TY - JOUR
AU - Kawano, Tomonori
AB - Abstract The impact of copper ions on the oxidative and calcium signal transductions, leading to cell death in plant cells, have been documented. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species and the stimulation of calcium channel opening allowing a transient increase in cytosolic calcium concentrations. These early events, completed within a few minutes after the contact with copper, are known to trigger the development of cell death. The effects of DNA fragments with copper-binding motifs as novel plant cell-protecting agents were assessed using cell suspension cultures of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. The addition of GC-rich double-stranded DNA fragments, prior to the addition of copper ions, effectively blocked both the copper-induced calcium influx and cell death. In addition, the DNA–Cu complex examined was shown to possess superoxide-scavenging catalytic activity, suggesting that DNA-mediated protection of the cells from copper toxicity is due to the removal of superoxide. Lastly, a possible mechanism of DNA–Cu interaction and future applications of these DNA fragments in the protection of plant roots from metal toxicity or in aid of phyto-remediation processes are discussed. Aequorin, cell death, copper phytotoxicity, metal toxicity, nucleozyme, ROS, Z-DNA. Introduction In most plants, exposure to elevated concentrations of heavy metals results in growth inhibition and severe damage although a certain amount of metal ions such as of iron, cobalt, copper, manganese, molybdenum, and zinc are required by all living organisms (Dietz et al., 1999). Visible symptoms of copper toxicity, such as chlorosis reflecting the loss of chlorophylls and necrotic lesions reflecting localized cell death, might largely be due to oxidative stress accompanying the direct and indirect actions of copper ions. Earlier work has suggested that treatment of living plants and tissues with excess copper (up to the sub-millimolar range) coincide with the production of, molecular responses to, and cellular damage by, reactive oxygen species (ROS) such as superoxide anion radicals ( O2. ), hydrogen peroxide (H2O2), and hydroxyl radicals (HO•), possibly by direct electron transfer involving metal cations, the formation of catalytic complexes with natural chelating agents such as small peptides, or as a consequence of metal-mediated inhibition of metabolic reactions (Dietz et al., 1999; Drążkiewicz et al., 2004; Tewari et al., 2006; Bona et al., 2007; Gonzalez et al., 2010; Yokawa et al., 2011a). It is well known that homeostasis in the levels of ROS and cross-talk among ROS and other signalling events are crucial to plant responses and to the adaptation to abiotic environmental factors that threaten the plants (Lin et al., 2006a; Kadono et al., 2010; Kunihiro et al., 2011; Yokawa et al., 2011b; Tran et al., 2013a, b). One of the key signalling events contributing to the above cross-talk would be a transient increase in the cytosolic free calcium ion concentration ([Ca2+]c). The induction of cell death by oxidative stresses that accompany the generation of ROS often involves the preceding influx of Ca2+ into the cytosolic space via the ROS-dependent activation of calcium channels on the vacuolar and/or plasma membranes (Lin et al., 2006a; Kadono et al., 2006, 2010). Our previous studies have revealed that various phytotoxic metal ions such as Al3+ and of lanthanides (Ln3+) induce an acute generation of ROS such as O2. by stimulating the activity (Kawano et al., 2001, 2002, 2003) and expression (Kunihiro et al., 2011) of plant NADPH oxidase. As a consequence, metal-dependently produced ROS stimulates the opening of ROS-responsive calcium channels in plant cells (Kawano et al., 2003, 2004; Lin et al., 2005). Conversely, the redox-responsive calcium channels could be blocked when Al3+ or Ln3+ (such as La3+, Nd3+, Eu3+, Sm3+, Gd3+ or Tb3+) is present at an excess level (Kawano et al., 2003; Lin et al., 2006b). Copper is also known to be a phytotoxic metal which induces both the oxidative burst and an increase in [Ca2+]c which is eventually followed by cell death in both aquatic plants (Gonzalez et al., 2010; Min et al., 2013) and terrestrial plant cells such as cultured tobacco cells (Inoue et al., 2005; Kagenishi et al., 2009). Interestingly, the possible mechanism of acute copper action may differ from the above metals which induce O2. generation via enzyme activation. According to earlier work (Chiou, 1983), copper ions damage the DNA molecules through the formation of HO• after reacting with H2O2. It has been proposed that reduction by H2O2 of Cu(II) to Cu(I) is followed by a further reaction between Cu(I) and H2O2, finally releasing the most violent ROS, HO• as below.  Cu(II)+H2O2−−−>Cu(I)+HO2+H+  Cu(I)+H2O2−−−>Cu(II)+OH–+HO• Note that O2. is readily derived from HO2 depending on pH, and, therefore, copper ions could be considered as efficient inducers of O2. where H2O2 is supplied by living cells. Furthermore, free copper ions and even the Cu derived from Cu/Zn-superoxide dismutase (SOD) catalyse the Fenton-type reactions for the production of HO•, if O2. , the source of H2O2, is provided in the presence of reducing sugars such as glucose (Kaneto et al., 1994). In fact, slow but continuous production of O2. reportedly occurs in dilute solutions of glucose and other reducing sugars (Kaneko et al., 2006), suggesting that the addition of copper ions to the mixture of similar organic chemicals or biomolecules results in the enhanced production of violent ROS members. Previously, monitoring of the Fenton-type reactions leading to the formation of HO• was performed by electron spin resonance spectroscopy and this reaction was shown to proceed in tobacco BY-2 cell suspension cultures after the addition of CuSO4 (Kawano and Muto, 2000). Due to the hyper-reactivity of HO• even against water molecules, HO• hardly migrates even a short distance in the aqueous phase, thus only the portion of HO• generated at the site adjacent to the DNA causes or enhances the degradation of, or damage to, DNA. According to earlier work, Cu2+ was shown to bind and to damage DNA (Chiou, 1983; Yokawa et al., 2011c), especially through affinity to the guanine (G) and cytosine (C) bases at physiological pH, eventually perturbing the neighbouring base pairs of adenine (A) and thymine (T) and disrupting the double-helical structure of DNA (Tajmir-Riahi et al., 1988). In addition, among three of the known biologically active forms of DNA, Z-DNA showing a non-Watson–Crick type structure (Bansal, 2003), possesses the highest affinity to the binding of Cu2+, especially at G bases (Geierstanger et al., 1991). Accordingly, the complex between Cu and Z-DNA domains readily results in damage to the DNA molecules (Geierstanger et al., 1991). The effect of a copper-binding short peptide derived from human prion protein on the protection of tobacco BY-2 cells from copper toxicity was investigated previously (Kagenishi et al., 2009). An excess of peptides (c. 5–10 times higher concentrations compared with that of copper ions) effectively blocked both Ca2+ influx and cell death induced by copper, possibly due to the removal of copper. By analogy, it was expected that Cu-binding DNA also prevents the toxicity of copper in plant cells. Since the major targets of copper-dependent damage to DNA are the AT-base pairs, the proposed model DNA should be rich in GC-base pairs. The use of a CGCGCG DNA hexamer, one of the minimal GC-rich Z-DNAs (Ban et al., 1996), is proposed here as one of the novel anti-copper molecules active in plant cell protection against the toxicity of copper. In the present study, we examined the effect of copper-binding DNA oligomers against the toxic actions of copper we examined by focusing on the Cu-responsive calcium signalling and cell death responses in suspension-cultured tobacco BY-2 cells expressing the Ca2+-sensitive luminescent protein aequorin. In addition, the removal of copper-dependently formed O2. by the DNA–Cu complex is discussed as the Cu-bound form of the CGCGCG DNA hexamer duplex is viewed as a novel antioxidant molecule with SOD-like catalytic activity. Materials and methods Plant material Suspension-cultured cells of tobacco (Nicotiana tabacum L. cv. Bright Yellow-2; cell line, BY-2) expressing apoaequorin exclusively in the cytosol were propagated as previously reported by Takahashi et al. (1997). Briefly, the culture was maintained in Murashige–Skoog (MS) liquid medium (pH 5.8) containing 0.2 μg ml–1 2,4-dichlorophenoxy acetic acid at 23 °C with shaking on a gyratory shaker in darkness and sub-cultured weekly with 3% (v/v) inoculum. Confluent cultures, sub-cultured at 1 week intervals, were used to inoculate the fresh MS liquid medium and were then cultured for 5 d. These 5-d-old cultures were harvested and used for the experiments. For preliminary study, a redox-sensitive cell line of tobacco (Bel-W3) was also used. Coelenterazine Coelenterazine, a luminophore required for the reconstitution of aequorin from apoaequorin (Shimomura and Johnson, 1978), was chemically synthesized by the group of Professor Isobe, Nagoya University, Nagoya, Japan (Isobe et al., 1994). DNA oligomers DNA oligomers, namely, CGCGCG, ATATAT, GGGGGG, and CCCCCC were synthesized (each 4mg) on demand at Genenet Co. Ltd. (Fukuoka, Japan). Treatments CuSO4 was first dissolved in water. The copper solution (10 μl) was added (0.03–10.0mM, final concentration) to the cells suspended in MS medium (190 μl). When required, DNA oligomers were mixed with the copper solution prior to addition to the cell suspension. Determination of cell death Copper-induced cell death in the cell-suspension culture was allowed to develop in the presence of the vital staining dye, Evans blue (0.1%, w/v). The dye was added to the cell suspension culture, 10min after the application of copper ions. The cells were further incubated for 1h to develop cell death fully as described by Kadono et al. (2006). After terminating the staining process by washing, the stained cells were observed under the microscope. For statistical analyses, four different digital images of cells under the microscope (each covering over 100 cells to be counted) were acquired and stained cells were counted. Monitoring of [Ca2+]c The changes in [Ca2+]c were monitored by Ca2+-dependent emission of blue light from aequorin as previously described by Kawano et al. (1998). The active form of aequorin was reconstituted by the addition of 1 μM coelenterazine to the apoaequorin-expressing tobacco cells, 8h prior to the measurements of [Ca2+]c. The aequorin luminescence was measured using a photometer (Luminescensor PSN AB-2200-R, Atto Corp., Tokyo, Japan). Detection of O2. For the detection of the O2. generated and/or maintained in the reaction mixture, an O2. -specific chemiluminescence probe, Cypridina luciferin analogue (CLA; 2-methyl-6-phenyl-3,7-dihydroimidazo [1,2-a] pyrazin-3-one), was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Generation of O2. was monitored by the chemiluminescence of CLA with a photometer (Luminescensor PSN AB-2200-R, Atto, Tokyo), and the yield of chemiluminescence was expressed as relative luminescence units (rlu), as previously described by Kawano et al. (1998). CLA is a specific probe for O2. (and singlet oxygen to a lesser extent), but not for other ROS members (Nakano et al., 1986). Generation of O2. Three different sources of O2. were used for assessing the catalytic activity of DNA oligomers (see cases 1, 2, and 3). For manifesting the short pulse of O2. increase, potassium superoxide (KO2) dissolved in dry DMSO was used as the standard source of O2. and injected into a potassium phosphate buffered (50mM, pH 7.0) DNA–Cu mixture in the photometric apparatus (case 1). For reproducing the continuous but slow release of O2. in the presence of copper ions and H2O2, a known path for Cu2+-mediated conversion of H2O2 to O2. via HO2 was demonstrated by injecting H2O2 into the Cu-containing reaction mixture in the photometric apparatus (case 2). For the preparation of a sono-photocatalytically activated O2. -rich medium, a titania-coated alumina fibre-equipped ultraviolet and ultrasonic wave-driven sono-photocatalytic apparatus designated as Ex-PCAW (WIPO No: WO10/032765), K2R Inc. (Kitakyushu, Japan) was used. It comprised a UV-A emitting bulb, ultrasonic wave-generating devices, sheets of titania-coated fibres, a pumping system, and dissolved O2 supply systems. With this apparatus, O2. concentration in the reaction mixture could be maintained at a desired range between 0.05 µM and 1 µM. DNA-mediated O2. removal was assessed under different O2. concentrations (case 3). Tetrazolium-based SOD assay kit In addition to the three cases of O2. removal by the DNA–Cu complex, a commercial SOD assay kit employing xanthine oxidase (XO) and water-soluble tetrazolium salt, WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulpho-phenyl)-2H-tetrazolium, monosodium salt), was obtained from Dojindo Laboratories (Kumamoto, Japan). The XO reaction and formation of formazan were performed as indicated by the vendor’s instruction. Spectroscopic assessment of the SOD-like action (a decrease in absorption corresponding to formazan) by the DNA–Cu complex was monitored with a spectrophotometer (Shimadzu UV-1800, Kyoto, Japan). Results and discussion Toxicity of cupric and ferric ions: a preliminary study It is well known that copper-dependent oxidative stress is not prevented by common reducing agents such as ascorbate (AsA), glutathione, and other thiols, since these reducing agents contribute to reproduce the Fenton-active catalyst Cu+ from Cu2+ (Yokawa et al., 2011c; Kawano et al., 2013). To confirm the above notions in tobacco cells, the impact of copper and iron ions was tested in combination with AsA which is used as the model reducing agent (Fig. 1). An excess of AsA added to the cell suspensions slightly increased the level of cell death (Fig. 1A). This increase was shown to be mediated by two of the known Fenton catalysts endogenously present in plant cells as the chelators of Cu+ and Fe2+ effectively blocked the cell-killing action of AsA (Fig. 1A). Therefore, one might predict that the toxicity of ferrous ions is higher than that of ferric ions and, similarly, a higher toxicity for cuprous ions than cupric ions is expected based on the performance of the above ions in Fenton-type reactions converting H2O2 to HO•. Interestingly, additions of ferric and cupric ions were shown to be much more highly toxic compared with the actions of ferrous and cuprous ions (Fig. 1B, C). Therefore, the overall roles must be considered of ferric/ferrous and cupric/cuprous redox cycles involving a series of redox interactions with various molecules covering both the oxidants and reductants, finally determining the status of redox stress and cellular damage. It is noteworthy that the severely toxic action of ferrous ions can be partially blocked by an excess of AsA, while the toxicity of cupric ions has a tendency to be highly enhanced in the presence of AsA (Fig. 1C). Hereafter, the focus was on the toxicity of cupric ions only, since cupric ions were shown to be more toxic than cuprous ions and more difficult to detoxify than ferric ions by the use of reducing agents. Fig. 1. View largeDownload slide Preliminary study assessing the toxic impacts of cupric, cuprous, ferric, and ferrous ions in a redox-sensitive tobacco cell line. (A) Ascorbate toxicity involving the ions of copper and iron. (B) Effects of ferric and ferrous ions on the induction of cell death. (C) Effects of cupric and cuprous ions on the induction of cell death. To elucidate the involvements of redox cycling in Fe/Cu toxicity, 1mM ascorbic acid (AsA) was used as both a reducing agent and an antioxidant. 2mM 2′,2-bipyridyl (Bipy) and o-phenanthroline (o-phen) were used as Fe and Cu chelators, respectively. Fig. 1. View largeDownload slide Preliminary study assessing the toxic impacts of cupric, cuprous, ferric, and ferrous ions in a redox-sensitive tobacco cell line. (A) Ascorbate toxicity involving the ions of copper and iron. (B) Effects of ferric and ferrous ions on the induction of cell death. (C) Effects of cupric and cuprous ions on the induction of cell death. To elucidate the involvements of redox cycling in Fe/Cu toxicity, 1mM ascorbic acid (AsA) was used as both a reducing agent and an antioxidant. 2mM 2′,2-bipyridyl (Bipy) and o-phenanthroline (o-phen) were used as Fe and Cu chelators, respectively. Prevention of Cu-induced cell death by DNA oligomers As previously demonstrated, the addition of CuSO4 resulted in acute cell death in tobacco BY-2 cells. Following the early events represented by ROS production and the ROS-mediated increase in [Ca2+]c induced by Cu treatment, which are probably completed within 5–10min following the application of Cu, the induction of cell death occurred during the additional 1h of static incubation. The dead cells, stained by Evans blue, were photographed under a microscope and counted in order to assess cell viability (Fig. 2A). Data suggested that copper induces cell death in a dose-dependent manner (Fig. 2B). Fig. 2. View largeDownload slide Visualization and quantification of copper-induced cell death in tobacco BY-2 cells. (A) Vital staining revealed the cupper-induced cell death and its inhibition by CG-repeated double-stranded DNA hexamer (CGCGCG). (B) Effect of copper concentration on the induction of cell death. Error bars represent S.D. (n=4). (This figure is available in colour at JXB online.) Fig. 2. View largeDownload slide Visualization and quantification of copper-induced cell death in tobacco BY-2 cells. (A) Vital staining revealed the cupper-induced cell death and its inhibition by CG-repeated double-stranded DNA hexamer (CGCGCG). (B) Effect of copper concentration on the induction of cell death. Error bars represent S.D. (n=4). (This figure is available in colour at JXB online.) Preliminary data (Fig. 1) strongly suggested that the use of known reducing agents, often considered to be effective in preventing the oxidative burst, is not a wise choice to prevent copper-mediated toxicity in tobacco cells. In turn, the likely approach, by analogy to previous work with a Cu-binding peptide (Kagenishi et al., 2009, 2011) is the removal or trapping of redox active copper ions by macromolecules. DNA hexamers harbouring a Cu-binding motif found in Z-DNA was chosen as a model molecule here. As expected, the addition to the cell suspension culture of the model GC-rich DNA hexamer with minimal Z-DNA structure, CGCGCG, prior to the addition of copper, effectively blocked the induction of cell death by copper (Fig. 3). On the other hand, the addition of a model AT-rich DNA hexamer (ATATAT) showed no protective action against the toxicity of copper in tobacco BY-2 cells (Fig. 3B). Both of the model DNA samples were designed to form the duplex structure (thus double-stranded). The above data strongly suggested that detoxification of copper ions requires the presence of GC base pairs in the double-stranded DNA. Fig. 3. View largeDownload slide Protection of tobacco BY-2 cells from copper-induced cell death by Z-DNA. (A) Model GC-rich and AT-rich double-stranded DNA oligomers used. (B) Effect of AT-repeated (ATATAT) and CG-repeated (CGCGCG) double-stranded DNA hexamers on cell death induction by 0.3mM CuSO4. Error bars represent S.D. (n=4). (This figure is available in colour at JXB online.) Fig. 3. View largeDownload slide Protection of tobacco BY-2 cells from copper-induced cell death by Z-DNA. (A) Model GC-rich and AT-rich double-stranded DNA oligomers used. (B) Effect of AT-repeated (ATATAT) and CG-repeated (CGCGCG) double-stranded DNA hexamers on cell death induction by 0.3mM CuSO4. Error bars represent S.D. (n=4). (This figure is available in colour at JXB online.) To test the hypothesis that GC pairs in the double-stranded DNA are essential for the protection of cells from copper-induced cell death, the effects were compared of four different combinations of DNA hexamers consisting of either C, G, or both (Fig. 4). Cell death induction by 0.3mM CuSO4 was shown to be inhibited by two different treatments with double-stranded GC-rich DNAs, namely a CGCGCG sequence and a 1:1 mixture of a CCCCCC sequence and a GGGGGG sequence. On the other hand, the addition of a CCCCCC or a GGGGGG sequence alone showed no inhibitory action against copper-induced toxicity, confirming that single-stranded DNA fragments are inactive in the detoxification of copper. Fig. 4. View largeDownload slide Structural requirement of GC-rich DNA hexamers for inhibition of copper toxicity in tobacco BY-2 cells. Concentration of DNA was fixed at 0.3mM, regardless of being single- and double-stranded forms. (A) Combinations of GC-rich model DNA oligomers used. (B) Effect of single-stranded and double-stranded GC-rich DNA hexamers on cell death induction by 0.3mM CuSO4. Error bars represent S.D. (n=4). Fig. 4. View largeDownload slide Structural requirement of GC-rich DNA hexamers for inhibition of copper toxicity in tobacco BY-2 cells. Concentration of DNA was fixed at 0.3mM, regardless of being single- and double-stranded forms. (A) Combinations of GC-rich model DNA oligomers used. (B) Effect of single-stranded and double-stranded GC-rich DNA hexamers on cell death induction by 0.3mM CuSO4. Error bars represent S.D. (n=4). Prevention of Cu-induced Ca2+influx by DNA oligomers Aequorin was reconstituted from apo-protein expressed in the cytosol of tobacco BY-2 cells by adding coelenterazine to the culture 8h prior to the experiments. The addition of CuSO4 (from 0.03mM to 10mM) resulted in a rapid and transient increase in aequorin luminescence reflecting the acute increase in [Ca2+]c in a dose-dependent manner (Fig. 5A, B). Fig. 5. View largeDownload slide Cu-induced [Ca2+]c elevation and its inhibition by the addition of the Cu-binding DNA hexamer in tobacco BY-2 cells expressing aequorin. (A) Typical traces of the Cu-induced increase in aequorin luminescence. (B) Effect of CuSO4 concentration ranging from 0.03mM to 10mM on the induction of [Ca2+]c increase. Effect of the CGCGCG DNA hexamer was examined at two different times, namely, 5min prior to and at the same time as the addition of 0.3mM CuSO4. Error bars represent S.D. (n=4). Fig. 5. View largeDownload slide Cu-induced [Ca2+]c elevation and its inhibition by the addition of the Cu-binding DNA hexamer in tobacco BY-2 cells expressing aequorin. (A) Typical traces of the Cu-induced increase in aequorin luminescence. (B) Effect of CuSO4 concentration ranging from 0.03mM to 10mM on the induction of [Ca2+]c increase. Effect of the CGCGCG DNA hexamer was examined at two different times, namely, 5min prior to and at the same time as the addition of 0.3mM CuSO4. Error bars represent S.D. (n=4). The Cu (0.3mM)-induced increase in [Ca2+]c was significantly lowered by a 0.3mM GCGCGC sequence (Fig. 5). On the other hand, ATATAT did not prevent Cu-induced Ca2+ influx (Fig. 6). These data form the first demonstration that an exogenously applied non-coding Z-DNA fragment can significantly modify a signalling event in the plant system. Fig. 6. View largeDownload slide Comparison of AT-repeated and CG-repeated DNA hexamers in the inhibition of the copper-induced increase in aequorin luminescence in tobacco BY-2 cells suspension culture. (A) Typical traces of a copper-induced increase in aequorin luminescence and its inhibition by DNA oligomers. (1) Water control, (2) addition of CuSO4 only (0.3mM, final concentration), (3) mixture of CGCGCG (0.3mM, final concentration) and CuSO4 (0.3mM, final concentration), (4) mixture of ATATAT (0.3mM, final concentration) and CuSO4 (0.3mM, final concentration). (B) Quantification of the action of two model DNA oligomers (CGCGCG, ATATAT) against the Cu-induced Ca2+ influx in tobacco BY-2 cells. Concentrations of both DNA and copper were identical with (A). Error bars represent S.D. (n=4). Fig. 6. View largeDownload slide Comparison of AT-repeated and CG-repeated DNA hexamers in the inhibition of the copper-induced increase in aequorin luminescence in tobacco BY-2 cells suspension culture. (A) Typical traces of a copper-induced increase in aequorin luminescence and its inhibition by DNA oligomers. (1) Water control, (2) addition of CuSO4 only (0.3mM, final concentration), (3) mixture of CGCGCG (0.3mM, final concentration) and CuSO4 (0.3mM, final concentration), (4) mixture of ATATAT (0.3mM, final concentration) and CuSO4 (0.3mM, final concentration). (B) Quantification of the action of two model DNA oligomers (CGCGCG, ATATAT) against the Cu-induced Ca2+ influx in tobacco BY-2 cells. Concentrations of both DNA and copper were identical with (A). Error bars represent S.D. (n=4). Removal of O2. by the DNA–Cu complex Up to now, facts have been accumulated that a minimal Z-DNA sequence prevents copper toxicity in tobacco cells. However, the mechanism is still obscure since minor doses, lower than that of the applied copper, also showed a significant effect in the protection of cells. For instance, Cu-binding peptides reportedly failed to prevent copper-induced cell death and stimulation of calcium influx when the ratio between peptides and copper ions was lower than 3:1 (Kagenishi et al., 2009). Thus, it is natural to speculate that there is alternative mechanism in addition to merely trapping the toxic ions. Our previous study revealed that the acute increase in [Ca2+]c is due to induced Ca2+ influx via stimulation of the Ca2+-permeable ion channels by Cu-dependently produced ROS, since both ROS scavengers and Ca2+ channel blockers effectively lowered the level of [Ca2+]c increase induced by copper ions (Kagenishi et al., 2009). It is, therefore, highly likely that the removal of ROS could be one of the key steps required for the prevention of copper toxicity leading to programmed cell death via the stimulation of the calcium signalling mechanism. Lastly, the possibility was tested that complexes formed between the GC-rich double-stranded DNA hexamers are catalytically capable of removing toxic ROS generated in the presence of copper ions through the Cu+/Cu2+ redox cycle. There was a need to find out if the mixture of Cu2+ and CGCGCG DNA hexamer removes or degrades the O2. . Three different sources of O2. were used here for assessing the catalytic activity of DNA oligomers. Case 1: For manifesting the short pulse of O2. increase, KO2 dissolved in dry DMSO was injected into the reaction mixture containing the CGCGCG DNA hexamer and CuSO4 (Fig. 7A). Following injection of 5 µM KO2 to the copper solution (10 µM CuSO4), a spike of CLA chemiluminescence reflecting the release of O2. was observed. In the presence of 1:1 v/v copper and CGCGCG DNA hexamer (each 10 µM), the KO2-induced increase in CLA chemiluminescence was shown to be lowered. In the absence of metal, DNA (CGCGCG) added alone showed no scavenging action at any concentration tested (between 10 and 250 µM) while copper alone only started to decay O2. at higher concentrations (100–250 µM). Since the DNA–Cu complex showed detectable activity at lower concentrations, the catalytic O2. -removing action of Cu could be enhanced in the DNA-bound form. Fig. 7. View largeDownload slide Removal of O2. by Cu-loaded CGCGCG DNA oligomers. (A) CLA-chemiluminescence reflecting the release of O2. after injection of KO2 into the reaction mixture containing copper and CGCGCG DNA oligomers. Inset, kinetic analysis (Lineweaver–Burk plot) of the O2. -removing activity in the DNA–Cu complex. (B) H2O2-dependent O2. production in copper solution. 1mM H2O2 was injected into the reaction mixture (pH 7.0, 50mM K-phosphate) containing 25 µM CuSO4 and/or 25 µM CGCGCG DNA hexamer. Data shown represent the mean of four replications. (C) Effect of the DNA–Cu complex concentration on the removal of sono-photocatalytically produced O2. . (D) Kinetic analysis of the O2. -removing activity in a DNA–Cu complex in the sono-photochemical apparatus. (E) Inhibition of XO-dependent WST-1 formazan formation by DNA–Cu complex. Difference in absorption between the control and three distinct treatments (either or both of CuSO4 and CGCGCG DNA, each 100 µM) are compared. (This figure is available in colour at JXB online.) Fig. 7. View largeDownload slide Removal of O2. by Cu-loaded CGCGCG DNA oligomers. (A) CLA-chemiluminescence reflecting the release of O2. after injection of KO2 into the reaction mixture containing copper and CGCGCG DNA oligomers. Inset, kinetic analysis (Lineweaver–Burk plot) of the O2. -removing activity in the DNA–Cu complex. (B) H2O2-dependent O2. production in copper solution. 1mM H2O2 was injected into the reaction mixture (pH 7.0, 50mM K-phosphate) containing 25 µM CuSO4 and/or 25 µM CGCGCG DNA hexamer. Data shown represent the mean of four replications. (C) Effect of the DNA–Cu complex concentration on the removal of sono-photocatalytically produced O2. . (D) Kinetic analysis of the O2. -removing activity in a DNA–Cu complex in the sono-photochemical apparatus. (E) Inhibition of XO-dependent WST-1 formazan formation by DNA–Cu complex. Difference in absorption between the control and three distinct treatments (either or both of CuSO4 and CGCGCG DNA, each 100 µM) are compared. (This figure is available in colour at JXB online.) Kinetic analysis with the Lineweaver–Burk (double-reciprocal) plot further evaluated the Vmax for the O2. -degrading action of the DNA–Cu complex, performed in the presence of various concentrations of KO2 (used as model substrate), to be 1.675 nmol mg–1 DNA s–1 (Fig. 7A, inset). Case 2: For reproducing the continuous but slow release of O2. from H2O2 with the aid of copper ions, a known path for Cu2+-mediated conversion of H2O2 to O2. via HO2, was demonstrated by injecting H2O2 into the Cu-containing reaction mixture in the photometric apparatus (Fig. 7B). The conversion of H2O2 into O2. is catalyzed by free copper and thus, no O2. is produced in the absence of copper (CGCGCG alone), and copper-dependently formed O2. is probably removed by the DNA–Cu complex. Again, the data are supporting that copper-mediated O2. production was inhibited by the CGCGCG DNA hexamer. Case 3: For the ease of controlled kinetic analysis, an O2. -rich medium was prepared via a sono-photocatalytic reaction using the titania-coated alumina fibre-equipped, ultra violet-irradiated, and ultrasonic wave-driven sono-photocatalytic apparatus as described in the Materials and methods. With this apparatus, O2. concentration in the reaction mixture could be maintained at a desired range between 0.05 and 1 µM. Activity of the DNA–Cu complex for the removal of O2. was assessed under controlled O2. concentrations. When the concentration of sono-photocatalytically generated O2. was controlled to be at c. 0.5 µM, the yield of CLA chemiluminescence reflecting the presence of O2. was lowered by the DNA (CGCGCG)–Cu complex in a dose-dependent manner (Fig. 7C). Contrary to case 2, O2. is continuously provided by the sono-photocatalytic system and, thus, in the absence of any element, a high level of chemiluminescence in the buffered water was continuously observed and this level is expressed as 100% of relative CLA chemiluminescence yield (Fig. 7C). The data with CGCGCG alone and copper alone clearly suggested that removal of O2. (lowering of CLA chemiluminescence) requires the presence of both CGCGCG and copper at the same time. The range of DNA–Cu linearly lowering the level of sono-photocatalytically generated O2. were shown to be equivalent to the range of Cu/Zn-SOD concentrations between c. 200 and 2000 units ml–1 (data not shown). By modulating the levels of sono-photocatalytically released O2. , the kinetic analysis could be performed with the Lineweaver–Burk plot to determine graphically the Vmax for the O2. -degrading action of the DNA–Cu complex to be 1.155 nmol mg–1 DNA s–1 (Fig. 7D). Case 4: Combination of tetrazolium salts and xanthine oxidase (XO) is often employed for visualizing the removal of O2. by SOD (Camejo et al., 2012). To clarify that CGCGCG–Cu complex, but not CGCGCG DNA alone nor copper alone, possesses the SOD-like action for the removal of O2. , a test has also been conducted using a commercial SOD assay kit using XO and the water-soluble tetrazolium salt known as WST-1 which produces a water-soluble formazan dye upon reduction with O2. . An increase in WST-1 formazan, reflecting the XO-dependent production of O2. , was shown to be inhibited in the presence of the DNA–Cu complex but not in the presence of DNA alone or copper alone (Fig. 7E). Mechanism of DNA action To our knowledge, the oxidative toxicity by copper ions is highly complex involving many interacting agents such as ROS and reducing chemicals, and concomitant signalling events activating the programmed cell death mechanism (Kawano et al., 2013). Here, the likely modes and putative roles of exogenously applied DNA oligomers with Cu-binding motifs are summarized and illustrated (Fig. 8). The primary role of such DNA, chiefly the Z-DNA minimal sequences, is the trapping of toxic copper ions. This may be the basal mechanism for protecting the cells from the toxicity of copper ions, similar to the reported actions of Cu-binding small peptides. Furthermore, upon binding to Cu, the model DNA hexamer (CGCGCG) showed catalytic activity for the removal of O2. . Taken together, by interfering with the early events in Cu-mediated redox-chain reactions, Cu-binding DNA might prevent the biochemical and signalling events afterwards, finally preventing the onset of cell death. Fig. 8. View largeDownload slide A model mechanism for DNA oligomer-mediated protection of tobacco cells from copper toxicity. (This figure is available in colour at JXB online.) Fig. 8. View largeDownload slide A model mechanism for DNA oligomer-mediated protection of tobacco cells from copper toxicity. (This figure is available in colour at JXB online.) The above studies are indicative of the hidden fact that the Cu-binding GC-rich DNA fragments may exhibit a novel function upon loading of Cu and such function may be related to the removal of ROS produced by free Cu, thus applicable for the protection of plant cells from copper toxicity. As the Vmax values obtained from independent assays fell in a similar range (Fig. 7A, D), it can be concluded that GC-rich double-stranded DNA has solid activity to form some catalytic complex for the removal of O2. . This molecule could be a good model for designing some novel inorganic element-centred artificial nucleozymes. The likely models of Cu-complex-mediated degradation of O2. should refer to the mode of action of Cu/Zn-SOD. However, further examination of this hypothesis is required in future experiments and this process may allow us to design better genetic or biochemical agents for the protection of plant cells from metal toxicity. Since the agent tested here is a DNA fragment, genetic modification of plants for the over-production and excretion of DNA fragments is one of the possible choices to minimize the phytotoxicities of various metals in future environments. 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TI - Protection of tobacco cells from oxidative copper toxicity by catalytically active metal-binding DNA oligomers
JF - Journal of Experimental Botany
DO - 10.1093/jxb/eru028
DA - 2014-03-21
UR - https://www.deepdyve.com/lp/oxford-university-press/protection-of-tobacco-cells-from-oxidative-copper-toxicity-by-0WNAJtb2es
SP - 1391
EP - 1402
VL - 65
IS - 5
DP - DeepDyve
ER -