Al³⁺-induced oxidative stress. Such chemicals of interest to be tested include zinc and manganese (Kawano et al., 2002).

Zinc is normally present in plants at high concentrations (Santa-Maria and Cogliatti, 1988) and its deficiency is one of the most widespread micronutrient deficiencies in plants, causing severe reductions in crop production (Cakmak, 2000). Increasing studies indicate that oxidative damage to cellular components caused in plants being challenged by ROS, is highly due to the deficiency of zinc (Pinton et al., 1994; Cakmak, 2000).

Previously, inhibitory action of Zn²⁺ against lanthanide-induced O₂^•– generation in tobacco cells have been reported (Kawano et al., 2002). Pretreatments with Zn²⁺ reportedly interferes the La³⁺- and Gd³⁺-induced O₂^•– generation in tobacco cells. In the tobacco model, Zn²⁺ was shown to minimize the earlier phase of lanthanide-induced O₂^•– production while allowing the release of O₂^•– in the later phase, thus causing the retardation of the lanthanide actions on O₂^•– generation.

Although this process is well known, if it is preserved in higher plants and the specific mechanism of action have is not still clear. For this reason, in the present study, effect of Zn²⁺ on Al³⁺-induced O₂^•– generation in the suspension cultures of tobacco BY-2 cells and rice (Oryza sativa L., cv. Nipponbare) cells, was examined. Furthermore, the regulation of the Ca²⁺ signaling, i.e., change in the cytosolic Ca²⁺ ion concentration ([Ca²⁺]_c), and the cross-talks among the elements which participate in the pathway were further explored. Finally the possible use of Zn²⁺ for protection of plant cells from Al³⁺ toxicity is discussed.

Materials and Methods

Chemicals

O₂^•–-specific chemiluminescence (CL) probe, Cypridina luciferin analog (CLA; 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one) designated as CLA was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Aluminum (III) chloride hexahydrate (AlCl₃·6H₂0), zinc sulfate heptahydrate (ZnSO₄·7H₂O), gadolinium chloride hexahydrate (GdCl₃·6H₂O), and salicylic acid (SA) were from Wako Pure Chemical Industries (Osaka, Japan). Lanthanum chloride heptahydrate (LaCl₃·7H₂O) was from Kanto Chemical Co., Inc (Tokyo, Japan). DPI chloride, Evans blue, 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron), N,N^′-dimethylthiourea (DMTU), were from Sigma (St. Louis, MO, USA). Coelenterazine was a gift from Prof. M. Isobe (Nagoya University).

Plant Cell Culture

Tobacco (Nicotiana tabacum L. cv. Bright Yellow-2) suspension-culture cells (cell line, BY-2, expressing the aequorin gene; Figure 1A) were propagated as previously described (Kawano et al., 1998). Briefly, the culture was maintained in Murashige–Skoog liquid medium (pH 5.8) supplemented with 3% (w/v) sucrose and 0.2 μg ml^–1 of 2,4-dichlorophenoxyacetic acid. The culture was propagated with shaking on a gyratory shaker in darkness at 23°C. For sub-culturing, 1.0 ml of confluent stationary culture was suspended in 30 ml of fresh culture medium and incubated at 23°C with shaking at 130 rpm on a gyratory shaker in darkness until used.

FIGURE 1

Figure 1. Microscopic images of plant cells used in this study. (A) Tobacco BY-2 cells. (B) Rice M1 cells. Scale bars, 50 μm.

Rice callus tissues (Oryza sativa L., cv. Nipponbare, cell line, M1; Figure 1B) were obtained from root explants derived from young seedlings and transferred in AA liquid medium to develop a suspension-culture. The cells were maintained and propagated at 23°C with shaking at 130 rpm on a gyratory shaker in darkness. For sub-culturing, with 2-week intervals, 10 ml of stationary culture was suspended in 100 ml of fresh culture medium.

Detection of O₂^•– with CLA

To detect the production of O₂^•– in plant cells, the 200 μl of plant cell suspension (either of tobacco or rice) was placed in glass cuvettes and CLA was added at final concentration of 2 μM (in tobacco cells) and 4 μM (in rice cells). The glass cuvettes containing 200 μl of plant cell suspension were placed in luminometers (CHEM-GLOW Photometer, American Instrument Co., Silver Spring, MD, USA; or PSN AB-2200-R Luminescensor, Atto, Tokyo). Generation of O₂^•– in cell suspension culture was monitored by CLA-CL, and expressed as relative chemiluminescence units (rcu) as previously described (Kawano et al., 1998). CLA-CL specifically indicates the generation of O₂^•– (and of ¹O₂ with a minor extent) but not that of O₃, H₂O₂ or hydroxy radicals (Nakano et al., 1986).

Aequorin Ca²⁺ Detection

To detect the changes in [Ca²⁺]_c in tobacco cells, 10 mL of plant cell suspension were pre-treated for 8 h with 10 μL of coelenterazine in the dark, then used for the experiments as previously described (Kawano et al., 1998). Also in this case, 200 μl plant of cell suspension was placed in glass cuvettes and placed in luminometers (as above). Increase in [Ca²⁺]_c, reflecting the induced Ca²⁺ into cells, was monitored as luminescence derived upon binding of Ca²⁺ to aequorin (the recombinant gene over-expressed in the cytosol) and expressed as rcu.

Treatments with Aluminum, Zinc, and Other Stimuli

Tobacco BY-2 cells were harvested various days after sub-culturing (as indicated), and used for experiments with CLA or aequorin. AlCl₃ was dissolved in distilled water and diluted with fresh culture media unless indicated, and 10–20 μl of the AlCl₃ solution was added to 180–190 μl of cell suspension in glass cuvettes, and level of [Ca²⁺]_c (aequorin experiment) or generation of O₂^•– (CLA experiment) were monitored. For comparison, effects of SA and hypo-osmotic shock (induced by dilution of media giving Δ100 mOsmol of hypo-osmolarity difference) on calcium homeostasis with and without zinc was monitored. Inhibition of events induced by Al³⁺ and other stimuli, monitored with CLA CL, aequorin luminescence, and cell death staining, was performed by addition of indicated concentration of Zn²⁺ to the cells prior to treatments with Al³⁺, SA, and hypo-osmotic shock.

Monitoring of Cell Death

Al³⁺-induced cell death in BY-2 tobacco cell suspension culture was allowed to develop in the presence of Evans blue, a lethal staining dye (0.1%, w/v). Evans blue was added to the cell suspension culture, 6 h after Al³⁺ application unless indicated or at the time indicated (0–8 h after Al addition). Then, the cells were further incubated for 1 h for fully developing and detecting the cell death as described (Kadono et al., 2006). After terminating the staining process by washing, stained cells were counted under microscopes. For statistical analyses, four different digital images of cells under the microscope (each covering 50 cells to be counted) were acquired and stained cells were counted.

Results and Discussions

Induction of O₂^•–

The effect of Al³⁺ concentration on induction of O₂^•– generation has been tested both in tobacco and rice cell suspension cultures (Figure 2). For this analysis, BY-2 tobacco cells have been tested 4 days after inoculation (DAI) unless indicated whereas the rice cell line M1 suspension culture was used 14 DAI, since the tobacco BY-2 cells grow at faster rate compared to rice M1 cells. In tobacco BY-2 cells, the active Al³⁺ concentrations for induction of O₂^•– generation ranged from 0.1 mM to 30 mM (optimally at 3 mM).

FIGURE 2

Figure 2. Effect of Al³⁺ concentration on O₂^•– induction. Typical records of Al³⁺-induced O₂^•– measured with CLA-CL in the cell suspension cultures of tobacco (A) and rice (B). Effect of Al³⁺ concentration on O₂^•– generation (C, tobacco; D, rice). Vertical error bars, SD; n = 3.

Notably, higher concentration of Al³⁺ was shown to be inhibitory to induction of O₂^•– generation in the tobacco cells (Figures 2A,C), while the rice cells showed only the proportional increase in generation of O₂^•– with the increase in Al³⁺ up to 100 mM (Figures 2B,D). In order to analyze the impact of Zn²⁺ against Al³⁺-induced O₂^•– generation, the concentration of Al³⁺ was fixed to at 3 mM for the tobacco cells and 100 mM for rice cells. Different concentrations have been chosen since the tobacco cells showed higher sensitivity to relatively lower concentrations of Al³⁺.

Effect of Pretreatment with Zn²⁺

To assess the effect of Zn²⁺, the cells of tobacco and rice were pre-treated with various concentration of ZnSO₄ for 5 min and then AlCl₃ was added to the cells (Figure 3). In tobacco cell, the O₂^•– generation induced by 3 mM Al³⁺ was significantly inhibited by 1 mM or higher concentrations of Zn²⁺, whilst in rice cell, 0.1 mM of Zn²⁺ was high enough to achieve a significant inhibition of O₂^•– generation induced by 100 mM Al³⁺. Although Zn²⁺-dependent retardation of lanthanide-induced O₂^•– production has been reported (Kawano et al., 2002), the Al³⁺-induced oxidative burst was simply inhibited without allowing the onset of slower increase in O₂^•– production.

FIGURE 3

Figure 3. Inhibition of the Al³⁺-induced generation of O₂^•– in the presence of Zn²⁺. Typical records of Al³⁺-induced O₂^•– generation in the absence and the presence of ZnSO₄ measured with CLA (A, tobacco; B, rice). Arrows indicate the time points for addition of AlCl₃ (3 mM, tobacco; 100 mM, rice). Effects of Zn²⁺ concentration on Al³⁺-induced O₂^•– generation (C, tobacco; D, rice). Vertical error bars, SD; n = 3.

The Zn²⁺-dependent inhibition was observed in both model cells selected from the monocots and dicots (rice and tobacco), suggesting that this phenomenon (Al³⁺/Zn²⁺ interaction) can be observed universally in the wide range of higher plants. Since the sensitivity was higher in tobacco BY-2 cells, this cell line was chosen to be used in the further experiments examining the mode of Al³⁺/Zn²⁺ interaction.

Effect of Culture Age on O₂^•– Production

Prior to treatment with Al³⁺, tobacco BY-2 cell suspension culture was aged for 1, 3, 5, 8, and 13 DAI of the fresh media (30 ml) with 0.5 ml of confluent culture (at 10 DAI). The cultures at 1 and 3 DAI were smooth and colorless. The 4 and 5 DAI cultures were also smooth but colored slightly yellowish. The 8 and 12 DAI cultures were highly dense and colored yellow. The growth of the culture was assessed by measuring the changes in fresh cell weight at each time point. Figure 4A shows a typical growth curve for tobacco BY-2 cell culture. Effect of culture age of tobacco BY-2 cells on the sensitivity to Al³⁺ was examined using the differently aged cultures (Figure 4B), and the high sensitivity to Al³⁺ was observed in 2 and 4 DAI of tobacco BY-2 cells.

FIGURE 4

Figure 4. Effect of culture age on Al³⁺-induced O₂^•– generation in tobacco cell suspension culture. (A) Growth curve for tobacco cells. (B) Four different patterns of CLA-CL reflecting the Al³⁺-induced O₂^•– generation.

Competition Between Zn²⁺ and Al³⁺

Application of double-reciprocal plot analysis for studying the behavior of living plants or cells, so-called in vivo Lineweaver–Burk plot analysis was carried out to assess the mode of Al³⁺/Zn²⁺ interaction according to the procedure described elsewhere (Kawano et al., 2003b). By making use of linear dose-dependency in the limited range of Al³⁺ concentrations (up to 3 mM) in 4 DAI culture of tobacco BY-2 cell, the in vivo kinetic analysis was carried out by assuming Al³⁺ as a ligand to the putative Al³⁺ receptors on the cells and Zn²⁺ as an inhibitor (Figure 5A). The reciprocals of the CLA-CL yields (1/CLA-CL) were plotted against the reciprocals of Al³⁺ concentrations (1/[Al³⁺]). Linear relationship between 1/CLA-CL and 1/[Al³⁺] were obtained both in the presence and absence of Zn²⁺ (Figure 5B). In the presence of Zn²⁺, the apparent K_m for Al³⁺ was elevated from 113 μM (control) to 376 μM (0.1 mM Zn²⁺; ca. 3.3-fold increase), while V_max for Al³⁺-induced CLA-CL was not drastically altered. V_max for Al³⁺-induced response in the absence of Zn²⁺ was calculated to be 14.3 rcu. In the presence of 0.1 mM Zn²⁺, V_max was 18.6 rcu (ca. 30% increase). Therefore, the mode of Zn²⁺ action against Al³⁺ can be considered as a typical competitive inhibition.

FIGURE 5

Figure 5. Competitive inhibition of the Al³⁺-induced O₂^•– generation by Zn²⁺ in tobacco cell suspension culture. (A) Effect of Zn²⁺ on Al³⁺-induced O₂^•– generation. (B) In vivo Lineweaver–Burk plot analysis.

According to Kawano et al. (2003a) the Al³⁺-induced generation of O₂^•– in tobacco cells is catalyzed by Al³⁺-stimulated NADPH oxidase which is sensitive to DPI. The cation-dependent enhancement in NADPH oxidase-catalyzed O₂^•– production is also known in human neutrophils in which binding of metal cations possibly results in spontaneous activation of the O₂^•–-generating activity of the membrane-bound enzyme (Cross et al., 1999). We can assume that NADPH oxidase itself, localized on the surface of cells (or other factors associated with NADPH oxidase), behaves as the receptor for Al³⁺ ions. The competitive mode of Zn²⁺ action against the Al³⁺-induced oxidative burst suggests us to consider that the binding site for Al³⁺ and Zn²⁺ on the NADPH oxidase or on the factors associated nearby must be identical.

Al³⁺-Induced Cell Death and its Inhibition by Zn²⁺

As shown in Figures 6A,B, treatment of tobacco BY-2 cells with various concentrations of AlCl₃ resulted in cell death induction. Notably, the presence of Zn²⁺ significantly protected the cells from the induction of cell death by Al³⁺ (Figure 6C), as predicted by the action of Zn²⁺ against Al³⁺-induced oxidative burst.

FIGURE 6

Figure 6. Al³⁺-induced cell death and its inhibition by addition of Zn²⁺ in tobacco BY-2 cells. (A) Effect of AlCl₃ concentration on cell death induction. Cell death was assessed by Evans blue staining 8 h after addition of AlCl₃. (B) Effect of post-Al³⁺ incubation (0.5–8 h) on development of cell death. (C) Effect of ZnSO₄ on Al³⁺-induced cell death. Cell death was assessed 6 h after Al³⁺ treatments. Vertical error bars, SD; n = 3.

Effect of Pretreatment with Mn²⁺

Manganese is another micronutrient possibly protecting the living cells from oxidative damage (Ledig et al., 1991) and reportedly blocks the lanthanide-induced oxidative burst (Kawano et al., 2002). In fact, Mn²⁺ is often employed as a scavenger of O₂^•– for preventing the biochemical reactions involving O₂^•– (Momohara et al., 1990).

Therefore, we tested the effect of MnSO₄ (up to 3 mM) for comparison. The results obtained suggested no inhibitory effect of Mn²⁺ against Al³⁺-induced generation of O₂^•–. Instead, low concentrations of Mn²⁺ slightly elevated the level of Al³⁺-induced oxidative burst (data not shown). For inhibition of Al³⁺-induced oxidative burst, much higher concentrations of MnSO₄ (10–100 mM) were required. Since the range of Mn²⁺ concentrations required for lowering the level of Al³⁺-induced generation of O₂^•– was at phytotoxic range (Caldwell, 1989) and thus inducing cell death even in the absence of Al³⁺ in BY-2 cells (ca. 40% of cells died in the presence of 30 mM MnSO₄), the use of Mn²⁺ is not suitable for preventing the production of O₂^•– induced by Al³⁺.

Anti-Oxidative Role for Zn²⁺

Plants require trace amounts of specific metals known as trace nutrients including Zn²⁺, supporting the essential functions of plant cells ranging from respiration to photosynthesis, and molecular biological studies on the mechanism for uptake of these metals by plants have been documented (Delhaize, 1996). One of the important roles for Zn²⁺ in living plants is anti-oxidative action against ROS (Kawano et al., 2002) as the present study successfully demonstrated that extracellular supplementation of Zn²⁺ inhibits the generation of O₂^•– (Figure 4) and cell death (Figure 6C) induced by Al³⁺.

In contrast to manganese, zinc is normally present in plants at high concentrations. For example, in roots of wheat seedlings, the cytoplasmic concentration of total Zn has been estimated to be approximately 0.4 mM (Santa-Maria and Cogliatti, 1988), and Zn-deficiency often results in inhibition of growth, as Zn reportedly protects the plants by preventing the oxidative damages to DNA, membranes, phospholipids, chlorophylls, proteins, SH-containing enzymes, and indole-3-acetic acid (Cakmak, 2000).

Here, Zn²⁺ at sub-mM concentrations showed strong inhibitory action against the toxicity of Al³⁺ (oxidative burst and cell death). The levels of Zn²⁺ naturally present in soil or plant tissues may be contributing to the prevention of Al³⁺-induced cellular damages but further studies on living plants are needed to evaluate this mechanism in living tissue and the possible applications to increase plant tolerance.

Oxidative and Calcium Crosstalk

Al³⁺ is known to inhibit plant calcium channels similarly to the action of various lanthanide ions (Lin et al., 2006b). The calcium channels sensitive to Al³⁺ could be identical to those involved in responses to ROS (Kawano et al., 2003a, 2004), cold shock (Lin et al., 2005, 2006a, 2007), and heat shock (Lin et al., 2007), but not responsive to osmotic shock (Lin et al., 2005, 2006b, 2007), as examined in transgenic cell lines of rice (Oryza sativa L., cv. Nipponbare) and tobacco (cell-lines, BY-2, Bel-B, and Bel-W3) all expressing aequorin in the cytosolic space.

To support the hypothesis that Al³⁺-induced distortion in [Ca²⁺]_c involves the members of ROS derived from the action of NADPH oxidase, and calcium channel opening leading to transient [Ca²⁺]_c elevation, the effect of DPI (NADPH oxidase inhibitor), ROS scavengers (Tiron, DMTU), and trivalent cations (La³⁺ and Gd³⁺) have been tested in tobacco cells expressing aequorin and compared with the antagonistic action of zinc protecting the cells (Figure 7A and inset).

FIGURE 7

Figure 7. Monitoring of Al³⁺-responsive [Ca²⁺]_c with aequorin luminescence in the presence and absence of various inhibitors. (A) Effect of high- and low-dose Zinc, DPI (NADPH oxidase inhibitor), ROS scavengers (Tiron, DMTU), and trivalent cations (La³⁺ and Gd³⁺) on Al-responsive [Ca²⁺]_c elevation in tobacco cell suspension culture expressing aequorin. (Inset) Quantitative comparison of the action of inhibitors against the peak height of Al-responsive [Ca²⁺]_c elevation (n = 3; error bars, SD). (B) Effect of Al³⁺ concentration on induction of [Ca²⁺]_c elevation. Typical records of Ca²⁺-responsive aequorin luminescence measured in the presence of different Aluminum concentrations are shown.

As expected, Tiron, DMTU, and high concentration of zinc (0.5 mM) effectively lowered the level of Al³⁺-induced [Ca²⁺]_c elevation. Especially, temporal patterns in which Al³⁺ induces an increase in [Ca²⁺]_c was shown to be sensitive to both zinc and DPI. In fact, these chemicals significantly retarded the Al³⁺-responsive calcium influx, thus, time required for attaining the peak of Al³⁺-responsive [Ca²⁺]_c elevation was shown to be longer, suggesting the zinc and DPI might share the common mode of action.

On the other hand, La³⁺ and Gd³⁺ strongly reduce the signal as we observed for high concentration of Al³⁺ (Figure 7A) supporting the view that they can concurrently act inhibiting the Ca²⁺ channel.

Previously, we have propose a model that Al³⁺ plays dual roles acting for and against the Ca²⁺ influx, by releasing O₂^•– and by inhibiting the Ca²⁺ channel(s), respectively (Kawano et al., 2003a). Al³⁺-dependent distortion in calcium signaling in plant cells can be dissected into two opposing modes of Al³⁺ actions, viz., (i) stimulation of ROS-responsive calcium channels via induction of O₂^•– and (ii) inhibition of calcium channels. At low Al³⁺ concentrations, the ROS-responsive Ca²⁺ influx potency is high but the driving force (due to ROS) is not sufficient. At high Al³⁺concentrations, the Ca²⁺ influx-driving force is at sufficient level but the channel’s Ca²⁺ permeability is low. This effect is showed in Figure 7B, where [Ca²⁺]_c elevation could be manifested only in the range of Al³⁺ concentration in which the two opposing effects eventually compromise (Kawano et al., 2003a). Zn²⁺ hardly blocks the calcium influx in model plant cells unless the event of interest is dependent on the ROS generating events (Figure 8). Therefore, we view here that Zn²⁺ might target only the earlier phase of Al³⁺ action involved in induction of O₂^•– as illustrated in Figure 9.

FIGURE 8

Figure 8. Effect of Al³⁺ and Zn²⁺ against SA- responsive and hypo-osmotic shock-responsive [Ca²⁺]_c elevation in tobacco cells. Error bars, SD; n = 3.

FIGURE 9

Figure 9. A model for Al³⁺-induced signaling leading to oxidative cell death and its inhibition by Zn²⁺ in model plant cells. Knowledge from present experiments using the aequorin-expressing cell lines of tobacco (BY-2) were strengthened by previously reported models. As suggested in the cells of Arabidopsis thaliana (Kunihiro et al., 2011), SA signaling pathway is activated in the downstream of ROS such as O₂^•– (and possibly of calcium signaling). The loop of ROS-stimulated the SA signaling and expression of NADPH oxidase which produces further ROS, may eventually lead to development of cell death. Zinc may block the loop by interfering the NADPH oxidase-catalyzed ROS production similarly to the model for lanthanide-induced oxidative burst and cell death in tobacco cells (Kawano et al., 2002). Arrows indicate the stimulation and the T-shaped lines indicate the steps inhibited by agent indicated. The numbers and the symbol “C” in small brackets next to arrows or T-shaped lines denote the source of knowledge, namely; C, data confirmed here; 1, Kawano et al. (2003a; BY-2 cells); 2, Kawano et al. (2004; BY-2 cells); 3, Lin et al. (2005; BY-2 cells); 4, Kunihiro et al. (2011; Arabidopsis thaliana); 5, Kawano et al. (2002; BY-2 cells); 6, Kawano et al. (1998; BY-2 cells); 7, Kawano et al. (2013; review on higher plants).

The Likely Signaling Paths

In Arabidopsis thaliana, Al³⁺-induced prolonged ROS generation requires the expression of AtrbohD coding for NADPH oxidase (Kunihiro et al., 2011). This work suggested that biosynthesis and signal transduction pathway for SA is involved in Al³⁺-mediated oxidative burst since the Al³⁺-induced AtrbohD expression and cell death were inhibited in the mutant and transgenic cell lines lacking SA biosynthesis, accumulation of SA, and SA-specific signaling components (sid2, NahG and npr1, respectively). It has been proposed that loop of SA signal transduction, involving the activity and further induction of NADPH oxidase, forms a signaling circuit enabling an amplification of SA-mediated signaling (Figure 9). This type of oxidative signal amplification was designated as SA/rboh loop (Kunihiro et al., 2011).

By analogy, there would be a similar mechanism in response to Al³⁺ in the cells of tobacco and rice since both the ROS production and cell death were commonly shown to be induced by Al³⁺ in these cells.

Lastly, we propose a likely mode of Zn²⁺ action against Al³⁺-induced cell death. Zn²⁺ may competitively antagonize the action of Al³⁺ by targeting the NADPH oxidase-catalyzed ROS production at upstream of SA signaling mechanism. As a consequence, activation of SA/rboh loop responsible for long-lasting oxidative burst releasing cytotoxic ROS could be prevented (Figure 9).

By assessing the action of Zn²⁺ against SA-induced [Ca²⁺]_c elevation which is known to be one of the key events in the SA-induced O₂^•–-mediated signaling path, involving the activation of Ca²⁺ channel identified as TPC1 channel (Kawano et al., 2013; Lin et al., 2005), we understood that target of antioxidant activity of zinc is not limited to the Al³⁺-induced NADPH oxidase-catalyzed mechanism (Figure 8). It is known that SA-induced rapid O₂^•– is catalyzed by extracellular (cell-wall bound) peroxidase, while SA-induced long-lasting oxidative burst requires the induction of rboh genes coding for NADPH oxidase (Kawano et al., 1998; Yoshioka et al., 2008). In contrast, Zn²⁺ failed to block the Ca²⁺ influx induced by hypo-osmotic shock possibly involving the mechanosensitive-cation channel (Takahashi et al., 1997).

Taken together, target of Zn²⁺ is specifically against the ROS-generating mechanisms (both NADPH oxidase-mediated and peroxidase-mediated) eventually leading to ROS-responsive calcium signaling (Figure 9).

Furthermore, the action of Al³⁺ may form a loop of repeated reaction involving the action of SA which further induces specific type of NADPH oxidase (in case of Arabidopsis thaliana, only AtrbohD is Al³⁺-responsive, Kunihiro et al., 2011).

Author Contributions

TK designed and supervised the experiments and some key data for plant age and ROS production were obtained by him. CL conducted most tobacco experiments (mostly calcium signaling and cell viability tests), AH and DC performed additional experiments. AH was in charge of rice cell experiments (both ROS detection and calcium signaling). DC and FB contributed on the analysis of data and writing of MS. All authors actively contributed in the discussion.

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.

Acknowledgment

This work was supported by Regional Innovation Cluster Program by Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Abbreviations

CL, chemiluminescence; CLA, Cypridina luciferin analog; DAI, days after inoculation; DPI, diphenylene iodonium; O₂^•–, superoxide anion radical; rcu, relative chemiluminescence units; ROS, reactive oxygen species; SA, salicylic acid.

References

Achary, V. M. M., Parinandi, N. L., and Panda, B. B. (2013). Calcium channel blockers protect against aluminium-induced DNA damage and block adaptive response to genotoxic stress in plant cells. Mutat. Res. 751, 130–138. doi: 10.1016/j.mrgentox.2012.12.008

PubMed Abstract | CrossRef Full Text | Google Scholar

Amenós, M., Corrales, I., Poschenrieder, C., Illéš, P., Baluška, F., and Barceló, J. (2009). Different effects of aluminum on the actin cytoskeleton and brefeldin A-sensitive vesicle recycling in root apex cells of two maize varieties differing in root elongation rate and aluminum tolerance. Plant Cell Physiol. 50, 528–540. doi: 10.1093/pcp/pcp013

PubMed Abstract | CrossRef Full Text | Google Scholar

Babourina, O., and Rengel, Z. (2009). Uptake of aluminium into Arabidopsis root cells measured by fluorescent lifetime imaging. Ann. Bot. 104, 189–195. doi: 10.1093/aob/mcp098

PubMed Abstract | CrossRef Full Text | Google Scholar

Cakmak, I. (2000). Possible roles of zinc in protecting plant cells from damage by reactive oxygen species. New Phytol. 146, 185–205. doi: 10.1046/j.1469-8137.2000.00630.x

CrossRef Full Text | Google Scholar

Caldwell, C. R. (1989). Analysis of aluminum and divalent cation binding to wheat root plasma membrane proteins using terbium phosphorescence. Plant Physiol. 91, 233–241. doi: 10.1104/pp.91.1.233

PubMed Abstract | CrossRef Full Text | Google Scholar

Cross, A. R., Erichson, R. W., Eliss, B. A., and Curnutte, J. T. (1999). Spontaneous activation of NADPH oxidase in a cell-free system: unexpected multiple effects of magnesium ion concentrations. Biochem. J. 338, 229–233. doi: 10.1042/bj3380229

PubMed Abstract | CrossRef Full Text | Google Scholar

Delhaize, E. (1996). A metal-accumulator mutant of Arabidopsis thaliana. Plant Physiol. 111, 849–855. doi: 10.1104/pp.111.3.849

PubMed Abstract | CrossRef Full Text | Google Scholar

Kadono, T., Yamaguchi, Y., Furuichi, T., Hirono, M., Garrec, J. P., and Kawano, T. (2006). Ozone-induced cell death mediated with oxidative and calcium signaling pathways in tobacco Bel-W3 and Bel-B cell suspension cultures. Plant Signal. Behav. 1, 312–322. doi: 10.4161/psb.1.6.3518

PubMed Abstract | CrossRef Full Text | Google Scholar

Kawano, T., Hiramatsu, T., and Bouteau, F. (2013). “Signaling role of salicylic acid in abiotic stress responses in plants,” in Salicylic Acid, eds S. Hayat, A. Ahmad, and M. N. Alyemeni (Dordrecht: Springer), 249–275.

Google Scholar

Kawano, T., Kadono, T., Fumoto, K., Lapeyrie, F., Kuse, M., Isobe, M., et al. (2004). Aluminum as a specific inhibitor of plant TPC1 Ca²⁺ channels. Biochem. Biophys. Res. Commun. 324, 40–45. doi: 10.1016/j.bbrc.2004.09.015

PubMed Abstract | CrossRef Full Text | Google Scholar

Kawano, T., Kadono, T., Furuichi, T., Muto, S., and Lapeyrie, F. (2003a). Aluminum-induced distortion in calcium signaling involving oxidative bursts and channel regulations in tobacco BY-2 cells. Biochem. Biophys. Res. Commun. 308, 35–42. doi: 10.1016/S0006-291X(03)01286-5

PubMed Abstract | CrossRef Full Text | Google Scholar

Kawano, N., Kawano, T., and Lapeyrie, F. (2003b). Inhibition of the indole-3-acetic acid-induced epinastic curvature in tobacco leaf strips by 2,4-dichlorophenoxyacetic acid. Ann. Bot. 91, 456–471. doi: 10.1093/aob/mcg043

PubMed Abstract | CrossRef Full Text | Google Scholar

Kawano, T., Kawano, N., Muto, S., and Lapeyrie, F. (2001). Cation-induced superoxide generation in tobacco cell suspension culture is dependent on ion valence. Plant Cell Environ. 24, 1235–1241. doi: 10.1046/j.1365-3040.2001.00766.x

CrossRef Full Text | Google Scholar

Kawano, T., Kawano, N., Muto, S., and Lapeyrie, F. (2002). Retardation and inhibition of the cation-induced superoxide generation in BY-2 tobacco cell suspension culture by Zn²⁺ and Mn²⁺. Physiol. Plant. 114, 395–404. doi: 10.1034/j.1399-3054.2002.1140309.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Kawano, T., Sahashi, N., Takahashi, K., Uozumi, N., and Muto, S. (1998). Salicylic acid induces extracellular superoxide generation followed by an increase in cytosolic calcium ion in tobacco suspension culture: the earliest events in salicylic acid signal transduction. Plant Cell Physiol. 39, 721–730. doi: 10.1093/oxfordjournals.pcp.a029426

CrossRef Full Text | Google Scholar

Kunihiro, S., Hiramatsu, T., and Kawano, T. (2011). Involvement of salicylic acid signal transduction in aluminum-responsive oxidative burst in Arabidopsis thaliana cell suspension culture. Plant Signal. Behav. 6, 611–616. doi: 10.4161/psb.6.5.14895

PubMed Abstract | CrossRef Full Text | Google Scholar

Lazof, D. B., Goldsmith, J. G., Rufty, T. W., and Linton, R. W. (1994). Rapid uptake of aluminum into cells of intact soybean root tips. A microanalytical study using secondary ion mass spectrometry. Plant Physiol. 106, 1107–1114.

PubMed Abstract | Google Scholar

Ledig, M., Tholey, G., Megias-Megias, L., Kopp, P., and Wedler, F. (1991). Combined effects of ethanol and manganese on cultured neurons and glia. Neurochem. Res. 16, 591–596. doi: 10.1007/BF00974879

PubMed Abstract | CrossRef Full Text | Google Scholar

Le Van, H., Kuraishi, S., and Sakurai, N. (1994). Aluminum-induced rapid root inhibition and changes in cell-wall components of squash seedlings. Plant Physiol. 106, 971–976.

PubMed Abstract | Google Scholar

Lin, C., Kadono, T., Suzuki, T., Yoshizuka, K., Furuichi, T., Yokawa, K., et al. (2006a). Mechanism for temperature-shift-responsive acute Ca²⁺ uptake in suspension-cultured tobacco and rice cells. Cryobiol. Cryotechnol. 52, 83–89.

Google Scholar

Lin, C., Kadono, T., Yoshizuka, K., Furuichi, T., and Kawano, T. (2006b). Effects of fifteen rare earth metals on Ca²⁺ influx in tobacco cells. Z. Naturforsch. 61, 74–80. doi: 10.1515/znc-2006-1-214

CrossRef Full Text | Google Scholar

Lin, C., Yoshizuka, K., and Kawano, T. (2007). Effect of rare earth elements on cold-responsive Ca²⁺ signaling in tobacco cells. Cryobiol. Cryotechnol. 53, 7–11.

Google Scholar

Lin, C., Yu, Y., Kadono, T., Iwata, M., Umemura, K., Furuichi, T., et al. (2005). Action of aluminum, novel TPC1-type channel inhibitor, against salicylate-induced and cold shock-induced calcium influx in tobacco BY-2 cells. Biochem. Biophys. Res. Commun. 332, 823–830. doi: 10.1016/j.bbrc.2005.05.030

PubMed Abstract | CrossRef Full Text | Google Scholar

Lukaszewski, K. M., and Blevins, D. G. (1996). Root growth inhibition in boron-deficient or aluminum-stressed squash may be a result of impaired ascorbate metabolism. Plant Physiol. 112, 1135–1140.

PubMed Abstract | Google Scholar

Ma, J. F., Yamamoto, R., Nevin, D. J., Matsumoto, H., and Brown, P. H. (1999). Al binding in the epidermis cell wall inhibits cell elongation of okra hypocotyl. Plant Cell Physiol. 40, 549–556. doi: 10.1093/oxfordjournals.pcp.a029576

CrossRef Full Text | Google Scholar

Markich, S. J., Warne, M. S. J., Westbury, A. M., and Roberts, C. J. (2002). A compilation of data on the toxicity of chemicals to species in Australasia. Part 3: metal. Aust. J. Ecotoxicol. 8, 1–137.

Google Scholar

Momohara, I., Matsumoto, Y., and Ishizu, A. (1990). Involvement of veratryl alcohol and active oxygen species in degradation of a quinone compound by lignin peroxidase. FEBS Lett. 273, 159–162. doi: 10.1016/0014-5793(90)81074-X

PubMed Abstract | CrossRef Full Text | Google Scholar

Nakano, M., Sugioka, K., Ushijima, Y., and Goto, T. (1986). Chemiluminescence probe with Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]-pyrazin-3-one, for estimating the ability of human granulocytes to generate O₂^–. Anal. Biochem. 159, 363–369. doi: 10.1016/0003-2697(86)90354-4

PubMed Abstract | CrossRef Full Text | Google Scholar

Panda, S. K., Baluska, F., and Matsumoto, H. (2009). Aluminum stress signaling in plants. Plant Signal. Behav. 4, 592–597. doi: 10.4161/psb.4.7.8903

PubMed Abstract | CrossRef Full Text | Google Scholar

Pinton, R., Cakmak, I., and Marschner, H. (1994). Zinc deficiency enhanced NAD(P)H-dependent superoxide radical production in plasma membrane vesicles isolated from roots of bean plants. J. Exp. Bot. 45, 45–50. doi: 10.1093/jxb/45.1.45

CrossRef Full Text | Google Scholar

Poschenrieder, C., Gunséa, B., Corralesa, I., and Barcelóa, J. (2008). A glance into aluminum toxicity and resistance in plants. Sci. Total Environ. 400, 356–368. doi: 10.1016/j.scitotenv.2008.06.003

PubMed Abstract | CrossRef Full Text | Google Scholar

Rengel, Z., and Zhang, W. H. (2003). Role of dynamics of intracellular calcium in aluminum-toxicity syndrome. New Phytol. 159, 295–314. doi: 10.1046/j.1469-8137.2003.00821.x

CrossRef Full Text | Google Scholar

Santa-Maria, G. E., and Cogliatti, D. H. (1988). Bidirectional Zn-fluxes and compartmentation in wheat seedling roots. J. Plant Physiol. 132, 312–315. doi: 10.1016/S0176-1617(88)80112-3

CrossRef Full Text | Google Scholar

Sanzonowicz, C., Smyth, T. J., and Israel, D. W. (1998). Calcium alleviation of hydrogen and aluminum inhibition of soybean root extension from limed soil into acid subsurface solutions. J. Plant Nutr. 21, 785–804. doi: 10.1080/01904169809365442

CrossRef Full Text | Google Scholar

Takahashi, K., Isobe, M., Knight, M. R., Trewavas, A. J., and Muto, S. (1997). Hypoosmotic shock induces increases in cytosolic Ca²⁺ in tobacco suspension-culture cells. Plant Physiol. 113, 587–594.

PubMed Abstract | Google Scholar

Yamamoto, Y., Kobayashi, Y., Devi, S. R., Rikiishi, S., and Matsumoto, H. (2002). Aluminum toxicity is associated with mitochondrial dysfunction and the production of reactive oxygen species in plant cells. Plant Physiol. 128, 63–72. doi: 10.1104/pp.010417

PubMed Abstract | CrossRef Full Text | Google Scholar

Yoshioka, H., Bouteau, F., and Kawano, T. (2008). Discovery of oxidative burst in the field of plant immunity: looking back at the early pioneering works and towards the future development. Plant Signal. Behav. 3, 153–155. doi: 10.4161/psb.3.3.5537

PubMed Abstract | CrossRef Full Text | Google Scholar