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Life in an oxygen-rich atmosphere has to deal with the danger of oxidative stress. Reactive oxygen species (ROS), such as superoxide (), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH), are produced during normal cell metabolism but their production is drastically enhanced when plants are exposed to natural abiotic stresses such as high light, low temperatures and drought, and to biotic stresses such as attack by pathogens or wounding (e.g. Scandalios, 2002). Environmental pollutants such as heavy metal ions, for example cadmium (Cd2+), are also known to induce oxidative stress (Schützendübel & Polle, 2002). Although ROS are considered to be damaging molecules, it is recognized that they play a major role in defense against pathogens, cellular signaling pathways and regulation of gene expression in a wide range of organisms, including plants (e.g. Apel & Hirt, 2004).
ROS are generated in a variety of reactions. These include the respiratory and photosynthetic electron transport chains and side reactions of enzymes such as peroxidases. In addition, specialized enzymes such as superoxide dismutases, xanthine oxidase and NADPH oxidases (NOXs) produce ROS (Halliwell & Gutteridge, 1999). ROS are not only produced in many different reactions but also in different compartments of the cell, including mitochondria, chloroplasts, peroxisomes, the cytosol and the apoplast.
Plant plasma membranes produce to the apoplastic side in response to different stimuli. This activity has been widely accepted to originate from plasma membrane-localized NOXs that reduce external O2 using cytoplasmic NADPH as the electron source, although other plasma membrane-localized or -associated enzymes may also contribute to production. Ten genes encode NOXs in Arabidopsis thaliana. These genes are termed respiratory burst oxidase homologs A to J (RbohA–J) because of their homology to the catalytic subunit gp91phox (Nox2) of the NOX complex of mammalian phagocytes (Torres & Dangl, 2005). The plant NADPH oxidases are predicted to have six transmembrane helices with two heme-binding sites, cytoplasmic binding sites for NADPH and FAD at the C-terminus and two calcium (Ca2+)-binding EF-hand motifs at the N-terminus (Torres & Dangl, 2005), a similarity with the NOX5 NADPH oxidase in mammals (Banfi et al., 2004). NOX-encoding genes are present in all plant species investigated so far, and they have distinctive expression patterns. Although the involvement of NOXs in important physiological processes has been demonstrated in plants (e.g. Foreman et al., 2003; Kwak et al., 2003; Potockýet al., 2007), their enzymatic properties have rarely been studied. It has been shown that the EF-hand motifs in plant NOXs bind 45Ca2+ (Keller et al., 1998) and that Ca2+ stimulates the NOX activity of isolated plasma membranes (Sagi & Fluhr, 2001). The activation of mammalian NOX5 by Ca2+ in a cell-free system is in the micromolar range (Banfi et al., 2004). Hence, in the plant plasma membrane micromolar Ca2+ concentrations can also be expected to lead to enhanced production.
Ca2+-binding sites can be probed by other metals such as lanthanides and cadmium. Cd2+ has been shown to block cysteine groups in enzymes, leading to their inactivation (Van Assche & Clijsters, 1990; Kabała et al., 2008), and to bind as a competitive inhibitor to Ca2+-binding motifs such as radish calmodulin (Rivetta et al., 1997) and the water-splitting complex of photosystem II (Faller et al., 2005). Cd2+ is also known to block proton transport in gp91phox (Henderson et al., 1988) and in complex III of the mitochondrial electron transport chain (Link & von Jagow, 1995). In addition, it has been reported that Cd2+ leads to increased production at complex III (Wang et al., 2004). It has been known for more than 30 yr that mitochondrial electron transport can lead to /H2O2 formation via the reaction of semiquinones with oxygen (e.g. Boveris & Chance, 1973; Cape et al., 2007; Zhang et al., 2007).
It has also been proposed that the plasma membrane NOXs may be implicated in the Cd2+-induced ROS production after short-term exposure to the metal (Olmos et al., 2003; Garnier et al., 2006). Long-term exposure of plants to Cd2+ in micromolar concentrations has been widely studied and the symptoms (e.g. increased ROS production caused by the failure of the cellular antioxidant system, cell death and growth defects) are well characterized (Romero-Puertas et al., 2004; Rodriguez-Serrano et al., 2006). In long-term exposure of plants to Cd2+, the molecular mechanism leading to increased production at the plasma membrane seems to require signaling pathways involving kinases, Ca2+ fluxes and de novo synthesis of NOX (Romero-Puertas et al., 2004; Rodriguez-Serrano et al., 2006; Van Belleghem, 2007).
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In the present study, Cd2+-dependent ROS production at the level of the plasma membrane and mitochondrial electron transport chain was investigated in vitro and in vivo. and H2O2 production was followed after exposure of isolated plasma membranes, mitochondria or intact roots to Cd2+. production by isolated plasma membranes was inhibited by Cd2+ competitively to Ca2+ (Fig. 4) while the addition of Cd2+ stimulated and H2O2 generation by mitochondria (Figs 5, 6). Upon exposure of intact roots to Cd2+, formation was inhibited (Fig. 7a,c) while H2O2 formation increased (Fig. 7b,d). Therefore, we conclude that the Cd2+-induced generation of ROS, namely H2O2, originates from inside the root cells, mainly from mitochondrial electron transport.
In our work with isolated plasma membranes, at least part of the was produced by a plasma membrane NADPH oxidase as this activity was inhibited by low concentrations of DPI (Ki = 1.5 µM) whereas KCN, an inhibitor of peroxidases, had no effect on the activity (Figs 2, 3). Potential -producing enzymes in the plasma membrane are NOXs (Torres & Dangl, 2005) and quinone oxidoreductases (Schopfer et al., 2008). Furthermore, the activity was stimulated by Ca2+ (Table 2) and inhibited by Cd2+ in a competitive manner (Fig. 4). The inhibition by low concentrations of DPI and the antagonistic effects of Ca2+ and Cd2+ indicate that the NADPH oxidase activity investigated in the present study can be attributed to a NOX activity. Sagi & Fluhr (2001) observed a twofold increase of production in isolated tobacco and tomato plasma membranes in response to millimolar Ca2+ concentrations, while in the present study production was only slightly (up to 30%) increased by 1–5 mM Ca2+. The maximum production activity obtained in soybean plasma membranes was similar to that reported previously for tobacco plasma membranes (Sagi & Fluhr, 2001). In contrast to the data reported by Sagi & Fluhr (2001), after depletion of the samples of cations using Chelex 100, the activity could be restored to the control level even with micromolar Ca2+ concentrations (Table 2). The human EF-hand motif-containing NOX5 was previously shown to be activated at low micromolar Ca2+ concentrations (Banfi et al., 2004), in accordance with the data presented here. In the present work, Mg2+ had similar effects to Ca2+ on production. Binding of Mg2+ and manganese (Mn2+) to the EF-hand motif of 13-4-4 proteins was reported previously (Athwal & Huber, 2002). It remains to be investigated whether these cations bind to the EF-hand motifs of NADPH oxidase and further whether the effect of Mg2+ and Mn2+ is restricted to plants, as the EF-hand motifs of the human NOX5 do not bind Mg2+ (Banfi et al., 2004).
It has been demonstrated that Cd2+ increases ROS formation in mitochondria from animals (Wang et al., 2004). Here, Cd2+ and antimycin A stimulated and H2O2 production in plant mitochondria respiring on succinate (Figs 5, 6). In animal tissue mitochondria are thought to be among the major targets of Cd2+ toxicity (Martel et al., 1990). Cd2+ and zinc (Zn2+) are well-known inhibitors of electron transport in mitochondria (Skulachev et al., 1967). Cd2+ blocks electron transfer between semiquinone and cytochrome b in respiratory complex III (Miccadei & Floridi, 1993) and causes the accumulation of semiquinone radicals, leading to the formation of (Wang et al., 2004). The binding site of Cd2+ in complex III is likely to be the same as that of Zn2+, that is, blocking a protonable group which is thought to be associated with deprotonation reactions of the quinol oxidation site in complex III (Link & von Jagow, 1995; Giachini et al., 2007).
We attribute most of the production measured in vivo to NOX activity because it was inhibited by Cd2+ and this inhibition was reversed by Ca2+ both in vitro and in vivo (Figs 4, 7a,c, 8). We can exclude the possibility that peroxidases are the source of production, because we found that isolated horseradish peroxidase was insensitive to Cd2+ at the concentration used here (data not shown). It has been proposed previously that the production of •OH involved in extension growth is initiated by NAD(P)H oxidase-catalyzed formation of at the plasma membrane (Schopfer et al., 2002; Liszkay et al., 2004) and involves peroxidases, which are abundant in the cell wall (Dunand et al., 2007). In the presence of the catalytic heme in peroxidases is converted into its so-called ‘compound III’. In this state peroxidases transform H2O2 into −OH and •OH (Chen & Schopfer, 1999; Schopfer et al., 2002). Thus, in the presence of H2O2, a measure of the production of •OH (Fig. 8) reflects the amount of produced in the apoplast in vivo. The , generated at the plasma membrane, is needed for the activation of the peroxidase to compound III. seems to be necessary in more than just catalytic amounts, because in the presence of Cd2+ production (Fig. 7a,c) and •OH production (Fig. 8) are strongly inhibited while H2O2 increases with time (Fig. 7b,d).
Cd2+-induced H2O2 production in vivo is likely to originate largely from mitochondrial electron transfer as Cd2+ increased ROS production in isolated mitochondria (Figs 5, 6). According to Varga et al. (2002), in cucumber, c. 70% of the extracellular Cd2+ can reach the cytoplasm, implying that a high percentage of the added Cd2+ will reach the mitochondria. H2O2 produced inside the mitochondria seems to be able to diffuse out of the cells as in the presence of antimycin A the amount of extracellular H2O2 was increased (Fig. 7b). In addition to H2O2 of mitochondrial origin, a basal level of peroxisomal H2O2 production (Corpas et al., 2001; del Río et al., 2002; Hänsch et al., 2006; Nyathi & Baker, 2006) may affect the total amount of H2O2 produced by intact roots.
Increased H2O2 production during short-term exposure of tobacco cell cultures to Cd2+ has been reported previously (Olmos et al., 2003; Garnier et al., 2006; Ortega-Villasante et al., 2007). Using video microscopy Ortega-Villasante et al. (2007) recently observed Cd2+-induced H2O2 generation inside and outside of roots, in agreement with our present conclusions. Olmos et al. (2003) and Garnier et al. (2006), however, concluded that NOX activity is responsible for Cd2+-induced H2O2 production, at least during the initial phase of the oxidative burst. Using tobacco cell cultures Olmos et al. (2003) measured Cd2+-induced H2O2 generation which was strongly inhibited by DPI and interpreted this as evidence for the involvement of a NOX-like enzyme in ROS production. Addition of DPI abolishes the Cd2+-induced generation of H2O2 in vivo and this is often taken as a sign of the involvement of a plasma membrane NOX-like enzyme in the Cd2+-induced oxidative burst (Olmos et al., 2003; Romero-Puertas et al., 2004; Rodriguez-Serrano et al., 2006). DPI is often regarded as a specific inhibitor of NOX at low micromolar concentrations (< 10 µM; Doussiere & Vignais, 1992) although it also inhibits complex I of the respiratory chain at equally low concentrations (Ragan & Bloxham, 1977). Therefore, DPI also inhibits mitochondrial ROS production in vivo. In addition, DPI inhibits other flavin-containing enzymes at higher concentrations (≥ 10 µM; Doussiere et al., 1992) and peroxidases at even higher concentrations (Frahry & Schopfer, 1998). In our hands, DPI inhibited Cd2+-induced H2O2 production in roots (data not shown) but because of the different sites of action of this inhibitor it seems to us to be impossible to draw conclusions from this fact.
Garnier et al. (2006) identified different phases of Cd2+-induced ROS production using tobacco cell cultures. A transient first wave was linked to the activity of plasma membrane NOX and a second longer lasting wave to production in mitochondria, and a third wave was characterized by lipid hydroperoxide accumulation concomitant with cell death. The first wave was completely abolished in an antisense construct of NtrbohD, implying that the NOXs of the plasma membrane were responsible for the first wave. We measured Cd2+-induced H2O2 production using mutants of atrbohC and atrbohD defective in NOX activity (Torres et al., 2005). ROS production is decreased in atrbohC (Foreman et al., 2003; Renew et al., 2005). In the atrbohC mutant, Cd2+ stimulated H2O2 production to the same extent as in wild type (Supplementary Material Fig. S1), indicating that the Cd2+-dependent H2O2 generation was not impaired in this mutant and was therefore not linked to NOX activity. In atrbohD, Cd2+-induced H2O2 production was abolished, in agreement with Garnier et al. (2006). This observation is in contradiction to the results presented here, which clearly indicate that Cd2+ induces mitochrondrial ROS production. This seems to indicate that some other processes involved in Cd2+ uptake or toxicity are impaired in the atrbohD mutant and in NtrbohD antisense strains (Garnier et al., 2006). Sagi et al. (2004) investigated Rboh antisense lines in tomato and reported major pleiotrophic effects on the phenotype of the plant and its reproductive organs. A total of 384 expressed sequence tags (ESTs) were down-regulated and 485 ESTs were up-regulated in the Rboh antisense line. According to Sagi et al. (2004), Rbohs play a role in redox-related cellular activities and they affect the level of the expression of ROS-dependent genes, signal transduction and developmental processes. Therefore, it seems to be difficult to draw conclusions from Rboh antisense lines. Further investigations must be performed on antisense lines to investigate in detail whether the lack of the Cd2+-induced ROS generation is attributable to alterations in the amount of ROS-detoxifying enzymes or to alteration of other metabolic processes.
A short time of exposure to the metal is crucial to study the primary effect of Cd2+ toxicity. After a long-term exposure of plants to Cd2+ many physiological processes are affected. Higher expression levels of Rbohs have been reported after long-term exposure of plants to Cd2+ (Van Belleghem, 2007) and chelation of Cd2+ takes place in the cytosol (Schützendübel & Polle, 2002). Furthermore, Cd2+ increases the concentration of abscisic acid (ABA) (Hsu & Kao, 2003), which is known to lead to the activation of the NOX (Kwak et al., 2003). Long-term exposure of plants to Cd2+ has been reported to activate and H2O2 production both in the cytoplasm and in the plasma membrane, where NOX is thought to be responsible for the activity (Romero-Puertas et al., 2004; Rodriguez-Serrano et al., 2006).
One problem limiting progress in understanding the biological role of ROS is that it is difficult to attribute an oxidative burst to a well-defined ROS-producing reaction. A new view of the primary sources of ROS produced in response to signaling is developing. Ashtamker et al. (2007) using tobacco cell cultures reported a first cryptogein-induced H2O2 burst in mitochondria, the endoplasmic reticulum and the nucleus followed by a second burst at the plasma membrane after a few seconds. In a mammalian system, mitochondrial H2O2 production in response to a stress signal partially mediated the activation of Nox1 after a lag time of a few hours (Lee et al., 2006). The availability of inhibitors of enzymatic reactions producing ROS in specific cell compartments, as shown here for Cd2+, may elucidate the complex interaction of ROS-producing reactions in vivo.