The toxicity of heavy metals in living organisms has become a major focus of research in recent decades as a result of the increased environmental pollution in industrial areas. Cadmium is one of the most dangerous heavy metals due to its high mobility in plants. This metal produces severe disturbances on plant metabolism affecting photosynthesis rate and water/nutrient balance, and also induces oxidative damages. By contrast with the enormous number of publications on the tolerance and accumulation of cadmium in plants, there is a remarkable lack of knowledge on the molecular mechanisms and signalling events underlying plant responses to Cd toxicity. The dual role of ROS and NO in heavy metal toxicity as both oxidative damage inducers and signalling molecules has been demonstrated in recent years. In this chapter we will analyze the contribution of different ROS and NO sources in the cell and their role in the regulation of cellular response to this metal.
21. 1. Introduction
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Heavy metals, such as Cd, Hg, Pb, and Al, are important environmental pollutants, particularly in industrial areas, as result of anthropogenic activities such as metal-working industries, cement factories, smelting plants and refineries, traffic and heating systems (Sanitá di Topi and Gabbrielli 1999). Many of the arable soil in the world have become moderately contaminated with Cd through the use of phospahte fertilizers, sludge, or irrigation water contaminated with Cd (Sanitá di Topi and Gabbrielli 1999). In polluted soils, Cd is generally present as a free ion or in other soluble forms, and its mobility depends on pH as well as the presence of chelating substances and other cations. Cadmium causes toxic effects on all living organism because it enters through the food chain being accumulated in edible part ingested by humans or domestic animals (Nordberg, 2004). Prolonged exposure of Cd to humans can cause renal dysfunction, lung damage, acute gastrointestinal effects or depression of immune system, increase cancer risk or anaemia (Nordberg, 2004). Cadmium accumulation in plants causes toxicity symptoms such as chlorosis, growth reduction and even cell death. The cellular toxicity of this metal can results from various direct and indirect effects on cell metabolism and can be explained by its chemical characteristics. Thus, Cd can bind to SH groups of proteins or enzymes, leading to misfolding, enzyme inhibition or interferences with redox regulation. Cadmium can also act displacing other cations from proteins or enzymes affecting their function (Van Assche and Clijsters 1990). Cadmium, like most of heavy metals, stimulates oxidative stress by inducing reactive oxygen species generation or producing disturbances in the antioxidative defences of cells, giving rise to oxidative damages to different molecules (Sandalio et al., 2009).
Cadmium is one of the most dangerous heavy metals due to its highly mobile nature and the low concentrations required to adversely affect the plant. During the last decade strong evidence have shown that oxidative stress imposed by reactive oxygen species (ROS) accumulation plays an important role in cellular Cd toxicity in different plant species, being the effects produced dose- and species-dependent (Benavides et al., 2005; Sandalio et al., 2009). However, ROS are double-faced molecules acting as signal molecules regulating a large gene network involved in cell response to biotic and abiotic stress. Nitric oxide (NO) is a gaseous reactive molecule with a pivotal signalling role in many developmental and cell response processes (Besson-Bard, Pugin and Wendehenne, 2008). This molecule can also interfere with ROS metabolism and during the last decade an increasing number of works have reported the effects of NO in alleviating the toxicity of heavy-metals, including Cd and As (Xiong, et al., 2010). Several defence strategies to avoid metal toxicity have been developed by plants, between them, preventing entry of metal by the exudation of metal-complexing agents (citrates and phytosiderophores) by roots, or metal immobilization in pectic sites and hystidyl groups in the cell wall (Sanitá di Toppi and Gabbrielli 1999 ; Clemens, 2006). A second line of defences involved the induction of specific peptides, called phytochelatins (PCs), which chelates the metal. PC-Cd complexes are transported into the vacuole to protect cells from toxicity derived from free Cd ions in the cytosol (Cobbett, 2000). The isolation of an Arabidopsis cad1 mutant, which is defective in PC activity and is hypersensitive to Cd, has demonstrated the importance of this mechanism in the defence against Cd (Howden et al., 1995). Cd and other metals can be also complexed by metallotioneins and nicotianamine (Sharma and Dietz, 2006).
21.2. Cadmium toxicity in plants
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The toxic effects of cadmium on several plant species have been reported by different authors (Sanitá di Toppi and Gabbrielli, 1999; Sandalio et al., 2001; Schutzendübel et al., 2001; Benavides, Gallego and Tomaro, 2005), although the mechanisms involved in cadmium toxicity are not yet fully understood. Cadmium inhibits seed germination, decreases plant growth, induces premature senescence and can even triggers cell death in cells suspension cultures (Fojtovà and KovaÅ™ik, 2000; McCarthy et al., 2001; Rodríguez-Serrano et al., 2009; De Michele et al., 2009). At cellular levels Cd produces alterations in membrane functionality by inducing changes in lipid composition and promoting lipid peroxidation (Ouariti et al., 1997; Hernández and Cooke, 1997; Sandalio et al., 2001), and produces disturbances in photosynthesis by affecting CO2 fixation and by inhibiting PSII photoactivation due to competition with essential Ca+2 sites (Faller, Kienzler and Krieger-Liszkay, 2005; Baryla et al., 2001). Cadmium toxicity is associated with disturbances in the uptake and distribution of macro- and micro-nutrients (Hernández et al., 1998; Rogers, Eide and Guerinot, 2000; Sandalio et al., 2001; Tsyganov et al., 2007) and, therefore, can compete with other cations for protein and transporter binding sites (Clemens, 2006). Cd uptake is carried out through the same plasma membrane transporters as those used for other cations, such as K+, Ca2+, Mg2+, Fe2+, Mn2+, or Cu2+ (Clemens, 2006). Cadmium leads to a reduction in Ca2+ content, which can then affect the activity of calmodulin-dependent proteins (Rivetta, Negrini and Cocucci, 1997; Rodríguez-Serrano et al., 2009). A relationship between Cd tolerance and Ca2+ homeostasis in a Cd-resistant pea mutant (SGECdt) has been observed (Tsyganov et al. 2007), and in radish and Arabidopsis seedlings calcium has been reported to alleviate Cd toxicity by reducing Cd uptake (Rivetta, Negrini and Cocucci 1997; Suzuki 2005).
The role of oxidative stress in Cd toxicity has been established in different plants species by analysing oxidative damages to proteins and lipids, as well as by studying disturbances in antioxidative defences imposed by the metal (Benavides, Gallego and Tomaro, 2005; Sandalio et al., 2009; Remans et al., 2010). Although Cd is a bivalent cation unable to participate in redox reactions in the cell, most transcriptome studies show up-regulation of genes encoding proteins involved in the defense against oxidative stress and ROS production (Suzuki et al., 2001; Zhao et al., 2009). These results suggest that oxidative stress is one of the primary effects of Cd exposure. Reactions involving oxygen free radicals are an intrinsic feature of plant senescence and they promote the process of oxidative deterioration that contributes to cell death (del Rio et al., 1998). Cd induces senescence symptoms and cell death in both cell culture and plant tissues, characterized by induction of glyoxylate cycle, oxidation of proteins and proteolytic activities (McCarthy et al., 2001; Romero-Puertas et al., 2002). Senescence is considered a type of plant cell death (PCD) and different approaches has indicated that Cd induces PCD in cell cultures (Fojtovà and Kovarik, 2000; de Michele et al., 2009). There are some evidences supporting dose dependence in the timing and intensity of the onset of the senescence process and of the final cell death event (De Michele et al., 2009). Condensation of chromatin, fragmentation of DNA, visualized by TUNEL assay, and induction of SAG12 expression are some of the symptoms of PCD and has been observed in Arabidopsis and tobacco cell cultures exposed to Cd (Fojtovà and KovaÅ™ik, 2000; de Michele et al., 2009). Cadmium-dependent senescence and PCD are regulated by ROS and NO although the mechanisms involved are not well understood (Yakimova et al., 2006; de Michele et al., 2009; Rodríguez-Serrano et al., 2009). However both, direct reaction of ROS and NO with proteins such as antioxidants, by oxidation and S-nitrosylation of proteins, or indirectly by regulating gene expression can take place (see section 21.6). Lipid signaling and Ca+2 play also an important role in Cd-induced cell death (Yakimova et al., 2006).
21.3 Sources of ROS in plants exposed to cadmium
Reactive oxygen species, singlet oxygen (1O2), superoxide radical (O2.-), hydroxyl radical (·OH) and hydrogen peroxide (H2O2) occur as by-products of the normal aerobic metabolism, such as respiration and photosynthesis, and their steady-state levels are determined by the interplay between different ROS-producing and ROS-scavenging mechanisms.This balance is kept by enzymes such as superoxide dismutase (SOD), which remove O2.- radicals, and catalase (CAT), peroxidase (POX) and peroxiredoxin which decompose H2O2, and metabolites such as glutathione (GSH) and ascorbate (ASC), which are coordinated to control ROS accumulation in different subcellular compartments. An excess of ROS is dangerous mainly due to reactions with lipids, proteins, and nucleic acids giving rise to lipid peroxidation, membrane leakage, enzyme inactivation, and DNA breaks or mutations, which can produce severe damage to cell viability. The subtle control of ROS production enable these species to act as signaling molecules, being involved in the regulation of several processes such as mitosis, tropism, cell death and cell response to biotic and abiotic stress. Compared with other ROS, H2O2 is a relatively long-lived molecule that is able to diffuse across cell membranes and acts as a signalling molecule during growth and development (Van Breusegem and Dat, 2006). However, despite our knowledge regarding the toxic effects of ROS induced by metals and the detoxification mechanisms, information on its role in regulation and signal transduction under metal stress is rather limited.
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The electron transfer chains associated to chloroplasts and mitochondria have been considered as prominent cellular sources of ROS, but this view has changed and the oxidative metabolism of peroxisomes has gained protagonism as demonstrated under different stress conditions (del Río et al., 2006, 2009). In peroxisomes purified from pea leaves, a Cd-dependent increase in the H2O2 concentration was observed which was mainly due to the activation of glycolate oxidase, a key enzyme of the photorespiration cycle (Romero-Puertas et al., 1999). In pea leaves, using a cytochemical approach it was demonstrated that Cd-dependent H2O2 production occurs in peroxisomes, in the outer mitochondrial membrane but mainly in the plasma membrane, where the NADPH oxidase is the main source of ROS (Romero-Puertas et al., 2004). In peroxisomes, H2O2 was present in locations in close contact with other organelles, which suggests possible cross-talk with other cell compartments (Romero-Puertas et al., 2004). In mitochondria, the Cd-dependent H2O2 produced could be due to an increased O2.- production at the complex III site of the electron transport chain, as has been reported in animals treated with Cd (Wang et al., 2004), and suggested in soybean roots (Heyno, Klose and Krieger-Lyzkay, 2008). H2O2 were also observed in the tonoplast from bundle sheet cells and plasma membrane from epidermal and transfer cells (Romero-Puertas et al., 2004). Cadmium-dependent superoxide radicals accumulation was demonstrated in tonoplast of bundle sheet cells, and plasma membrane from mesophyll cells, although the source has not been identified (Romero-Puertas et al., 2004). Accumulation of both H2O2 and O2.- were also observed in vascular tissues from Cd-treated pea plants by confocal lasser microscopy and electron microscopy cytochemistry (Romero-Puertas et al., 2004; Rodríguez-Serrano et al., 2006; 2009a). This fact is associated with lignifications processes that are very active in this tissue under physiological conditions and are also induced under metal toxicity (Schützendübel et al., 2001; Rodríguez-Serrano et al., 2009a). Results obtained with inhibitors or modulators of signal transduction processes showed that the earliest control point in ROS production induced by Cd is at the level of phosphorylation/dephosphorylation of proteins, in fact a comparative transcriptomics study using different metals and sodium chloride in Arabidopsis thaliana, show that Cd specifically induced genes coding kinases (Zhao et al., 2009), which demonstrate the importance of this processes in the regulation of cell response to Cd. Calcium ions are also important in the regulation of ROS production induced by Cd, as well as the cGMP, probably by causing a transient elevation of Ca+2 concentration (Romero-Puertas et al., 2004).
NADPH oxidases (NOXs) are located in the plasma membrane and catalyse the production of O2.-, which can be converted in H2O2 spontaneously or in the reaction catalysed by SOD. Ten genes encoding NOXs has been described in Arabidopsis and are termed respiratory burst oxidase homolog A to J (rbohA-J) because their homology to de catalytic subunitgp91phox (Nox2) of the NOX complex of mammalian phagocytes (Torres and Dangl, 2005). The role of NADPH-oxidase as the main source of ROS under Cd stress has also been demonstrated in tobacco cell cultures (Olmos et al. 2003; Garnier et al., 2006; Horemans et al., 2007) and alfalfa roots (Ortega-Villasante et al., 2005). In Arabidopsis plants the analysis of transcript levels of different NADPH oxidases showed a transient increase in the expression of rbohF in response to Cd, while the expression of rbohC and rbohD did not change (Horemans et al., 2007). However the contribution of NADPH oxidases on cadmium-induced ROS production are controverter (Heyno et al., 2008). In tobacco cell cultures, Cd induced cell death, which was preceded by three successive waves of ROS production. The first wave was due to an NADPH oxidase, followed by an accumulation of O2.- and fatty acid hydro peroxides (Garnier et al., 2006). Preceding the first oxidative burst induced by Cd, a rapid and transient induction of cytosolic Ca2+ concentration requiring protein phosphorylation and IP3-mediated release of calcium from internal stores (Garnier et al., 2003). Downstream from the cytoplasmic calcium mobilization, protein phosphorylation, calmodulin and Ca2+ might also directly regulate NtrbohD activity (Garnier et al., 2006). The mitochondrial electron transport chain disturbances induced by cadmium promotes a second wave of ROS production possible by an increase of the semi-ubiquinone radical concentrationn (Garnier et al., 2006). The third wave of ROS, concomitant with cell death, consisted in membrane peroxidation as result of previous increase of ROS production by mitochondria (Garnier et al., 2006). Recently Remans et al (2010) have demonstrated a Cd-dependent induction of NADPH oxidase and the differential regulation of gene expression by Cd and Cu in Arabidopsis plants, and they suggested a link between NADPH oxidase and lipoxygenase gene expression. A scheme of the different subcellular locations of ROS production is shown in Fig. 1.
The inhibition of antioxidative enzymes is also often described as a potential mechanism leading to cadmium-mediated increase in cellular ROS levels (Sandalio et al., 2001; Romero-Puertas et al., 2002; Schützendübel and Polle, 200; Benavides et al., 2005; Sandalio et al., 2009).One of the consequences of exposure of plant cells to cadmium is the drastic consumption of GSH for both sequestration of the metal and for synthesis of PCs, which gives rise the limitation of the GSH level necessary for keeping the redox balance of the cell leading then to ROS accumulation (Romero-Puertas et al., 2007).