The relationship between metal toxicity and cellular redox imbalance

Review

The relationship between metal toxicity and cellular redox imbalance Shanti S. Sharma1 and Karl-Josef Dietz2 1 2

Department of Biosciences, Himachal Pradesh University, Shimla 171 005, India Biochemistry and Physiology of Plants – W5-135, Bielefeld University, 33501 Bielefeld, Germany

The relationship between cellular redox imbalances leading to oxidative stress and metal toxicity in plants has been studied intensely over the past decades. This interdependency was often considered to reflect a rather indirect metal effect of cellular disregulation and progressive secondary damage development. By contrast, recent experiments revealed a clear relationship between metal stress and redox homeostasis and antioxidant capacity. Analysis of plants expressing targeted modifications of components of the antioxidant system, the comparison of closely related plant species with different degrees of toxic metal sensitivity and effector studies with, for instance, salicylic acid have established a link between the degree of plant tolerance to metals and the level of antioxidants. Heavy metal toxicity Heavy metals (HMs), added to the soil largely through diverse anthropogenic activities, constitute one of the major environmental contaminants that restrict plant productivity. Their non-biodegradability results in prolonged persistence in the environment, which is coupled with the tendency for bio-enrichment through food chains [1]. HMs such as Cd and Pb, but also metalloids such as As, generally intrude into plant cells at the expense of essential inorganic ions on account of similar properties, such as ionic radii [2]. Specific transporters might be involved in metal ion uptake, such as in the case of Cd uptake in Alpine Penny-cress (Thlaspi caerulescens) [3]. If they are allowed to accumulate in crop plants, toxic metals pose a threat to human health. The phytotoxicity of some metals is long established [4]. Metal toxicity is ascribed to three main reasons (Figure 1): (i) direct interaction with proteins due to their affinities for thioyl-, histidyl- and carboxyl-groups, causing the metals to target structural, catalytic and transport sites of the cell; (ii) stimulated generation of reactive oxygen species (ROS) that modify the antioxidant defence and elicit oxidative stress; and (iii) displacement of essential cations from specific binding sites, causing functions to collapse. For example, Cd2+ replaces Ca2+ in the photosystem II reaction centre, causing the inhibition of PSII photoactivation [5]. In view of the differences in the chemical properties of metals and their concomitant distinct behaviour in biological systems [6], these three mechanisms might not exclusively account for their toxicity. Corresponding author: Dietz, K.-J. ([email protected]).

The role of oxidative stress in metal toxicity has been assessed by measuring alterations in the redox metabolic components of stressed plants [7–10]. Over the past few years major progress has been achieved, particularly by comparing metal tolerant and/or metal hyperaccumulator genotypes with their non-tolerant relatives and by using transgenic plants that overexpress or lack specific redox elements. These approaches provided novel insight into the relationship between metal sensitivity and cellular redox imbalance. Sources of ROS and their toxicity in metal-stressed plants Subcellular sites of ROS generation A fraction of the metabolized O2, for example 1–5% of that consumed by isolated mitochondria, is converted to ROS [11,12]. Thus, singlet oxygen (1O2), superoxide (O2), hydrogen peroxide (H2O2) and hydroxyl radicals (OH) are produced as a result of spin inversion and one-, twoand three-electron-transfer reactions to O2, respectively. Cellular ROS production is stimulated in response to metabolic imbalances imposed by abiotic and biotic stresses and proceeds through similar mechanisms [12,13]. The electron transfer activities of chloroplasts and mitochondria and oxidative metabolism in the peroxisomes represent the predominant sources of ROS (Figure 2). The contribution of chloroplasts was considered larger than that of mitochondria, but this view has changed [14]. The peroxisomal activities of xanthine oxidase and NADPH-dependent oxidase generate O2, and glycolate oxidase, flavin oxidases and b-oxidation yield H2O2, which is metabolized by catalase (CAT) [15,16]. Abiotic stresses, including Cd, stimulate peroxisome biogenesis [17,18]. Also, plasma-membrane-bound NADPH oxidase is involved in HM-induced ROS generation, for example in Ni-treated durum wheat (Triticum durum D.) [19], Cdtreated pea (Pisum sativum) [20] and Pb-treated Vicia faba [21]. Pharmacological approaches indicate the involvement of Ca2+ and protein kinases in HM-induced activation of NADPH oxidase [20–23]. The NADPH oxidase activity is associated with Cd2+-induced, but not Cu2+-induced, mitogen-activated protein kinase (MAPK) activation in rice (Oryza sativa) roots. The Cd-induced MAPK activation might confer Cd tolerance in rice plants because the Cdtolerant cultivars possessed substantially higher MAPK activities [23]. The onset of ROS generation is fast, for example in Cdtreated (50 mM, 6 h) Scots pine (Pinus sylvestris) seedlings

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Figure 1. Principal mechanisms of heavy metal (HM)-induced damage development in sensitive plants and points of interaction and counteraction during acclimation in tolerant plants. Redox-active HMs directly elicit reactive oxygen species (ROS) generation. As a general property, HMs bind to and interfere with targets or compete for binding sites (-SH represents thiols, -COO represents carboxylic acids and imidazole represents histidyl residues), thereby altering target protein functions, which in turn causes changes in cell metabolism or triggers signalling events that can lead to acclimation. Activation of acclimation responses causes feedback loops with various sites of HM action, leading to, for example, the repair of damaged macromolecules, strengthening of the antioxidant defence system and lowering of HM concentrations in plasmatic compartments.

Figure 2. Pathways of heavy metal (HM)-dependent ROS generation. HM uptake by transporters and distribution to organelles is followed by ROS generation, stimulated either by HM redox activity or by HM effects on metabolism in a subcellular site-specific manner. HM-dependent activation of plasma-membrane-localized NADPH oxidase also contributes to the release of ROS. Excess ROS causes redox imbalances and disturbances in signalling processes (such as the MAPK pathways) that inhibit plant growth and might cause cell damage. Red dots symbolize the distribution of HM in the cell and apoplast.

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Review [8]. Cell imaging by using fluorescent probes of Cd- and Hgtreated (6–24 h) Medicago sativa confirmed the rapid accumulation of peroxides and depletion of glutathione (GSH) and homoglutathione (hGSH) causing redox imbalance [24]. The Cd-induced cell death in bright yellow-2 (BY2) tobacco (Nicotiana tabacum) cells was preceded by NADPH-oxidase-dependent accumulation of H2O2 followed by cellular O2 and fatty acid hydroperoxide accumulation [22]. The rapid O2 generation occurred concomitantly with enhanced superoxide dismutase (SOD) activities in the apoplast of Cu-treated (1–6 h) wheat roots [25]. The cellular redox perturbation seems to be an essential prerequisite for development of HM-dependent phytotoxicity symptoms. ROS reactions with biomolecules In contrast to the physiologically non-redox-active HMs, such as Zn2+ and Cd2+, the redox-active HMs Fe, Cu, Cr, V and Co enable redox reactions in the cell, for example Cu+ $ Cu2+ + e and Fe2+ $ Fe3+ + e [26]. They are involved in the formation of OH from H2O2 via HaberWeiss and Fenton reactions and initiate non-specific lipid peroxidation [7]. Lipid peroxidation is also specifically induced by HM-dependent activation of lipoxygenases (LOXes), for example 9-LOX in Cd-treated Arabidopsis thaliana [27]. Oxidized polyunsaturated fatty acids are precursors for signalling molecules like jasmonic acid, oxilipins and volatile derivatives [28,29]. ROS react with cellular constituents depending on properties such as chemical reactivity, redox potential, half-life and mobility within the cellular compartments. OH radicals are the most reactive and short-lived (1 ns) [12] and will oxidize biomolecules within diffusion distance. ROS induce reversible as well as irreversible oxidative modifications of proteins. This activity might differ in an organelle-specific manner. For example, in Cd-treated Pisum sativum, the proportionally greater degree of protein carbonylation in isolated peroxisomes compared to the whole-plant extracts implies a higher Cd-dependent ROS production in these organelles [30]. Likewise, DNA is oxidized mainly by OH and 1O2, which have been reported to affect guanine, but less by H2O2 and O2, and it also reacts with reactive nitrogen species [31]. Cellular antioxidant defence and its status under HM stress Antioxidative enzymes Antioxidant defence mechanisms keep the routinely formed ROS at a low level and prevent them from exceeding toxic thresholds [32]. Abiotic stresses disrupt the equilibrium between ROS generation and detoxification [12]. The antioxidant network consists of enzymatic and non-enzymatic components: O2 scavenging by SOD and H2O2 decomposition by ascorbate peroxidase (APX), peroxiredoxins (PRXes) and CAT are predominantly associated with the maintenance of cellular redox steady state. Isoforms of SOD, APX and PRX are localized in several subcellular compartments, whereas CAT is mostly localized in the peroxisome. They strongly differ in their substrate affinities and ensure a tight control of H2O2 concentrations at very low levels [33]. Other enzymes,

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such as glutathione peroxidase (GPX) and glutathione-Stransferases (GSTs), also contribute to the redox poise of the cell and change, for example, in Cu-treated Arabidopsis seedlings [34]. Antioxidant enzyme activities in HM-stressed plants reveal stimulation, no-effect and suppression depending on the plant species, metal ion, concentration and exposure duration (summarized in Ref. [9]). Despite the lack of distinct pattern(s), the responses reflect the modified redox status of the cells induced by HMs. Enhanced transcript levels of APX, CAT and GPX in barley (Hordeum vulgare) treated with Cd under varying nutritional conditions have been demonstrated [35–37]. However, these observations do not provide a basis sufficient for defining mechanistic relationships. Open questions concern the identification of primary versus secondary mechanisms of damage and thus the distinction between, on the one hand, principal defence and tolerance-associated molecular systems and, on the other hand, non-specific secondary stress responses (Figure 1). Glutathione (GSH)–ascorbate couple GSH and ascorbate accumulate to millimolar concentrations in chloroplasts and mitochondria owing to the ascorbate–GSH cycle, which also operates in peroxisomes [38]. The redox state of GSH and ascorbate is maintained through glutathione reductase (GR), monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR). They have a pivotal role in defence against ROSinduced oxidative damage. GSH functions as an HM-ligand [39] and an antioxidant. Upon HM exposure, GSH concentrations drop as a consequence of initiated phytochelatin (PC) biosynthesis. This causes oxidative stress and in turn short-term toxicity [9,40]. The constitutively elevated GSH biosynthesis in different Thlaspi species is a strong determinant of their tolerance to excess Ni via alleviation of Nidependent oxidative stress [41]. When comparing the effects of overexpression of A. thaliana PC synthase (AtPCS1) or Caenorhabditis elegans PC synthase (CePCS) in tobacco, AtPCS1 transformants, in contrast to wild type (WT) and CePCS, were found to be Cd-hypersensitive. The plants exhibited strong depletion of GSH, accumulation of g-glutamylcysteine and higher oxidative stress [42]. Apparently, the ability of high rate PC synthesis is insufficient to cope with the metal load if the functionality of the antioxidant system is simultaneously hampered. Similarly, the GSH content was inversely linked to Cd sensitivity when comparing ten pea genotypes showing differing Cd sensitivity [43]. HM-induced oxidative stress is invariably stronger in sensitive genotypes The role of oxidative stress in metal toxicity has recently been assessed in metal-tolerant and -sensitive plants ranging from lower to flowering plants. The dataset can be categorized in supportive correlative analysis and genetic studies, both strengthening the direct link between the degree of oxidative stress and metal toxicity (Table 1). For example, the lower sensitivity of the alga Dunaliella tertiolecta to Cu, evident by a three times lower lipid peroxidation than in the sensitive D. salina, corresponded with 45

Review

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Table 1. Summary of the experimental evidence in support of the conclusion that a strong antioxidant defence system is needed or is at least a beneficial trait in heavy metal tolerancea Organism Yeast Cryptococcus liquefaciens Algae Dunaliella tertiolecta, D. salina Ferns Pteris vittata, P. ensiformis, Nephrolepis exaltata Higher plants Arabidopsis halleri, A. thaliana

Phenotype

Salient redox attribute in tolerant counterpart

Refs

Metal tolerant

High SOD activity

[87]

Cu tolerant or sensitive

High APX activity

[44]

As tolerant, hyperaccumulator or sensitive

High SOD, CAT and APX activity

[46]

Upregulation of APX and MDAR4 expression; high APX and class III peroxidases activity High SOD and CAT activity; high GSH concentrations

[47] [48]

High SOD and CAT activity High GSH concentrations driven by SAT activity

[49] [41]

Silene cucubalus

Zn and Cd hyperaccumulator or nonhyperaccumulator Cd tolerant, hyperaccumulator or nonhyperaccumulator Ni hyperaccumulator or non-accumulator Differentially Ni tolerant and/or hyperaccumulator Metal tolerant or sensitive genotype

[88]

Salix viminalis Triticum aestivum Pisum sativum Arabidopsis thaliana

Metal tolerant or sensitive genotype Al tolerant or sensitive Cu tolerant or sensitive Transgenic with enhanced Ni tolerance

Brassica napus

Transgenic with enhanced Al tolerance

Relatively less Cu-dependent membrane damage (ion leakage) High SOD activity High CAT and GST activity High peroxisomal Mn-SOD and CAT activity High OAS, Cys and GSH concentrations due to overproduction of G. goesingense SAT Overexpression of Ta-Mn-SOD

Thlaspi caerulescens, Nicotiana tabacum Alyssum bertolonii, N. tabacum Thlaspi species

[89] [53] [55] [41] [54]

a

The table gives the phenotype in relation to metal tolerance and hyperaccumulation, the observed biochemical feature and the reference.

higher APX activity [44]. The Chinese brake fern (Pteris vittata), a versatile fast growing As hyperaccumulator [45], showed no toxicity symptoms, low levels of lipid peroxidation and high activities of SOD, CAT and APX in response to elevated As concentrations when compared with two other ferns, namely Pteris ensiformis and Nephrolepis exaltata, implicating the antioxidative defence in As tolerance [46]. Among the flowering plants, studies on three species, namely Arabidopsis halleri, T. caerulescens and Alyssum bertolonii, have provided insight into the mechanisms involved in metal hyperaccumulation. Remarkably, all of them exhibited stronger antioxidative capability when compared with their respective metal non-accumulator relatives. A comparison of the gene expression of the Zn and Cd hyperaccumulator A. halleri with that of the nonhyperaccumulator A. thaliana using A. thaliana cDNA microarrays revealed that, in addition to particular metal transporters, metallothioneins 2b and 3, APX and MDAR4 in the ascorbate–GSH pathway are expressed at higher levels in A. halleri than in A. thaliana. The activities of APX and class III peroxidases were also highly elevated in A. halleri [47]. Likewise, the Cd-tolerant T. caerulescens exhibited a 200- to 300-fold higher CAT activity than the nonaccumulator N. tabacum [48]. Significantly higher SOD activity and GSH concentrations were measured in T. caerulescens. Subsequently, the root growth of N. tabacum in response to Cd treatment seemed to be arrested concomitant with a fivefold increase in H2O2 levels, whereas that of T. caerulescens was maintained with much lower H2O2 levels [48]. In a similar comparison, roots of A. bertolonii, an Ni hyperaccumulator, possessed >500-times greater CAT activity and 2.4-times greater SOD activity, as compared with N. tabacum. Correspondingly, the H2O2 increase due to Ni treatment was higher in the latter than in the former [49]. 46

Ni tolerance and hyperaccumulation in Thlaspi species is linked to the constitutive ability to accumulate higher concentrations of GSH, Cys and O-acetyl L-serine (OAS) [41]. The elevated GSH concentrations in T. goesingense were driven by constitutively elevated activities of serine acetyl transferase (SAT), which provides the C skeleton for Cys synthesis. The causality was proven in a transgenic approach: the overproduction of T. goesingense SAT in the nonaccumulator A. thaliana led to the accumulation of OAS, Cys and GSH [41] and coincided with strongly enhanced resistance to Ni-induced growth inhibition and oxidative stress. Along with high GSH levels, T. goesingense also possessed constitutively high activities of GR and CAT. However, the heritable metal tolerance in other species (e.g. Pteris vittata [50] and Silene vulgaris [51]) was not found to be caused by the constitutively high GSH concentrations, implying the species-specificity of GSH functions. Al toxicity restricts plant productivity in acidic soils and is mitigated largely through organic acids [52]. Oxidative stress is evidently involved in the development of Alinduced toxic symptoms. Thus, Al-tolerant lines of wheat developed by in vitro microspore selection produced lower amounts of ROS in response to Al and grew better in comparison with the Al-sensitive genotype. The Al tolerance could tentatively be ascribed to the higher activity of CAT and GST observed in the tolerant lines [53]. Mn-SOD is also likely to have a role in Al tolerance; compared with WT plants, transgenic Brassica napus overexpressing mitochondrial bread wheat (Triticum aestivum) Mn-SOD (Ta-Mn-SOD) showed reduced malondialdehyde accumulation and growth inhibition in response to Al [54]. The TaMn-SOD transgenic plants showed 1.5- to 2.5-fold greater SOD activity than WT plants. Likewise, Cu tolerance in pea correlated with increased activities of peroxisomal SOD and CAT [55].

Review Three broad patterns have emerged from these studies on contrasting species or ecotypes. (i) High metal sensitivity often is linked to inferior constitutive antioxidative defence. This is in contrast to other commonly acknowledged plant responses to HMs; for instance, the biosynthesis of PCs is activated by specific metals to a varying extent and therefore could not explain the heritable tolerance in Silene vulgaris [56]. (ii) A strong component of the antioxidative superiority of HM-tolerant and hyperaccumulating genotypes is a constitutive high level of H2O2-decomposing enzymes, particularly CAT and APX. This indicates a contribution of H2O2 as a mediator of HM toxicity. Coincidentally, H2O2 has a sufficiently long half-life (1 ms) [12] to diffuse through and between the cells and is even membrane-mobile (transmembrane transport of H2O2 is facilitated by aquaporins [57]). (iii) The observed elevated constitutive SOD levels in HMtolerant hyperaccumulators seem to comprise part of the strategy to lower the O2 burden via enhanced O2 detoxification. Emerging additional players in the antioxidant network against HM toxicity Peroxiredoxins (PRXes) PRXes are robust peroxide-decomposing enzymes with several organelle-specific variants, including mitochondrial and chloroplastic variants. Their function in plant cellular redox homeostasis is clearly established [58,59]. A tDNA insertion mutant of A. thaliana lacking the expression of mitochondrial PrxIIF (AtPrxIIF-knockout [KO]) was more sensitive to Cd in terms of root growth than the WT, signifying the involvement of PrxIIF in cellular detoxification of Cd such that root growth is maintained under Cd stress up to a certain threshold [60]. Proline Free L-proline accumulates in plants exposed to HMs (reviewed in Ref. [61]). Studies involving in vitro assays and transgenic plants revealed that proline (Pro) acts as an antioxidant. An evaluation of the Promediated tolerance to Cd was made in transgenic Chlorella reinhardtii that overexpressed moth bean (Vigna aconitifolia) delta-1-pyrroline-5-carboxylate synthetase (P5CS), the committing enzyme of the Pro synthesis pathway, and accumulated higher Pro levels [62]. Under Cd treatment, Pro levels directly correlate with the reduction state of GSH. In Cd-treated WT with low Pro, the GSH pool was fourfold more oxidized than in the transgenic algae, with high Pro indicating an antioxidant action of Pro. As a consequence, increased GSH might be allocated to PC biosynthesis for Cd sequestration. Transgenic Pro-overproducing tobacco plants produced by antisense suppression of proline dehydrogenase, the committed step of Pro catabolism, were resistant to Pb, Ni and Cd [63]. Several in vitro assays using electron spin resonance (EPR) revealed that Pro scavenges OH radicals and 1O2 and inhibits freeradical-mediated grafting of methyl acrylate (MA) onto the cellulose backbone [64–66]. Similar antioxidant behaviour of Pro is expected in vivo in the light of millimolar cytosolic Pro concentrations [61].

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a-Tocopherol Tocopherols and tocotrienols are synthesized and accumulate in plants. They react efficiently with 1O2 and act as terminators of chain reactions during lipid peroxidation [67]. Their involvement in plant responses to HM stress has only very recently been addressed [68]; a-tocopherol accumulates in leaves of A. thaliana treated with either Cu or Cd for a seven-day period. Interestingly, the increase in a-tocopherol was initially slower in the presence of Cu but then increased rapidly between days four and seven to sixfold the control value. In a converse manner, a-tocopherol levels increased without a lag phase in Cd-treated plants, doubling by day two and increasing fourfold by day seven. Both transcript and protein levels of hydroxypyruvate dioxygenase (HPPD), an enzyme associated with tocopherol biosynthesis, exhibited a consistent increase in response to the Cd treatment. In the same study, the hypersensitivity of the tocopherol-deprived A. thaliana mutant (vte1) to HM was observed [68]. The mutant vte1 was much more sensitive towards Cu and Cd than WT. In contrast to vte1, the ascorbate-deficient mutant vtc2 exhibited the WT response, an observation that proved the link between a-tocopherol and HM stress alleviation to be specific. The a-tocopherol-dependent protection against Cu- and Cd-induced oxidative disorders has also been reported in animal systems [69]. Carbon monoxide In plants, as in animals, carbon monoxide (CO) is synthesized from heme by the activity of heme oxygenases (HOs) [70], and in animals it serves as a bioactive messenger molecule. CO synthesis in animals is enhanced under various stress regimes [71], and the reported effects indicate an antioxidative action of CO [72]. HO is associated with antioxidative defence in Cd-stressed soybean (Glycine max) [73]. More recently, the alleviation of Cdinduced oxidative damage by CO in Medicago sativa roots has been demonstrated [74]. Cd induced the production of endogenous CO in roots, and CO pretreatment decreased the Cd-dependent oxidative damage, mainly via modulation of enzymes associated with GSH metabolism. HM-induced signalling HMs interfere with cell signalling pathways. In fact, it might be hypothesized that HM-induced disregulation of signalling events significantly participates in the HM toxicity response, as well as in damage development. In animals and humans, HMs activate the transcription factors nuclear factor kB (NF-kB) and activating protein 1 (AP-1), which control cell survival, differentiation, inflammation and growth (reviewed in [26]). In plants, convincing evidence demonstrates interference of Cu, Pb, Zn and Cd with mitogen kinase signalling cascades. Excess Cu rapidly activates the mitogen-activated kinases SIMK, MMK2, MMK3 and SAMK in Medicago sativa, but the same set of kinases responded to Cd exposure only with a considerable time delay [75]. The differential signalling response is discussed in relation to the redox activity of Cu versus the only indirect redox effects of Cd. MAPK pathways integrate diverse signalling stimuli, and specific elements are also activated by ROS [76]. The use of 47

Review inhibitors in rice gave circumstantial evidence for the involvement of NADPH oxidase and functional mitochondria in the Cd-dependent activation of the rice MAPK pathway [23], similar to their involvement in Pb-induced cell death in rice [77]. Plant jasmonic acid, salicylic acid and ethylene levels increase upon exposure to HMs [20,36,78]. The roles of these plant hormones in plant HM tolerance are controversial, and both beneficial effects (the counteraction of Cd and Mn toxicity by the activation of antioxidant defence systems [36,79]) and negative effects (enhancement of H2O2 generation [80]) have been reported. By modulating components of the cellular signalling network, mostly through application of established inhibitors or effectors, Romero-Puertas et al. [81] defined a working scheme of how redox components and signalling elements, such as Ca2+, cGMP and NO, positively or negatively modulate the expression of antioxidant genes during long-term Cd stress in pea. Conclusions and perspectives Efficient metal transport and binding are crucial mechanisms in safe cellular management of toxic HMs [61,82]. For example, in a recent study, expression of the metal transporter HMA4 (heavy metal ATPase 4) was proven to be essential for manifesting the Zn hyperaccumulation and full hypertolerance phenotype in A. halleri [83]. In addition to these mechanisms of cellular metal management, redox homeostasis and avoidance of oxidative stress are additional determinants of metal phytotoxicity. In this context, three main questions still need to be answered. (i) Which mechanisms link HM stress intimately to redox imbalances and oxidative stress? Metal exposure inevitably increases available metal concentrations in plasmatic compartments. Even in the presence of effective scavenging and export mechanisms, metals will bind to high-affinity targets. Proteins of the photosynthetic and respiratory electron transport chains and metabolic and antioxidant enzymes are thioredoxin (Trx) targets and known elements of the thiol-disulfide redox network [84], making them likely to be binding partners of HMs. HM-binding can disturb metabolism, stimulate ROS generation, inhibit ROS detoxification and suppress anti-oxidative-stress signalling. A systematic knowledge of involved mechanisms is unavailable and certainly should be a prime aim for future research. (ii) Does the upregulation of diverse antioxidants enhance metal tolerance with similar efficiency? Plants rely on a buffered and redundant antioxidant defence system that is sufficient to cope with an adversely changing environment. The available data (Table 1; [9]) provide an extremely restricted view on single elements of the antioxidant system. Nevertheless, they suggest that there are multiple sensitive sites in the cell that need to be protected against redox imbalances under HM exposure. More information is needed on the response of the antioxidant defence from a more global viewpoint. Contributing parameters are likely to vary in a species-specific manner owing to factors such as differences in transporter properties and activities, target site 48

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sensitivity or signalling pathways. Transgenic poplar accumulating GSH to a level 3.5-fold higher than WT exhibited unaltered Cd sensitivity [85]. To explain such differences in metal response, we can tentatively assume that plant cells with strong antioxidant defence and a strong redox homeostasis system can cope with extra metals; however, a better understanding of the redox regulatory network of the plants cell is needed. (iii) Why do the redox-linked signalling cascades fail to activate appropriate compensation mechanisms when subjected to HM stress? It can be hypothesized that HMs acting as, for instance, thiol ligands strongly interfere with redox signalling, for example by mimicking reduced states of sensor proteins even when redox imbalances and oxidative stress develop. Therefore, constitutively elevated levels of antioxidants might allow the cell to maintain a functional metabolism during metal exposure. Other future directions of research might concern the redox responses to multi-metal stress [86] because single metal contamination is virtually non-existent in the environment. In addition, novel and mechanistic insight might be gained from the response of anaerobic organisms; their O2-independent metabolism, which results in a lack of or low levels of ROS, might render them less sensitive to toxic metals than their aerobic counterparts. All these observations suggest that the development of suitable plant variants for potential application in phytoremediation might require an additional focus on the antioxidative defence capability of the plant species concerned. Acknowledgements We gratefully acknowledge support from the Indian Department of Science and Technology (DST), the German Science Foundation (DFG) and the German Academic Exchange Service (DAAD). The article in part originates from a DBT overseas associateship (S.S.).

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