Ozone-Induced Cell Death Mediated with Oxidative and Calcium Signaling Pathways in Tobacco Bel-W3 and Bel-B Cell Suspension Cultures

Abstract

Ozone (O 3 )-induced cell death in two suspension-cultured cell lines of tobacco (Nicotiana tabacum L.) derived from Bel-W3 (hyper-sensitive to O 3 ) and Bel-B (highly tolerant to O 3 ) varieties were studied. By exposing the newly prepared cell lines to the pulse of ozonized air, we could reproduce the conditions demonstrating the difference in O 3 sensitivity as observed in their original plants, depending on the exposure time. Since O 3 -induced acute cell death was observed in the dark, the requirement for photochemical reactions could be eliminated. Addition of several ROS scavengers and chelators inhibited the cell death induced by O 3 , indicating that singlet oxygen ( 1 O 2 ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical and redox-active metals such as Fe 2+ play central roles in O 3 -induced acute damages to the cells. As expected, we observed the generation of 1 O 2 and H 2 O 2 in the O 3 -treated cells using chemiluminescent probes. On the other hand, an NADPH oxidase inhibitor, superoxide dismutase (SOD), and some SOD mimics showed no inhibitory effect. Thiols added as antioxidants unexpectedly behaved as prooxidants drastically enhancing the O 3 -induced cell death. It is noteworthy that some ROS scavengers effectively rescued the cells from dying even treated after the pulse of O 3 exposure, confirming the post-ozone progress of ROS-dependent cell death mechanism. Since one of the key differences between Bel-B and Bel-W3 was suggested to be the capacity for ROS detoxification by catalase, the endogenous catalase activities were compared in vivo in two cell lines. As expected, catalase activity in Bel-B cells was ca. 7-fold greater than that in Bel-W3 cells. Interestingly, Ca 2+ chelators added prior to (not after) the pulse of O 3 effectively inhibited the induction of cell death. In addition, increases in cytosolic Ca 2+ concentration sensitive to Ca 2+ chelators, ion channel blockers, and ROS scavengers were observed in the transgenic Bel-W3 cells expressing aequorin, suggesting the action of Ca 2+ as a secondary messenger initiating the oxidative cell death. The O 3 -induced calcium response in Bel-W3 cells was much greater than Bel-B cells. Based on the results, possible pathways for O 3 -dependent generation of the lethal level of ROS and corresponding signaling mechanism for induction of cell death were discussed.

Increased Anion Channel Activity Is an Unavoidable Event in Ozone-Induced Programmed Cell Death

“…However, O 3 also induced cell shrinkage, another hallmark of the PCD process in both plant and animal cells [35], [59]. These data fulfill the widely accepted criteria for PCD and confirm that our model responds to acute O 3 exposure in the same way as previously described for other plants [3], [4], [5], [10].…”

“…Moreover, addition of Ca 2+ chelators or Ca 2+ channel blockers resulted in a significant inhibition of an O 3 -induced [Ca 2+ ] cyt increase and subsequent cell death suggesting that the uptake of extracellular Ca 2+ via the activation of PM Ca 2+ channels is required for the induction of active cell death [4], [10]. A change in [Ca 2+ ] cyt was detected in Arabidopis cells expressing aequorin in response to O 3 , as shown in Figure 3A.…”

Prevention of Copper-Induced Calcium Influx and Cell Death by Prion-Derived Peptide in Suspension-Cultured Tobacco Cells

“…Evans blue was added to the cell suspension culture, following 10 min of calcium measurements after Cu application. Cells were further incubated for 1 h in order to fully develope and detect the cell death as described by Kadono et al (2006). After terminating the staining process by washing, stained cells were counted under microscopes.…”

“…Induction of cell death by oxidative stresses accompanying the generation of reactive oxygen species (ROS) is often mediated by early signaling events such as calcium infl ux via ROS-mediated activation of calcium channels on the plasma membrane (Kadono et al, 2006). Our previous studies have revealed that various phytotoxic metals such as Al and 15 rare earth elements induce an acute generation of ROS, such as superoxide anion radicals, by stimulating the plant NADPH oxidase activity (Kawano et al, 2001(Kawano et al, , 2002(Kawano et al, , 2003 and, as a consequence, ROS stimulates the opening of ROS-responsive calcium channels on the surface of plant cells (Kawano et al, 2003(Kawano et al, , 2004.…”

Mitigation of copper toxicity by DNA oligomers in green paramecia

“…[1][2][3] One of key signaling events contributing to crosstalk in plant responses to abiotic stress would be transient increase in cytosolic free calcium ion concentration stimulated by generation of ROS through ROS-dependent activation of calcium channels on the vacuolar and/or plasma membranes. 1,4 Impact of various ions of transition metals, chiefly of copper, on oxidative and calcium signal transductions leading to cell death in plant cells have been documented to date. Among such metals, copper is known to be a phytotoxic metal which reportedly induces a series of biological and chemical reactions in plant cells including the burst of reactive oxygen species production and stimulation of calcium channel opening allowing a transient increase in cytosolic calcium concentrations.…”