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Mechanisms of metal detoxification in photosynthetic organisms 1 . Biological Effects of Metals: Emphasis in Algae1.1. Positive Effects: Essentiality Whereas the biological effects of synthetic organic chemicals are generally negative, uptake of metals and metalloids can be harmless (i.e., no effects), toxic or even beneficial. For instance, many metals and metalloids are essential for the normal growth, development and reproduction of organisms. These include not only abundant elements (essential macronutrients) such as calcium, magnesium, potassium, and sodium, but also trace elements (essential micronutrients) such as chromium, cobalt, copper, iron, manganese, molybdenum, nickel, selenium, vanadium, and zinc. Elements such as copper, iron, manganese, and molybdenum can exist in more than one oxidation state in cells (e.g., Fe(II) versus Fe(III)) and can catalyze essential life processes that involve electron transfer (e.g., photosynthesis, respiration, and nitrogen fixation). Elements such as nickel and zinc do not readily undergo oxidation state changes but can catalyze essential life processes that involve hydrolytic transformation (e.g., hydration and dehydration of carbon dioxide) (Stumm and Morgan, 1996; Butler, 1998). The metalloid selenium acts as a strong antioxidant in animals. Falchuk et al. (1975) reported that growth in zinc deficient media inhibits cell division in Euglena gracilis. Zinc is essential for the biochemical events of the premitotic state which include initiation of DNA synthesis, DNA synthesis and progression from G-2 to mitosis. Zn is also essential for the folding of TWCA1, the major Zn-requiring isoform of carbonic anhydrase in the marine diatom Thalassiosira weissflogii. Carbonic anhydrase is one of the main enzymes in nature as it is responsible of catalysing the first step of carbon assimilation in photosynthetic organisms. Lane and Morel (2000) provide strong evidence of in vivo metal substitution of Co for Zn in carbonic anhydrase, showing crucial roles for Co and Zn in expression and regulation of carbonic anhydrase. Recent studies have also found that cadmium, which was usually considered a nonessential element, can be essential under certain circumstances. For instance, in zinc-depleted coastal and marine waters, cadmium can play a direct role in the enzyme carbonic anhydrase in diatoms (Price and Morel, 1990; Lee, Roberts & Morel, 1995; Lane and Morel, 2000). The essentiality of these metals and metalloids results in a bell-shaped dose-response curve, with deficiency symptoms occurring at low concentrations and toxic effects occurring at high concentrations. Between the two extremes there is generally an optimal concentration range within which an organism experiences optimal growth, development and reproduction. When the environmental concentration of an essential element is within the optimal concentration range, organisms can regulate their internal concentrations of the element through binding and detoxification by metallothioneins in animals and bacteria (Roesijadi, 1992) or phytochelatins in algae, plants, and some fungi (Grill, Winnacker & Zenk, 1985; Ahner and Morel, 1995). This is fundamentally different from synthetic organic chemicals for which there is generally no deficiency and no optimal effects. 1.2. No Effects: Tolerance and Adaptation Non-essential metals and metalloids such as lead and antimony generally do not have explicit positive effects to organisms because they do not have direct nutritional or biochemical functions. Nevertheless, adverse effects may not be observed at low metal levels due to detoxification and adaptation. Since metals and metalloids have been in existence on the Earth since the formation of the planet, organisms emerging and evolving on the Earth have more or less adapted to background levels of metals and metalloids in their habitats. In addition to functioning in the regulation of essential metals, metallothioneins (in animals and bacteria) and phytochelatins (in algae, plants, and some fungi) are also involved in the detoxification of nonessential metals because of their high affinity for these metals (Roesijadi, 1992; Ahner and Morel, 1995). When organisms are exposed to elevated levels of metals, these low-molecular-weight proteins and peptides are induced to bind and detoxify excess metals. Organisms can also detoxify metals by isolating the metals and metalloids within their tissues as granules, as insoluble metal precipitates, or by excretion. For instance, some crustaceans (i.e., organisms such as crabs and shrimp) can accumulate metals in their chitinous exterior, which is periodically shed as they grow larger and molt. Because of detoxification, organisms may show some degree of resistance or tolerance to the toxicity of metals and metalloids. If the resistance to a metal or metalloid is heritable, exposure to the metal/metalloid will likely act as a selective agent, resulting in an increased resistance of the population in subsequent generations, in other words, resulting in adaptation to metals and metalloids. Such adaptation can occur rapidly and simply without appreciable cost to the organism (Chapman et al., 1998a). Adaptation to elevated metal concentrations has been well documented (Klerks and Weis, 1987; Chapman et al., 1998a). 1.3. Adverse Effects: Toxicity and Deficiency At elevated concentrations when the influx of metals and metalloids exceeds the regulation (for essential elements) or detoxification capacity (for nonessential elements), toxic effects may occur. However, adverse effects occur not only at elevated concentrations, for essential elements they also occur at very low concentrations due to deficiency. Slow cell growth and low anabolic activity have been widely described in cultures of photosynthetic microorganisms ( ). 1.3.1. Toxic effect of heavy metals on photosynthesis Although heavy metals are essential for photosynthetic organisms, many of them have been increasing in the environment to high concentrations (Cu2+, Cd2+, Ni2+, Hg2+, Pb2+, etc.) having adverse effects on metabolism and growth of photosynthetic organisms. Among the most severe effects, reduction in chlorophyll content, degeneration of chloroplasts and reduction of photosynthesis are included (Shanker 1996). Copper ions are directly involved in the photosynthetic electron transport chain as a plastocyanin constituent. Copper is a constituent of chloroplast Cu-Zn-SOD (superoxide dismutase) and also of the light harvesting Chl a/b-protein complex (LHC) of PSII with unknown function. Cu2+-ions, an essential micronutrient for photosynthetic organisms, may indirectly influence the chloroplast ultrastructure, pigment, and lipid biosynthesis. However, it has been reported that at high concentrations (1-10 mM) copper causes breakdown of chlorophylls and carotenoids and inhibits PSI and PSII electron transport. Inhibition of electron transport generates formation of toxic oxygen species and thus causes peroxidative breakdown of pigments and membrane lipids in Scenedesmus cells. Excess copper causes lower accumulation of some extrinsic polypeptides of the oxygen-evolving complex and a decrease in PSII activity (Mostowska 1996). Even lower concentrations ( mM) inhibited significantly photosynthesis and endogenous respiration in Chlamydomonas reinhardtii cells. A significant inhibition on PSII by copper has also been reported (Reiriz et al. 1994). There are not many data on the toxic effect of nickel on photosynthetic apparatus. It appears that carbon metabolism is the primary target of Ni2+ and that the effect of this metal on the primary photochemical processes is indirect and is a consequence of disturbances caused by Ni2+ in Clavin cycle reactions (Mostowska 1996). Cadmium, which is a long-range transported heavy metal pollutant, inhibits the synthesis of chlorophyll and carotenoids in many species, being the extent of the inhibition greater with cadmiun than with copper. In microalgae, nitrogen metabolism has been shown to be a main target of Cd2+ which produces competitive inhibition of glutamine synthetase with Mn2+ thus blocking nitrogen incorporation into carbon skeletons ( ). Cadmium and nickel lead to decline in CO2-fixation rates and have pronounced effects on Calvin cycle enzymes. Heavy metals slow down Calvin cycle reactions and limit ATP and NADPH consumption (Shanker 1996). To investigate adaptive responses to metal stress at the subcellular level, the oxidative balance in isolated chloroplasts was evaluated for the first time in the unicellular alga Gonyaulax polyedra exposed to the toxic metals Hg(2+), Cd(2+), Pb(2+), and Cu(2+). Different antioxidant responses were verified according to the metal and model of stress applied. Cells chronically exposed to metals exhibited high activity of the antioxidant enzymes superoxide dismutase and ascorbate peroxidase, high glutathione content, and decrease of peridinin levels, whereas no significant changes were detected for beta-carotene levels. In contrast, cells subjected to acute metal stress displayed twice as much beta-carotene but only a slight increase in superoxide dismutase and ascorbate peroxidase activities. The correlation of acute metal treatment and oxidative stress was inferred from the higher oxygen uptake and decreased reduced glutathione pool found in treated cells. By acting at the subcellular site, where oxidative stress is triggered, induction of such chloroplast antioxidants might be crucial for cell survival during exposure to heavy metals (Okamoto OK et al., 2001). 1.3.2. Effects on cell growth and cell structure in algae Different authors have shown growth inhibition of different algae by the presence of metals. Cadmium induces reduction of growth, reduction of chlorophyll content, and lethality in Chlamydomonas reinhardtii (Collard and Matagne 1990). Cell growth has been described to be significantly affected by cadmium in other microalgae including Scenedesmus obliquus in which young cultures have been shown to accumulate less cadmium than older cultures approaching stationary growth phase (Cain et al. 1980). Toxic effects of cadmium on the growth of Euglena gracilis were reported by Einicker-Lamas et al. (1996). It has been found that Cr(VI) inhibits cell proliferation and the formation of coenobia in Scenedesmus acutus (Corradi and Gorbi 1993). Although vanadium is essential for cell growth and chlorophyll formation in green algae, reveals toxic influences on cell division of Chlorella pyreneidosa, these disturbances arising in the same range of V-concentrations as the known possitive effects of the trace metal (Meisch and Benzschawel 1978). Franklin et al. (2000) reported that pH influences the metal concentration required to inhibit growth. The authors explained this in terms of the possible mechanism of competition between hidrogenions and Cu in Chlorella sp. The ultrastructure of many algae has been shown to change in the presence of metals. Visviki and Rachlin (1994) showed that cadmium has a greater impact on the ultrastructural changes of Dunaliella salina and Chlamydomonas bullosa than copper, the metal stress affecting a variety of cellular parameters including total cell volume, pyrenoid, nucleus, starch granules, polyphosphate bodies, lipids, vacuoles, cell wall and periplasmalemmal space. Also the ultrastructure of Euglena gracilis showed significant changes in the presence of cadmium including myelin-like structures in mitochondria, chloroplasts altered in shape, and thylakoid arrangement and increase of osmiophilic plastoglobuli (Duret et al. 1986; Einicker-Lamas et al. 1996). The authors conclude that such alterations indicate that respiratory processes are the initial target of cadmium in this alga (Duret et al. 1986). Special attention has been paid in literature to membrane alterations. The degenerative process described in Phaeodactylum tricornutum cells growing in the presence of copper have been reported to be closely related with alterations or disorders in membrane systems (Cid et al. 1996). Thus, flow cytometry was used to show an increase in the cytoplasmic membrane potential and in the mitochondrial membrane potential in Phaeodactylum tricornutum cells growth in the presence of copper. As described by Demidchik et al. (2001) copper has been found to inhibit plasma-membrane ATPase activity. 2. Mechanisms for heavy metal detoxification and tolerance There is much contention in the literature over the possible mechanisms of metal tolerance. This could indicate a general lack of understanding of metal toxicity issues but is just as likely to reflect the complex nature of higher plants responses to metal toxicity. Tolerance to heavy metals in plants may be defined as the ability to survive in a soil that is toxic to other plants, and is manifested by an interaction between a genotype and its environment (Macnair et al., 2000), although the term is frequently used more widely in the literature to include changes that may occur experimentally in the sensitive response to heavy metals. In a number of thorough genetic studies, such adaptive metal tolerance has been shown to be governed by a small number of major genes with perhaps contributions from some more minor modifier genes (Macnair, 1993; Macnair et al., 2000; Sach et al., 2000). The question of whether this means that only a single biochemical or molecular change is required to produce tolerance to a specific metal remains to be resolved. Related to this question is the occurrence of multiple tolerance and co-tolerance where plants can grow on soils enriched in combinations of several heavy metals. This tolerance could result from a less specific mechanism that confers a broad resistance to several different metals (co-tolerance) or may involve a series of independent metal-specific mechanisms (multiple tolerance) (Schat et al., 2000). However, the evidence for co-tolerance is not strong, suggesting that specific mechanisms are involved for each metal present at a toxic concentration (Macnair et al., 2000; Schat et al., 2000). It is quite likely that different species may have evolved different mechanisms to tolerance excess metals and that even within the one plant species more than one mechanism could be in operation. Plants have both constitutive (utilised by tolerant phenotypes only) mechanisms to withstand excess metals (Meharg, 1994). There are a number of strategies the plants could possibly employ to combat high external metal concentrations. These can be classified in two main categories, i.e. firstly, restriction of uptake or transport and secondly, internal tolerance mechanisms. 2.1. Restriction of uptake or transport 2.1.1. Exclusion One mechanism of preventing the toxicity effects of metals is preventing excess metals from entering the plant. Two main mechanisms have been described in which a plant could do this, either by precipitating or by complexing metals in the plant environment. The binding properties of the cell wall and its role as a mechanism of metal tolerance has been reviewed (Ernst et al., 1992). As an example, it has been reported that the heavy-metal tolerant Silene vulgaris ssp. Humilis accumulated a range of metals in the epidermal cell walls, either bound to a protein or as silicates (Bringezu et al., 1999). One related process concerns the role plant exudates in metal tolerance. Plant exudates have a variety of roles (Marschner, 1995) including that of metal chelators that may enhance the uptake of certain metals. In an investigation into the role of Ni-chelating exudates in Ni hyperaccumulating plants, it was observed that the Ni-chelating histidine and citrate accumulated in the root exudates of non-hyperaccumulating plants, and thus could help to reduce Ni uptake and so play a role in a Ni-detoxification strategy (Salt et al., 2000). Since the range of compounds exuded is wide, other exudates could play a role in tolerance to other metals. The clearest example of a role for root secretions in tolerance is in relation to organic acids and the detoxification of the light metal Al (Ma et al., 2001). Buckwheat, for example, secretes oxalic acid from the roots in response to Al stress, and accumulates non-toxic Al-oxalate in the leaves (Ma et al., 1997); thus detoxification occurs both externally and internally. Examples of metal complexation by exudates have been described also in marine microorganisms. As example, in the marine diatom Skeletonema costatum (Grev.) complexation of Zn by algal exudates has been reported (Schintu M. et al., 1999). Many of these cations are essential and so complete cellular exclusion of the metal is not possible; selective efflux may be more realistic. In bacteria, for example, most resistance systems are based on the energy-dependent efflux of toxic ions (Silver, 1996). The number of examples of exclusion or reduced uptake mechanisms in higher plants is quite limited. The clearest example of reduced uptake as an adapted tolerance mechanism is in relation to arsenic toxicity (Meharg and Macnair, 1990, 1992). In Holcus lanatus roots, phosphate and arsenate appear to be taken up by the same systems. However, an arsenate-tolerant genotype showed a much lower rate of uptake for both anions than the non-tolerant genotype, and also showed an absence of the high-affinity uptake system. The altered phosphate and arsenate uptake system was genetically correlated to arsenate tolerance (Meharg and Macnair, 1992). Further work has suggested that arsenate tolerance in H. Lanatus requires both this adaptive suppression of the high affinity transport system, together with constitutive phytochelatin (PC) production since arsenate can still accumulate to high levels in tolerant plants (Hartley-Whitaker et a., 2001a). An alternative strategy for controlling intracellular metal levels at the plasma membrane involves the active efflux of metal ions, although there is very little direct evidence for such a process in plants. In bacteria, for example, efflux pumping is the basis of most toxic ion resistance systems, involving transporters such as P-type ATPases or cation/H+ antiporters (Silver and Ji, 1994; Silver, 1996); efflux pumping systems have been identified for Cu, Cd, Zn, Co and Ni (Silver, 1996). Although there is no direct evidence for a role for plasma membrane efflux transporters in heavy metal tolerance in plants, recent research has revealed that plant posses several classes of metal transporters that must be involved in metal uptake and homeostasis in general, and thus could play a key role in tolerance. These include the heavy metal CPx-ATPases, the Nramps, and the CDF (cation diffusion facilitator) family (Williams et al., 2000), and the ZIP family (Guerinot, 2000). Recently, a role for the Nramps in Fe and Cd uptake has been reported (Thomine et al., 2000); interestingly, disruption of an AtNramp 3 gene slightly increased Cd resistance, whereas overexpression resulted in Cd hypersensitivity in Arabidopsis. In the Zn/Cd hyperaccumulator Thlaspi caerulescens, Pence et al. cloned a transporter, ZNT1, that mediates high-affinity Zn uptake as well as low affinity Cd uptake, and is expressed at high levels in the roots and shoots (Pence et al., 2000). Increased expression, resulting from changes in the plant Zn status, led to increased Zn infflux in the roots. However, the transport function, specificity and cellular location of most of these proteins in plants is as yet unknown. 2.1.2. Protection of plasma membrane integrity The plant plasma membrane may be regarded as the first structure that is a target for heavy metal toxicity. Plasma membrane function may be rapidly affected by heavy metals by an increased leakage from cells in the presence of high concentrations of metals, particularly of Cu. For example, it was shown that Cu, but not Zn, caused increased K+ efflux from excised roots of Agrostis capillaris (Wainwright and Woolhouse, 1977). Similary, others concluded that damage to the cell membrane, monitored by ion leakage, was the primary cause of Cu toxicity in roots of Silene vulgaris, Mimulus guttatus, and wheat, respectively (De Vos et al., 1991; Strange and Macnair, 1991; Quartacci et al., 2001). Such damage could result from various mechanisms including the oxidation and cross-linking of proteins thiols, inhibition of key membrane proteins such as the H+ -ATPase, or changes to the composition and fluidity of membrane lipids (Meharg, 1993). Certainly direct effects of Cu and Cd treatments on the lipid composition of membranes have been reported (Fodor et al., 1995; Hernández and Cooke, 1997; Quartacci et al., 2001) which may have a direct effect on membrane permeability. Thus tolerance may involve the protection of plasma membrane integrity against heavy metal damage that would produce increased leakage of solutes from cells. However, there is little evidence to show how this might be achieved. For example, metal-tolerant plants do not appear to posses enhanced tolerance to free radicals or reactive oxygen species, but rather rely on improved mechanisms for metal homeostasis (Dietz et al., 1999). Again these effects on membranes are metal-specific since, in contrast to Cu, Zn protects membranes against oxidation and generally does not cause membrane leakage (Ernst et al., 1992; Cakmak, 2000). Another factor that may be involved in the maintenance of plasma membrane integrity in the presence of heavy metals could be enhanced membrane repair after damage (Salt et al., 1998). This could involve heat shock proteins or metallothioneins. 2.2. Compartmentation, complexing and reparation within the cell 2.2.1. Compartmentation within vacuoles Efflux of ions at the plasma membrane or transport into the vacuole are two ways of reducing the levels of toxic metals in the cytosol and so are potentially important mechanisms for heavy metal tolerance. One well-documented example, the accumulation of Cd and PCs in the vacuole involving an ABC transporter, has already been described ( ), but there is evidence that vacuole may be important in the accumulation of other metals involving other tonoplast transport systems. Earlier studies showed that the vacuole is the site for the accumulation of a number of heavy metals including Zn and Cd (for reviews see Ernst et al., 1992; De, 2000). Apart from the Cd-PC accumulation process, the best evidence for a role of vacuolar accumulation in relation to metal tolerance is for Zn. For example, uptake analysis using Zn65 with barley leaves suggested that rapid compartmentation of Zn into the vacuole was an important mechanism for dealing with high levels of Zn (Brune et al., 1994). Further studies on barley levels showed that, although Cd, Zn and Mo were found mainly in the vacuole, Ni was primarily found in the cytosol and this appeared to be related to the development of leaf damage (Brune et al., 1995); however, compartmentalization in the roots was not examined. Analysis of transport systems at the tonoplast has added support to a vacuolar mechanism of tolerance. Verkleij et al. isolated tonoplast vesicles from roots of Zn-tolerant and –sensitive ecotypes of Silene vulgaris (Verkleij et al., 1998). They showed that at high Zn concentrations, Zn transport was 2,5 times higher into vesicles from the tolerant lines than from the sensitive ones, suggesting that the tonoplast plays an important role in naturally selected Zn tolerance. More recently, an Arabidopsis gene (ZAT) was isolated that is closely related to the animal ZnT (Zn transporter) genes (Van der Zaal et al., 1999). ZAT mRNA seemed to be expressed constitutively throughout the plant and was not induced by higher Zn concentrations. However, overexpression of ZAT in transgenic plants led to a significant increase in Zn resistance and an enhanced accumulation in the root under high Zn treatments. Thus the Zn transporter could be envolved in sequestration of Zn in the vacuole and thus in Zn tolerance in plants. The marine diatom Skeletonema costatum was used to study mechanisms of detoxification when submitted to cadmium and copper contamination. Copper resulted more toxic for S. costatum than cadmium. Heavy cellular damages were observed for cadmium and copper toxic concentrations. Exposure to these concentrations induced a migration of inclusions from the peripheral cytoplasm to the vacuole. Electron energy loss spectroscopy (EELS) investigations demonstrated that Cd and Cu were specifically trapped in these inclusions. However, Cu was less sequestered than cadmium in the vacuole. EELS determination of oxidation states evidenced that trace metals were sequestered as Cd2+ and Cu2+. Nitrogen and sulfur are involved in metallic storage, especially in the case of cadmium contamination (Nassiri Y. Et al, 1997). 2.2.2. Complexing by metallothioneins Higher plants contain two major types of cysteine-rich, metal-binding peptides, the metallothioneins (MTs) and the phytochelatins (PCs). MTs are gene-encoded plypeptides that are usually clssified into two groups. Class 1 MTs posses cysteine residues that align with a mammalian (equine) renal MT; Class 2 MTs also posses similar cysteine clusters but these cannot be easily aligned with Class 1 MTs (de Miranda et al., 1990; Robinson et al., 1993; Prasad, 1999). MT genes have now been identified in a range of higher plants (Prasad, 1999) including Arabidopsis where, in addition to Class 1 and Class 2 MT genes, MT3 and MT4 types have been recognized (Goldsbrough, 2000). In plants, there is a lack of information concerning the metals likely to be bound by MTs, although Cu, Zn and Cd have been the most widely studied (Tomsett and Thurman, 1988; Robinson et al., 1993; Goldsborough, 2000). Although MTs can be induced by Cu treatments and there is evidence for a role in heavy metal tolerance in fungi and animals (Hamer, 1986), the role of MTs in heavy metal detoxification in plants remains to be established (Zhou and Goldsbrough, 1994; Zenk, 1996; Giritch et al., 1998; Schat et al., 2000). However, it has been reported that MT2 mRNA was strongly induced in Arabidopsis seedlings by Cu, but only slightly by Cd and Zn (Zhou and Goldsbrough, 1994); when genes for MT1 and MT2 from Arabidopsis were expressed in an MT-deficient yeast mutant, both genes complemented the mutation and provided a high level of resistance to Cu. Van Vliet et al. showed that MT genes can be induced by Cu, and that the expression of MT2 RNA is increased in a Cu-sensitive mutant of Arabidopsis that accumulates high concentrations of Cu (van Vliet et al., 1995). More evidence is needed to establish a relationship between Cu sensitivity and MT production. By contrast, Haag-Kerwar et al. (1999) argued against a role for MT2 relative to PCs in Cd detoxification. The role of MTs remains to be established. They could clearly play a role in metal metabolism (Hamer, 1986) and a possible role as antioxidants has been reported (Dietz et al., 1999). As they are the first food-chain step in the sea nature, microalgae are one of the most important organisms in our ecosystems being seriously affected by metal pollution. MTs are among the resistance mechanisms to metals found in microalgae. The microalgae Tetraselmis suecica (Kylin) Butch is tolerant to cadmium. Class III metallothioneins were investigated for their involvement as a possible tolerance mechanism in this microalga when exposed to cadmium. The T. suecica cells were able to synthesize class III metallothioneins of three to six subunits of (g-Glu-Cys) (Perez-Rama M. et al., 2001). 2.2.3. Complexing by phytochelatins Chelation of metals in the cytosol by high-affinity ligands is potentially a very important mechanism of heavy-metal detoxification and tolerance. Potential ligans include amino acids and organic acids, and two classes of peptides, the phytochelatins and the metallothioneins (Rauser, 1999; Clemens, 2001). The phytochelatins have been the most widely studied in plants, particularly in relation to Cd tolerance (Cobbet, 2000; Goldsbrough, 2000). The phytochelatins (PCs) are a family of metal-complexing peptides that have a general structure (γ- Glu Cys)n-Gly where n=2-11, and are rapidly induced in plants by heavy metal treatments (Rauser, 1995; Zenk, 1996; Cobbett, 2000; Goldsbrough, 2000). PCs are synthesized non-translationally using gluthatione as a substrate by PC synthase (Grill et al., 1989; Rauser, 1995), an enzyme that is activated in the presence of metal ions (Cobbett, 2000). The genes for PC synthase have now been identified in Arabidopsis and yeast (Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et al., 1999). Evidence has been presented both for and against a role for PCs in heavy metal tolerance (for reviews see Ernst et al., 1992; Meharg, 1994; Zenk, 1996; Cobbett, 2000; Goldsbrough, 2000). However, a clear role in Cd detoxification has been supported by a range of biochemical and genetic evidence. Howden et al. isolated a series of Cd-sensitive mutants of Arabidopsis that varied in their ability to accumulate PCs; the amount of PCs accumulated by the mutants correlated with the degree of sensitivity to Cd (Howden et al., 1995a, b). Using Brassica juncea, it has been shown that Cd accumulation is accompanied by a rapid induction of PC biosynthesis and that the PC content was theoretically sufficient to chelate all Cd taken up; this protects photosynthesis but did not prevent a decline in transpiration rate (Haag-Kerwer et al., 1999). Using Arabidopsis, Xiang and Oliver showed that treatment with Cd and Cu resulted in increased transcription of the genes for gluthatione synthesis, and the response was specific for those metals thought to be detoxified by PCs (Xiang and Oliver, 1998). Zhu et al. overexpressed the γ-glutamylcysteine synthetase gene from E. Coli in Brassica juncea resulting in increased biosynthesis of glutathione and PCs and an increased tolerance to Cd (Zhu et al., 1999). A similar approach was taken with Arabidopsis; γ-glutamylcysteine synthetase was expressed in both sense and antisense orientations resulting in plants with a wide range of glutathione levels (Xiang et al., 2001). Plants with low glutathione levels were hypersensitive to Cd, although elevating the levels above wild-type did not increase metal resistance. The gene for PC synthase (CAD1) has been identified in Arabidopsis as well as an homologous gene in Schizosaccharomyces pombe (Ha et al., 1999); a mutant of the latter with a targeted deletion of this gene was PC-deficient and Cd-sensitive. To compare the involvement of PCs in metal detoxification, the sensitivity of the cad 1-3 mutant was tested for sensitivity to a range of heavy metals in both Arabidopsis and S. Pombe (Ha et al., 1999). PCs appeared to be important in the detoxification of Cd and arsenate, but played no role in the detoxification of Zn, Ni and selenite ions. In contrast to the S. Pombe mutant, cad 1-3 showed slight sensitivity to Cu and Hg. A possible role for PCs in Cu tolerance had also been suggested (Salt et al., 1989) from studies on copper-tolerant Mimulus guttatus; exposure to Cu in the presence of buthionine sulphoximine (BSO), a potent inhibitor of γ-glutamylcysteinil synthetase, caused a considerable reduction in root growth that was not seen in the presence of inhibitor alone. The role of PCs in Cu tolerance remains to be resolved. An involvement of PCs in arsenate tolerance has also been proposed (Hartley-Whitaker et al., 2001a, b). Although evidence for the role for PCs in detoxification is strong, especially for Cd, these peptides may play other important roles in the cell, including essential heavy-metal homeostasis, sulphur metabolism or, perhaps, as anti-oxidants (Rauser, 1995; Dietz et al., 1999; Cobbett, 2000). Their participation in the detoxification of excess concentrations of some heavy metals may be a consequence of these other functions (Steffens, 1990). Certainly the role of PCs in adaptative tolerance has been questioned (Meharg, 1994; Schat et al., 2000). It was suggested that the general lack of examples of co-tolerance indicates that adaptive tolerance is unlikely to be produced by changes in relatively non-specific binding compounds such as PCs (or metallothioneins or organic acids) (Macnair et al., 2000). 2.2.4. Synthesis of phytochelatins in phytoplancton Phytochelatins quantification is a difficult task which requires very precise and sensitive methods. Using a very sensitive HPLC method, it is have been quantified phytochelatins from phytoplankton in laboratory cultures at environmentally relevant metal concentrations and in marine field samples. Intracellular concentrations of phytochelatin, in the diatom Thalassiosira weissflogii, exhibit a distinct dose-response relation with free Cd2+ concentration in the medium and are detectable even when the free Cd2+ concentration is less than 1 pM. Ambient phytochelatin concentrations thus appear to provide a measure of the metal stress resulting from the complex mixture of trace metals and chelators in natural waters (Ahner et al., 1994). Phytochelatins have been found to be synthetised also in response to the presence of metal mixtures including metals other than cadmium. Interestingly, Stigeoclonium sp. exposed to high concentrations of individual metals (Zn, Pb and Cd available as free cations) synthesised a great amount of phytochelatins (PC2-PC4). The order of PC induction by the studied metals in the Stigeoclonium sp. was Cd > Pb > Zn. The PC production in algae of the genus (Pawlik-Skowronska B., 2001). The production of phytochelatins have been shown to occur in the presence of individual metals other than cadmium. As example, in Stichococcus bacillaris, an ubiquitous green microalga, in the studied concentration range 0.1-20 mM, inorganic lead caused a significant production of induced thiol peptides: PC (n=2-4) and some other unidentified oligopeptides, probably (GluCys)n. The time of appearance and the concentration of individual oligomers of phytochelatins were dependent on the external Pb concentration and time of metal exposure. The rapid formation of these peptides in S. bacillaris in response to Pb, and their elimination (by about 90%) when algae were placed into the Pb-free solution reveal a tight regulation of GSH and phytochelatin pools in the algal cells exposed to toxic metals. The authors suggest that both PCs and GSH are the primary line of defence against the Pb toxicity (Pawlik-Skowronska B., 2000). Cd-exposed riverine diatom Thalassiosira pseudonana made more phytochelatins than Cu-exposed cells. It is hypothesized that T. pseudonana has evolved an effective detoxification mechanism as a result of a more severe exposure to toxic metals in rivers and estuaries. In contrast, Ditylum brightwellii, a marine-estuarine species, cannot adjust well to metal exposure. Its poor defense against metal toxicity was marked by low SH- contents. (Rijstenbil J.W. et al., 1994). 2.2.5. Complexing by organic acids and amino acids Carboxylic acids and amino acids such as citric, malic and histidine are potential ligands for heavy metals and so could play a role in tolerance and detoxification(for reviews see Rauser, 1999; Clemens, 2001); however strong evidence for a function in tolerance has not been reported. A 36-fold increase was reported in the histidine content of the xylem sap on exposure to Ni in the Ni-hyperaccumulating plant Alyssum lesbiacum (Kramer et al., 1996). In addition, supplying histidine to a non-accumulating species greatly increased both its Ni tolerance and the capacity for Ni transport to the shoot. 2.6. Reparation with Heat Shock Proteins Heat shock proteins (HSPs) characteristically show increased expression in response to the growth of a variety of organisms temperatures above their optimal growth temperature. They are found in all groups of living organisms, can be classified according to molecular size and are now known to be expressed in response to a variety of stress conditions including heavy metals (Vierling, 1991; Lewis et al., 1999); they act as molecular chaperones in normal protein folding and assembly, but may also function in the protection and repair of proteins under stress conditions. There have been several reports of an increase in HSP expression in plants in response to heavy metal stress. Tseng et a. Showed that, in rice, both heat stress and heavy-metal stress increased the levels of mRNAs for low molecular mass HSPs (16-20 kDa) (Tseng et al., 1993), while Neumann et al. indicated that HSP17 is expressed in roots of Armeria maritima plants grown on Cu-rich soils (Neuman et al., 1995). Small heat shock proteins (e.g. HSP17) were also shown to increase in cell cultures of Silene vulgaris andLycopersicon peruvianum in response to a range of heavy metal treatments (Wollgiehn and Neumann, 1999); however no or very low amounts of HSPs were found in plants growing on metalliferous soils, suggesting that HSPs are not responsible for the heritable metal tolerance of Silene. Working with cell cultures of L. Peruvianum, it was shown that a larger HSP (HSP70) also responds to Cd stress (Neumann et al., 1994). It is of interest that antibody localization showed that HSP70 could be involved in the protection of membranes against Cd damage. Expression of HSP70 also increased in the seaweed Enteromorpha intestinalis after exposure to a variety of stressors including Cu (Lewis et al., 2001). Thus, in relation to earlier discussions of tolerance mechanisms involving a more resistant plasma membrane or improved repair mechanisms, HSPs could have an important role in this respect. Interestingly, it was reported that a short heat stress given prior to heavy-metal stress induces a tolerance effect by preventing membrane damage, as judged by ultrastructural studies (Neumann et al., 1994). Clearly more molecular evidence is required to support such an important repair or protective role. The heat-shock response of Euglena gracilis was studied by cell labeling at both the normal growth temperature (23 degrees C) and an elevated temperature (35 degrees C). Analysis of the labeled proteins by two-dimensional polyacrylamide gel electrophoresis indicated that the rate of synthesis of two polypeptides p55 (55 kDa) and p40 (40 kDa) increased in cells labeled at the highest temperature studied. These polypeptides are also overexpressed in Cadmium-resistant Euglena gracilis cells labeled at the normal growth temperature (23 degrees C). On the basis of these results, p55 and p40 appear to be heat-shock proteins involved in some steps of the acquired Cd-resistance process in Euglena gracilis cells (Barque J.P. et al., 1994). 3. Applications 3.1. Metal bioremoval by microalgae Bioremoval is defined as the accumulation and concentration of pollutants from aqueous solutions by the use of biological materials, thus allowing the recovery and/or environmentally acceptable disposal of the pollutants. Either plant or microbial biomass can be used for this purpose; the latter is more commonly used (Gadd 1988; Volesky 1990). The idea of using microalgae in bioremoval processes was initially proposed by Oswald and Gotaas (1957) but gained interest in recent years. Biological systems to remove metal ions from polluted waters could emerge as the potential alternative to chemical treatments (Volesky 1990; Maeda and Sakaguchi 1990). Nowadays the process included the addition of chemicals for metal precipitation or exchange resins to bind them. Other less frequently employed methods are activated carbon adsorption, electrodialysis and reverse osmosis. One of the main interests of microalgae in biotechnology is focussed on their use for heavy metals and radionuclide removal from effluents and wastewaters. In parallel to detoxification it is possible to recover valuable elements such as gold and silver after appropriate treatment of the loaded microbial biomass. Many types of biomass in non-living form have been studied for their heavy metal uptake capacities and suitability to be used as bases for biosorbent development. These include bacteria (Strandberg et al., 1981; Scott and Karanjkar, 1992; Sag andKutsal, 1995), fungi (Tobin et al., 1984; Huang et al., 1991; Fourest et al., 1994; Matheickal and Yu, 1997), yeast (Huang et al., 1990; Volesky et al., 1993; Matheickal and Yu, 1996), fresh water algae (Crist et al., 1981; Ozer et al., 1994), marine algae (Holan et al., 1993; Chong and Volesky, 1995; Fourest and Volesky, 1996; Matheickal and Yu, 1996; Matheickal et al., 1997, 1998) and others (Freer et al., 1989; Deshkar et al., 1990; Schneider et al., 1995). In general, the heavy metal uptake capacities varied significantly for different types of biomass studied. For divalent heavy metal ions, the reported values for bacterium biomass typically ranged from 0.05 to 0.2 mmol/g; for fungi and yeast, 0.2 to 0.5 mmol/g; for fresh water algae, 0.5 to 1.0 mmol/g and for marine algae, 1.0 to 1.5 mmol/ g. Among these values, the capacities of the biomass of a few species of marine macro algae, commonly known as brown algae, were much higher than those of other types of biomass. They were also much higher than those of activated carbon and natural zeolite and were comparable to those of synthetic ion exchange resins (Matheickal et al. 1997). Marine macro algae are harvested/cultivated in many parts of the world. They are readily available in large quantities for the development of highly effective biosorbent materials. However, considering the large number of macro-algae species identified so far, only a few have been studied for their heavy metal uptake properties. Most of these studies were limited to the Ascophyllum and Sargassum species (Holan et al., 1993; Volesky, 1994; Chong and Volesky, 1995; Fourest and Volesky, 1996). Metal contamination of the aquatic environment affects organisms at the biochemical, cellular, community and population level. Algae have been commonly used as test organisms because of their ecological importance as primary producers of most aquatic food chains and account for much of the production base of freshwater and marine ecosystems. Microalgae have applications in heavy metals removal from aquatic environments since they have a high capability for accumulating dissolved metals without dying (Rai L.C. et al., 1981; Sandau E. et al., 1996; Vílchez C. et al., 1997). In microalgae there are two main types of mechanisms for metal removal processes. First, passive uptake, also called biosorption. This mechanism is metabolically independent. Biosorption is reversible and very rapid (it is completed in 5–10 min). The amount of metal accumulation per unit of biomass is proportional to the concentration of metal ion in the solution. In addition, biosorption can be affected by pH and the presence of other ions in the medium that may precipitate heavy metals as insoluble salts, but it is unaffected by metabolic inhibitors or light/dark cycles. Second, active uptake is metabolically- dependent and more effective than biosorption for low concentrations of heavy metals (below 1 ppm). It may involve metal ion consumption for algal growth and/or intracellular accumulation of heavy metals. In addition, heavy metals may be precipitated by excreted secondary metabolites. Both mechanisms can work simultaneously in microalgae, and their importance may depend on the algal specie, culture conditions and the metal chemical properties. In Ankistrodesmus braunii and Chlorella vulgaris cadmium binding to cell walls accounted for approximately 80% of total uptake. Biosorption was the major uptake component in C. vulgaris with respect to a wide range of other metals including uranium. In Eremosphaera viridis, however, intracellular uptake comprised the majority of total uptake. A wide range of metal ions is bonded by living cells of microalgae including Cu2+, Zn2+, Ba2+, Mn2+, Co2+, Cd2+, Ni2+, Sr2+, Hg2+ and Ag+ (Rai et al. 1994). Both mechanisms can work simultaneously in algae cells. However, biosorption is the major uptake component in some algae as C. vulgaris (Bedell G.W. et al., 1990; Kuyucak N. et al., 1990; Vílchez C. et al., 1997; Wilde E.W. et al., 1993). Culture medium pH is an important factor directly affecting the toxicity of metals in algae. Acidity or alkalinity of the medium can, in turn, moderate the toxicity of heavy metals. Lower pH may increase the bioavailability of metal ions resulting in increased toxicity but for instance favouring metal accumulation by algae (Franklin N.M. et al., 2000; Macfie S.M. et al., 1994; Bedell G.W. et al., 1990; Kuyucak N. et al., 1990; Rachlin J.W. et al., 1993). Biosorption of heavy metals from aqueous solutions is a relatively new technology for the treatment of industrial wastewater (Volesky, 1990). Adsorbent materials (biosorbents) derived from suitable biomass can be used for the effective removal and recovery of heavy metal ions from wastewater streams. The major advantages of the biosorption technology are its effectiveness in reducing the concentration of heavy metal ions to very low levels and the use of inexpensive biosorbent materials. Biosorption processes are particularly suitable for the treatment of waste water streams containing dilute heavy metal ion concentrations, or when very low concentrations of heavy metals are required (Volesky, 1990). The limitations of the technology include that large-scale production of effective biosorbent materials has not been established and that the technology has only been tested for limited practical applications. 3.1.1. Cadmium Cadmium toxicity is a major problem affecting crop productivity world-wide. The presence of cadmium in the rhizosphere can cause alterations in many physiological processes including nitrogen metabolism (Hernández et al. 1997), photosynthesis (Krupa et al. 1993), carbohydrate metabolism (Greger and Bertell, 1992) and sulphate assimilation (Nussbaum et al. 1988). The dramatic decrease in the biomass production reported in plants fed with cadmium (Chaoui et al. 1997) may be a consequence of a reduced uptake and transport of several essential nutrients within the plant (Ouariti et al. 1997). Cadmium could also have a direct influence on metabolism through the inactivation of several enzymes (Chugh et al. 1992; Ju et al. 1997). Many papers have been published that report cadmium sequestration by microalgae. Cadmium sequestration by Chlamydomonas has been extensively studied on base of the synthesis of intracellular cadmium chelators, phytochelatins (Hu et al. 2001; Howe and Merchant 1992). Chorella homosphaera cells immobilized in alginate supply a good system to remove cadmium together with zinc and gold from water. When the initial concentration of heavy metals ranged between 20-720 mg.l-1, up to 99% of cadmium and zinc were removed after 60 min and 90% after 30 min (40% associated with matrix) (da Costa and Leite 1991). Cadmium removal was also reported by Volesky and Prasetyo (1994) using a new biosorbent material derived from a brown marine alga (Ascophyllum nodosum) in a packed-bed flow-through column. It reached a 99.98% removal from an effluent containing 10 mg Cd.l-1. Dead biomass of microalgae can directly be used for metal biosorption, working the cell wall like a metal ion exchanger (Rai et al. 1994). Cadmium absorption by non-living biomass of microalgae (Fehrmann and Pohl 1993) and the use of algal biomass for mercury extraction from groundwaters (Barkley 1991) can be cited as example. Algal cells were immobilized in a permeable polymeric matrix. The product so-called AlgaSORB, packed into adsorption columns, exhibited excellent flow characteristics and acted like a “biological” ion-exchange resin. Mercury also accumulated in immobilized C. emersonii cells (Wilkinson et al. 1989). 3.1.2. Lead Screening tests of different marine algae biomass types revealed a high passive biosorptive lead uptake up to 270 mg Pb g-1 biomass. This limit was increased to 370 mg Pb g-1 in cross-linked Fucus vesiculosus and Ascophyllum nodosum; however ion- exchange resin Amberlite IR-120 presents a higher lead uptake than biosorbent materials (Holan and Volesky 1994). Accumulation of lead and zinc from metalliferous spoil tip stream has been reported by Pyatt et al (1993) by using differnet algae. 3.1.3. Copper Although copper is important as a micronutrient for algal growth, it is highly toxic at elevated concentrations in the ionic forms (Sunda and Guillard 1996). Photosynthesis of unicellular algae was inhibited by copper (Overnell 1976). Thomas et al. (1980) described effects on the morphology of phytoplankton. The effects of different copper (II) species on algae have recently been reviewed by Gledhill et al. (1997). Copper uptake in microalgae can occur at the extend to milimolar ( ). Different experiments about the accumulation capacity for copper and zinc have been reported in the marine microalgae Nannochloropsis gaditana Lubian (Eustigmatophyceae). Accumulation differences between free and immobilized microalgal cells were investigated finding no differences for copper, and little differences for zinc. Free cells accumulated practically 100% Cu or Zn in the media under experimental conditions. Experiments in order to compare the accumulation capacity of living vs. dead cells were designed too, obtaining the largest accumulation levels for both metals in the beads containing immobilized living microalgae. In all experiments the authors reported that the calcium alginate beads showed strong affinity for Cu (Moreno-Garrido et al. 2002). 3.1.4. Iron Iron is an important micronutrient to algae, as web as to vascular plants, since it is constituent of many enzymes and cytochromes and essential for chlorophyll synthesis (Raven 1988). Recently it has been suggested that iron is one of the major limiting elements for primary productivity in the open ocean (Geider and La Roche 1994). Large quantities of iron are present in seawater as ferric hydroxide, bound to particulate matter or are precipitated (Suzuki et al. 1995) and are therefore not available to marine organisms. 3.1.5. Mercury Biological systems to remove metal ions from polluted waters could emerge as the potential alternative to chemical treatments (Volesky 1990; Maeda and Sakaguchi 1990). Nowadays the process included the addition of chemicals for metal precipitation or exchange resins to bind them. Other less frequently employed methods are activated carbon adsorption, electrodialysis and reverse osmosis. One of the main interests of microalgae in biotechnology is focussed on their use for heavy metals and radionuclide removal from effluents and wastewaters. In parallel to detoxification it is possible to recover valuable elements such as gold and silver after appropriate treatment of the loaded microbial biomass. 3.1.6. Uranium Freshwater Chlorella, Scenedesmus and Chlamydomonas sp were capable of taking up significant amounts of uranium (Muraleedharan et al. 1991). Green algae and cyanobacteria were found to have fewer reactive surfaces than diatoms. 3.2. Algae as metal bioindicators The capacity of metal binding of photosynthetic organisms it has been found to display seasonal variations. Two species of macroalgae, Porphyra spp. and Enteromorpha spp., display similar and marked seasonal variations in the concentration factor (CF) of Cu, Pb, Cd and Hg in field conditions (Vasconcelos and Leal 2001). The CF variations are specific for each metal and reproducible over several years. The authors showed that the maximum binding capacity of the algae was not significantly dependent on the season for Pb, Cd and Hg. All the tested metals promoted the liberation of exudates, both cysteine- and glutathione-like ligands were exuded in the presence of Cu, only cysteine-like ligands in the presence of Pb, and only glutathione-like ligands in the presence of Cd, the rise depending of the season of the year, particularly for Cu. Highest levels were produced in the presence of added Pb. Other algae have been shown as good metal bioindicators due to its specific patterns of metal binding. The uptake kinetics of four metals (Cd, Cr, Se and Zn) have been reported in two marine macroalgae (the green alga Ulva lactuca and the red alga Gracilaria blodgettii) which could have utility as bioindicators ( ). The authors show that metal uptake generally displayed a linear pattern with increasing exposure time. With the exception of Cr, which exhibited comparable uptake rate constants at different concentrations, uptake rate constants of Cd, Se and Zn decreased with increasing metal concentration, indicating that the seaweeds had a higher relative uptake at lower metal concentration. Uptake of Cd and Zn was higher in U. lactuca than in G, blodgettii, whereas uptake of Cr and Se was comparable between the two species. Only Cd and Zn uptake in U. lactuca was significantly inhibited by dark exposure. A decrease in salinity enhanced the uptake of Cd, Cr, Se and Zn in U. lactuca. In G. blodgettii, Cd uptake increased twofold when salinity was decreased, whereas uptake of Cr and Zn was not significantly affected by salinity change. The predicted bioconcentration factor was 3 x 10(4) for Cd, 2 x 10(3) for Cr, 40 to 150 for Se, and 1 to 2 x 10(4) for Zn in U. lactuca. The kinetic study suggested that U. lactuca would be a good biomonitor of Cr and Zn contamination in coastal waters. In recent years interesting papers have been published that report different methods of application in bioindication for metals. In cultures of Selenastrum capricornutum the addition of low concentrations of copper significantly stimulates a unique ascorbate-dependent peroxidase activity of this organism. This enzymatic system could be used as a sensitive bioindicator for copper in fresh-water (Sauser et al. 1997; Wong et al. 2001). The pulse-amplitude-modulation (PAM) fluorimetric method was used in the past as a rapid method for assesing toxic effect of pollutants in plants. Recently, PAM has been reported to provide a very sensitive method in copper bioassay (Juneau et al. 2002). Franklin et al. (2001) showed that esterase activity is a sensitive indicator of copper toxicity in S. capricornutum. 3.3. Enhancement of secondary metabolites production The presence of metals has been shown to enhance the byosinthesis of secondary metabolites in different photosynthetic organisms. Meisch et al. (1978) demonstrated that the trace element vanadium has a considerable possitive influence on the synthesis of d-aminolevulinic acid in the autotrophically grown green alga Chlorella pyreneidosa. It was suggested by the authors that vanadium acts as a catalyst in the conversion of 4,5-dioxovaleric acid to d-aminolevulinic by transamination.
Bibliography
Ahner BA, Morel FMM. Phytochelatin production in marine algae. Limnol. Oceanogr. 1995; 40, 658-665.
Ahner BA, Price NM, Morel FM. Phytochelatin production by marine phytoplankton at low free metal ion concentrations: laboratory studies and field data from Massachusetts Bay. Proc Natl Acad Sci U S A 1994 Aug 30;91(18): 8433-6.
Arazi T, Sunkar R, Kaplan B, Fromm H. A tobacco plasma membrane calmodulin-binding transporter confers Ni2+ tolerance and Pb2+ hypersensitivity in transgenic plants. Plant J. 1999 Oct;20(2):171-82.
Barque JP, Chacun H, Marouby S, Bonaly J. Cadmium resistance of achlorophyllous Euglena gracilis cells: constitutive overexpression of two heat-shock proteins. Biochem Biophys Res Commun 1994 Aug 30;203(1):540-4.
Bedell GW, Darnall DW. Immobilization of nonvi-able, biosorbent, algal biomass for the recovery of metal ions, in: Volesky B. (Ed.), Biosorption of heavy metals, CRC Press, Boca Raton, 1990, pp. 313–326.
Bilgrami KS, Kumar S. Effects of copper, lead and zinc on phytoplankton growth, Biol. Plant. 1997;39, 315–317.
Blechert S, Brodschelm W, Holder S, Kammerer L, Kutchan TM, Mueller MJ, Xia ZQ, Zenk MH. The octadecanoic pathway: signal molecules for the regulation of secondary pathways. Proc Natl Acad Sci U S A. 1995 May 9; 92(10): 4099-105.
Bozhkov AI. Three dose-dependent stages of the effect of copper ions on functional activity of biological systems. Biochemistry (Mosc) 1997 Feb; 62(2): 149-57.
Bringezu K, Lichtenberger O, Leopold I, Neumann D. Heavy metal tolerance of Silene vulgaris. Journal of Plant Physiology 1999; 154, 536-546.
Brune A, Urbach W, Dietz K-J. Compartmentation and transport of zinc in barley primary leaves as basic mechanisms involved in zinc tolerance. Plant, Cell and Environment 1994; 17,153-162.
Brune A, Urbach W, Dietz K-J. Differential toxicity of heavy metals is partly related to a loss of preferential extraplasmic compartmentation: a comparison of Cd-, Mo-, Ni- and Zn-stress. New Phytologist 1995; 129, 403-409.
Butler, A. Acquisition and utilization of transition metal ions by marine organisms. Science 1998; 281, 207 210.
Cain JR, Paschal DC, Hayden CM. Toxicity and bioaccumulation of cadmium in the colonial green alga Scenedesmus obliquus. Arch Environ Contam Toxicol 1980; 9(1):9-16.
Cakmak I. Possible roles of zinc in protecting plant cells from damage by reactive oxygen species. New Pohytologist 2000; 146, 185-205.
Chapman PM. Selenium - A potential time bomb or just another contaminant. Human Ecol. Risk Assess. 1999; 5, 1123-1138.
Chapman PM, Wang F, Janssen C, Persoone G, Allen HE. Ecotoxicology of metals in aquatic sediments: binding and release, bioavailability, risk assessment and remediation. Can. J. Fish. Aquat. Sci. 1998a; 55, 2221-2243.
Charbonnel-Campaa L, Lauga B, Combes D. Isolation of a type 2 metallothionein-like gene preferentially expressed in the tapetum in Zea mays. Gene 2000; 254, 199-208.
Chardonnens AN, Koevoets PLM, van Zanten A, Schat H, Verkleij JAC: Properties of enhanced tonoplast transport in naturally selected zinc-tolerant Silene vulgaris. Plant Physiol 1999, 120:779-785.
Chong KH, Volesky B. Description of two-metal biosorption equilibria by Langmuir-type models. Biotechnol. Bioeng. 1995; 47, 451-460.
Cid A, Fidalgo P, Herrero C, Abalde J. Toxic action of copper on the membrane system of a marine diatom measured by flow cytometry. Cytometry 1996 Sep 1; 25(1): 32-6.
Clemens S. Molecular mechanisms of plant metal tolerance and homeostasis. Planta 2001; 212, 475-486.
Clemens S, Kim EJ, Neumann D, Schroeder JI: Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J 1999, 18:3325-3333.
Cobbet CS. Phytochelatin bioshyntesis and function in heavy-metal detoxification. Current Opinion in Plant Biology 2000; 3, 211-216.
Collard JM, Matagne RF. Isolation and genetic analysis of Chlamydomonas reinhardtii strains resistant to cadmium. Appl Environ Microbiol 1990 Jul;56(7):2051-5.
Corradi MG, Gorbi G. Chromium toxicity on two linked trophic levels. II. Morphophysiological effects on Scenedesmus acutus. Ecotoxicol Environ Saf 1993 Feb; 25(1):72-8.
Crist RH, Oberholser K, Shank N, Nguyen M. Nature of bonding between metallic ions and algal cell walls. Environ. Sci. Technol. 1981; 15, 1212- 1217.
Davies KL, Davies MS, Francis D. The influence of an inhibitor of phytochelatin synthesis on root growth and root meristematic activity in Festuca rubra L. In response to zinc. New Phytologist 1991ª ; 118, 565-570.
Davies KL, Davies MS, Francis D. Zinc-induced vacuolation in root meristematic cells of Festuca rubra L. Plant, Cell and Environment 1991b ; 14, 399-406.
De DN. Plant cell vacuoles. Collingwood, Australia: CSIRO Publishing; 2000.
De Knecht JA, Koevoets PLM, Verkleij JAC, Ernst WHO. Evidence against a role for phytochelatin in naturally selected increased cadmium tolerance in Silene vulgaris (Moench) Garcke. New Phytologist 1992; 122, 681-688.
De Knecht JA, van Dillen M, Koevoets PLM, Schat H, Verkleij JAC, Ernst WHO. Phytochelatins in cadmium sensitive and cadmium-tolerant Silene vulgaris. Chain lenght distribution and sulfide incorporation. Plant Physiology 1994; 104, 255-261.
Demidchik V, Sokolik A, Yurin V. The effect of Cu2+ on ion transport systems of the plant cell plasmalemma. Plant Physiology 1997; 114, 1313-1325.
Demidchik V, Sokolik A, Yurin V. Characteristics of non-specific permeability and H+-ATPase inhibition induced in the plasma membrane of Nitella flexilis by excessive Cu2+. Planta 2001 Mar; 212(4): 583-90.
Dietz K-J, Baier M, Krämer U. Free radical and reactive oxygen species as mediators of heavy metal toxicity in plants. In: Prasad MNV, Hagemeyer J, eds. Heavy metal stress in plants: from molecules to ecosystems. Berlin: Springer-Verlag 1999; 73-97.
De Miranda JR, Thomas MA, Thurman DA, Tomsett AB. Metallothioneins genes from the flowering plant Mimulus guttatus. FEBS Letters 1989; 260, 277-280.
De Vos CHR, Schat H, De Waal MAM, Vooijs R, Ernst WHO. Increased resistance to copper-induced damage of the root cell plasmalemma in copper tolerant Silene cucubalus. Physiologia Plantarum 1991; 82, 523-528.
Deshkar AM, Bokada SS, Dara SS. Modified Hardwickia binata bark for adsorption of mercury(II) from water. Water Res. 1990; 24, 1011- 1016.
Dietz K-J, Baier M, Krämer U. 1999. Free radicals and reactive oxigen species as mediators of heavy metal toxicity in plants. In: Prasad MNV, Hagemeyer J, eds. Heavy metal stress in plants: from molecules to ecosystems. Berlin: Springer-Verlag, 73-97.
Duret S, Bonaly J, Bariaud A, Vannereau A, Mestre JC. Cadmium-induced ultrastructural changes in Euglena cells. Environ Res 1986 Feb; 39(1): 96-103.
Einicker-Lamas M, Soares MJ, Soares MS, Oliveira MM. Effects of cadmium on Euglena gracilis membrane lipids. Braz J Med Biol Res. 1996; Aug; 29(8): 941-8.
Ernst WHO, Verkleij JAC, Schat H. Metal tolerance in plants. Acta Botanica Neerlandica 1992; 41, 229-248.
Falchuk KH, Drishan A, Vallee BL. DNA distribution in the cell cycle of Euglena gracilis. Cytofluorometry of zinc deficient cells. Biochemistry 1975 Jul 29; 14(15): 3439-44.
Fodor E, Szabó-Nagy A, Erdei L. The effects of cadmium on the fluidity and H+-ATPase activity of plasma membrane from sunflower and wheat roots. Journal of Plant Physiology 1995; 147, 87-92.
Fourest E, Canal C, Roux JC. Improvement of heavy metal biosorption by mycelial dead biomass (Rhizopus arrhizus, mucor miechei and Pencillium chrysogenum): pH control and cationic activation. FEMS Microbiol. Rev. 1994; 14, 325- 332.
Fourest E, Volesky B. (1996) Contribution of sulfonate groups and alginate to heavy metal biosorption by the dry biomass of Sargassum fluitans. Environ. Sci. Technol. 30, 277- 282.
Franklin NM, Adams MS, Stauber JL, Lim RP. Development of an improved rapid enzyme inhibition bioassay with marine and freshwater microalgae using flow cytometry. Arch Environ Contam Toxicol 2001 May; 40(4): 469-80.
Franklin NM, Stauber JL, Markich SJ, Lim RP. pH-dependent toxicity of copper and uranium to a tropical freshwater alga (Chlorella sp.). Aquatic Toxicol. 2000 Mar 1; 48(2-3): 275-289.
Freer J, Baeza J, Maturana H, Palma G. Removal and recovery of uranium by modified Pinus radiata D. Don Bark. J. Chem. Tech. Biotechnol. 1989; 46, 41- 48.
García-Hernández M, Murphy A, Taiz L. Metallothioneins 1 and 2 have distinct but overlapping expression patterns in Arabidopsis. Plant Physiology 1998; 118, 387-397.
Giritch A, Ganal M, Stephan UW, Baumlein H. Structure, expression and chromosomal localization of the metallothionein-like gene family of tomato. Plant Molecular Biology 1998; 37, 701-714.
Goldsbrough P. Metal tolerance in plants: the role of phytochelatins and metallothioneins. In: Terry N, Banuelos G, eds. Phytoremediation of contaminated soil and water. CRC Press LLC 2000; 221-233.
Gries GE, Wagner GJ. Association of nickel versus transport of cadmium and calcium in tonoplast vesicles of oat roots. Planta 1998; 204, 390-396.
Grill E, Loffler S, Winnacker E-L, Zenk MH: Phytochelatins, the heavymetal-binding peptides of plants, are synthesized from glutathione by a specific -glutamylcysteine dipeptidyl transpeptidasse (phytochelatin synthase). Proc Natl Acad Sci USA 1989, 86:6838-6842.
Grill E, Winnacker E-L, and Zenk MH. Phytochelatins: The principal heavy-metal complexing peptides of higher plants. Science 1985; 230, 674-676.
Guerinot ML. The ZIP family of metal transporters. Biochimica et Biophysica Acta 2000; 1465, 190-198.
Ha S-B, Smith AP, Howden R, Dietrich WM, Bugg S, O’Connell MJ, Goldsbrough PB, Cobbett CS: Phytochelatin synthase genes from Arabidopsis and the yeast, Schizosaccharomyces pombe. Plant Cell 1999, 11:1153-1164.
Haag-Kerwer A, Schäfer HJ, Heiss S, Walter C, Rausch T. Cadmium exposure in Brassica juncea causes a decline in transpiration rate and leaf expansion without effect on photosynthesis. J. Exp. Bot. 1999; 50: 1827-1835.
Hamer DH. Metallothionein. Annu. Rev. Biochem., Jan 1986; 55: 913 - 951.
Hartley-Whitaker J, Ainsworth G, Meharg AA. Copper- and arsenate- induced oxidative stress in Holcus lanatus L. Clones with differential sensitivity. Plant, Cell and Environment 2001b; 24, 713-722.
Hartley-Whitaker J, Ainsworth G, Vooijs R, Ten Bookum W, Schat H, Meharg AA. Phytochelatins are involved in differential arsenate tolerance in Holcus lanatus. Plant Physiol. 2001; 126: 299-306.
Hernandez LE, Cooke DT. Modification of the root plasma membrane lipid composition of cadmium-treated Pisum sativum. J. Exp. Bot. 1997; 48: 1375-1381.
Himelblau E, Amasino RM. Delivering copper within plant cells. Current Opinion in Plant Biology 2000; 3, 205-210.
Hirschi KD, Zhen RG, Cunningham KW, Rea PA, Fink GR. CAX1, an H+/Ca2+ antiporter from Arabidopsis. PNAS, Aug 1996; 93: 8782 - 8786.
Holan ZR, Volesky B, Prasetyo I. Biosorption of cadmium by biomass of marine algae. Biotech. Bioeng. 1993; 41, 819- 825.
Howden R, Andersen CR, Goldsbrough PB, Cobbett CS. A cadmium-sensitive, glutathione-deficient mutant of Arabidopsis thaliana. Plant Physiology, Apr 1995; 107: 1067 - 1073.
Howden R, Goldsbrough PB, Andersen CR, Cobbett CS: Cadmiumsensitive, cad1, mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol 1995, 107:1059-1066.
Huang JP, Huang CP, Morehart AL. Removal of heavy metals by fungal (Aspergillus oryzae) adsorption. In: Heavy Metals in the Environment, ed. J. P. Vernet, 1991; pp. 329- 349. Elsevier, London.
Huang JP, Huang CP Morehart AL.The removal of Cu(II) from dilute aqueous solutions by Saccharomyces cerevisiae. Water Res. 1990; 24, 433- 499.
Ilangovan K, Salazar M, Dash S, Monroy O, Ramos A. Interaction of cadmium, copper and zinc in Chlorella pyrenoidosa Chick. Environ. Technol. 1992; 13, 195–199.
Inouhe M, Ito R, Ito S, Sasada N, Tohoyama H, Joho M. Azuki bean cells are hypersensitive to cadmium and do not synthesize phytochelatins. Plant Physiol. 2000; 123: 1029-1036.
Juneau P, El Berdey A, Popovic R. PAM fluorometry in the determination of the sensitivity of Chlorella vulgaris, Selenastrum capricornutum, and Chlamydomonas reinhardtii to copper. Arch Environ Contam Toxicol. 2002 Feb; 42(2): 155-64.
Kessler E. Limits of growth of five Chlorella species in the presence of toxic heavy metals, Arch. Hydrobiol. 1986; 73, 123–128.
Klerks PL, Weis JS. Genetic adaptation to heavy metals in aquatic organisms: a review. Environ. Pollut. 1987; 45, 173-205.
Krämer U, Cotter-Howells J D, Charnock JM, Baker AJM, Smith JAC. Free histidine as a metal chelator in plants that accumulate nickel. Nature 1996; 379, 635-638.
Kuyucak N, Volesky B, Biosorption by algal biomass, in: Volesky B. (Ed.), Biosorption of Heavy Metals, CRC Press, Boca Raton, 1990, pp. 173–198.
Lam PKS, Wut PF, Chan ACW, Wu RSS. Individual and combined effects of cadmium and copper on the growth response of Chlorella vulgaris, Environ. Toxicol. 1999; 14, 347–353.
Lane TD, Morel, FMM. A biological function for cadmium in marine diatoms. Proc. Natl. Acad. Sci. USA 2000; 97, 4627-4631.
Lane TW, Morel FM. Regulation of carbonic anhydrase expression by zinc, cobalt, and carbon dioxide in the Marine Diatom Thalassiosira weissflogii. Plant Physiol 2000 May; 123(1): 345-52.
Lee JG, Roberts SB, Morel, FMM. Cadmium: A nutrient for the marine diatom Thalassiosira weissflogii. Limnol. and Oceanogr. 1995; 40, 1056-1063.
Lewis S, Donkin ME, Depledge MH. Hsp70 expression in Enteromorpha intestinalis (Chlorophyta) exposed to environmental stressors. Aquat Toxicol 2001 Jan;51(3):277-91.
Lewis S, Handy RD, Cordi B, Billinghurst Z, Depledge MH. Stress proteins (HSPs): methods of detection and their use as an environmental biomarker. Ecotoxicology 1999; 8, 351-368.
Ma JF, Ryan PR, Delhaize E. Aluminium tolerance in plants and the complexing role of organic acids. Trends in plant science 2001; 6, 273-278.
Ma JF, Zheng SJ, Matsumoto H. Detoxifying aluminium with buckwheat. Nature 1997; 390, 569-570.
Macfie SM, Tarmohamed Y, Welbourn PM. Effects of cadmium, cobalt, copper and nickel on growth of the green alga Chlamydomonas reinhardtii: The influ-ence of the cell wall and pH, Arch. Environ. Contam. Toxicol. 1994; 27, 454–458.
Macnair MR. The genetics of metal tolerance in vascular plants. New Phytol. 1993; 124, 541–559.
Macnair MR, Tilstone GH, Smith SE. The genetics of metal tolerance and accumulation in higher plants. In: Terry N, Banuelos G, eds. Phytoremediation of contaminated soil and water. CRC Press LLC. 2000; 235-250.
Marschner H. Mineral nutrition of higher plants, 2nd edn. London: Academic Press. 1995.
Meharg AA. The role of the plasmalemma in metal tolerance in angiosperms. Physiologia Plantarum 1993; 88, 191-198.
Matheickal JT, Feltham J, Yu Q. Cu(II) binding by marine alga Ecklonia radita biomaterial. Environ. Technol. 1997; 18, 25- 34.
Matheickal JT, Yu Q. Biosorption of lead from aqueous solutions by marine alga Ecklonia radita. Water Sci. Technol. 1996; 34, 1- 7.
Matheickal JT, Yu Q. Biosorption of lead(II) from aqueous solutions by Phellinus badius. Miner. Eng. 1997; 10, 947- 957.
Matheickal J. T., Yu Q., Woodburn G. M. (1998) Biosorption of cadmium(II) from aqueous solutions by pre-treated biomass of marine algae Durvillaea potatorum, Wat. Res., in press.
Meharg AA. Integrated tolerance mechanisms: constitutive and adaptive plant responses to elevated metal concentrations in the environment. Plant Cell Environ. 1994; 17, 989–993.
Meharg AA, Macnair MR. An altered phosphate uptake system in arsenate-tolerant Holcus lanatus. New Phytologist 1990; 116, 29-35.
Meharg AA, Macnair MR. Genetic correlation between arsenate tolerance and the rate of influx of arsenate and phosphate in Holcus lanatus. Heredity 1992; 69, 336-341.
Meisch HU, Bauer J. The role of vanadium in green plants. IV. Influence on the formation of delta aminolevulinic acid in Chlorella. Arch Microbiol 1978 Apr 27; 117(1): 49-52.
Meisch HU, Benzschawel H. The role of vanadium in green plants. III. Influence on cell division of Chlorella. Arch Microbiol 1978 Jan 23; 116(1): 91-5.
Murphy A, Taiz L. A new vertical mesh transfer technique for metal-tolerance studies in Arabidopsis (Ecotypic Variation and Copper-Sensitive Mutants). Plant Physiology, May 1995; 108: 29 - 38.
Murphy A, Taiz L. Comparison of metallothionein gene expression and nonprotein thiols in ten Arabidopsis ecotypes (correlation with copper tolerance). Plant Physiology, Nov 1995; 109: 945 - 954.
Nassiri Y, Mansot JL, Wery J, Ginsburger-Vogel T, Amiard JC. Ultrastructural and electron energy loss spectroscopy studies of sequestration mechanisms of Cd and Cu in the marine diatom Skeletonema costatum. Arch Environ Contam Toxicol 1997 Aug;33(2):147-55.
Neumann D, Lichtenberger O, Gunther D, Tschiersch K, Nove L. Heat-shock proteins induce heavy-metal tolerance in higher plants. Planta 1994; 194, 360–367.
Neumann D, Nieden UZ, Lichtenberger O, Leopold I. How does Armeria maritima tolerate high heavy metal concentrations? Journal of Plant Physiology 1995; 146, 704-717.
Okamoto OK, Pinto E, Latorre LR, Bechara EJ, Colepicolo P. Antioxidant modulation in response to metal-induced oxidative stress in algal chloroplasts. Arch Environ Contam Toxicol 2001 Jan;40(1):18-24.
Ortiz DF, Kreppel L, Speiser DM, Scheel G, McDonald G, Ow DW. Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar mem-brane transporter. EMBO J. 1992 11, 3491–3499.
Ozer N, Aksu Z, Kutsal T, Caglar A. Adsorption isotherms of lead(II) and chromium(VI) on Cladophora crispata. Environ. Technol. 1994; 15, 439- 448.
Palmiter RD, Findley SD. Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J., Feb 1995; 14: 639 - 649.
Pawlik-Skowronska B. Phytochelatin production in freshwater algae Stigeoclonium in response to heavy metals contained in mining water; effects of some environmental factors. Aquat Toxicol 2001 May;52(3-4):241-9.
Pawlik-Skowronska B. Relationships between acid-soluble thiol peptides and accumulated Pb in the green alga Stichococcus bacillaris. Aquatic Toxicol 2000 Sep 1;50(3):221-230.
Pence NS, Larsen PB, Ebbs SD, Letham DLD, Lasat MM, Garvin DF, Eide D, Kochian LV. The molecular physiology of heavy metal transport in the Zn/Cd hyperaccumulator Thlaspi caerulescens. PNAS, Apr 2000; 97: 4956 - 4960.
Perez-Rama M, Herrero Lopez C, Abalde Alonso J, Torres Vaamonde E. Class III metallothioneins in response to cadmium toxicity in the marine microalga Tetraselmis suecica (Kylin) Butch. Environ Toxicol Chem 2001 Sep;20(9):2061-6.
Persans MW, Yan X, Patnoe J-MML, Krämer U, Salt DE. Molecular dissection of the role of histidine in nickel hyperaccumulation in Thlaspi goesingense (Hálácsy). Plant Physiology, Dec 1999; 121: 1117 - 1126.
Petris
MJ, Mercer JF, Culvenor JG, Lockhart P, Gleeson PA, Camakaris J.
Prassad MNV. 1999. Metallothioneins and metal binding complexes in plants. In: Prasad MNV, Hagemeyer J, eds. Heavy metal stress in plants: from molecules to ecosystems. Berlin: Springer-Verlag, 51-72.
Price NM, Morel FMM. Cadmium and cobalt substitution for zinc in a zinc- deficient marine diatom. Nature 1990; 344, 658-660.
Quartacci MF, Cosi E, Navari-Izzo F. Lipids and NADPH-dependent superoxide production in plasma membrane vesicles from roots of wheat grown under copper deficiency or excess. J. Exp. Bot. 2001; 52: 77-84.
Rachlin JW, Grosso A. The growth response of the green alga, Chlorella vulgaris to combined divalent cation exposure, Arch. Environ. Contam. Toxicol. 1993; 24, 16–20. Rai L.C., Gaur M.J., Po H.D., Phycology and heavy metal pollution, Biol. Rev. 1981; 56, 99–151. Rauser WE. Phytochelatins and related peptides (Structure, biosynthesis, and function). Plant Physiology, Dec 1995; 109: 1141 - 1149.
Rauser WE: Structure and function of metal chelators produced by plants; the case for organic acids, amino acids, phytin and metallothioneins. Cell Biochem Biophys 1999, 31:19-48.
Rea PA, Li Z-S, Lu Y-P, Drozdowicz YM, Martinoia E. From vacuolar gs-x pumps to multispecific ABC transporters. Annu. Rev. Plant Physiol. Plant. Mol. Biol., Jan 1998; 49: 727 - 760. Reiriz S, Cid A, Torres E, Abalde J, Herrero C. Different responses of the marine diatom Phaeodactylum tricornutum to copper toxicity. Microbiologia 1994 Sep; 10(3): 263-72.
Rijstenbil JW, Sandee A, Van Drie J, Wijnholds JA. Interaction of toxic trace metals and mechanisms of detoxification in the planktonic diatoms Ditylum brightwellii and Thalassiosira pseudonana. FEMS Microbiol Rev 1994 Aug; 14(4): 387-96.
Robinson NJ, Tommey AM, Kuske C, Jackson PJ. Plant metallothioneins. Biochemical Journal 1993; 295, 1-10.
Roesijadi G. Metallothinoeins in metal regulation and toxicity in aquatic animals. Aquat. Toxicol. 1992; 22, 81-114.
Ros ROC, Cooke DT, Burden RS, James CS. Effects of the herbicide MCPA and the heavy metals, cadmium and nickel on the lipid composition, Mg2+ -ATPase activity and fluidity of plasma membranes from rice, Oryza sativa (cv. Bahia) shoots. Journal of Experimental Botany 1990; 41, 457-462.
Sag Y, Kutsal T. Biosorption of heavy metals by Zoogloea ramigera: Use of adsorption isotherms and a comparison of biosorption characteristics. Chem. Eng. J. Biochem. Eng. J. 1995; 60, 181- 188.
Salt DE, Kato N, Krämer U, Smith RD, Raskin I. The role of root exudates in nickel hyperaccumulation and tolerance in accumulator and nonaccumulator species of Thlaspi. In: Terry N, Banuelos G, eds. Phytoremediation of contaminated soil and water. CRC Press LLC 2000; 189-200.
Salt DE, Rauser, WE. MgATP-dependent transport of phytochelatins across the tonoplast of oat roots. Plant Physiol. 1995; 107, 1293–1301.
Salt DE, Smith RD, Raskin I: Phytoremediation. Annu Rev Plant Physiol Plant Mol Biol 1998, 49:643-668.
Salt DE, Thurman DA, Tomsett AB, Sewell AK. Copper phytochelatins of Mimulus guttatus. Proceedings of the Royal Society B, London 1989; 236, 79-89.
Salt DE, Wagner GJ. Cadmium transport across tonoplast of vesicles from oat roots. Evidence for a Cd2+/H+ antiport activity. J. Biol. Chem, Jun 1993; 268: 12297 - 12302. Sandau E, Sandau P, Pulz O. Heavy metal sorption by microalgae, Acta Biotechnol. 1996; 16, 227–235. Sauser KR, Liu JK, Wong TY. Identification of a copper-sensitive ascorbate peroxidase in the unicellular green alga Selenastrum capricornutum. Biometals 1997 Jul; 10(3): 163-8.Schat H, Kalff MMA. Are phytochelatins involved in differential metal tolerance or do they merely reflect metal-imposed strain? 1992; 99, 1475-1480.Schat H, Llugany M, Bernhard R. Metal-specific patterns of tolerance, uptake and transport of heavy metals in hyperaccumulating and nonhyperaccumulating metallophytes. In: Terry N, Banuelos G, eds. Phytoremediation of contaminated soil and water. CRC Press LLC 2000; 171-188.
Schintu M, Koussih L, Chevolot L, Amiard JC, Robert JM. Monitoring of labile zinc in cultures of Skeletonema costatum using a salt groundwater. Ecotoxicol Environ Saf 1999 Mar;42(3):207-11.
Schneider IAH, Rubio J, Misra M, Smith RW. Eichhornia crassipes as biosorbent for heavy metal ions. Miner. Eng. 1995; 9, 979-988.
Scott JA, Karanjkar AM. Repeated cadmium biosorption by regenerated Enterobactor aerogenes biofilm attached to activated carbon. Biotechnol. Lett. 1992; 14, 737- 740.
Silver S. Bacterial resistance to toxic metal ions- a review. Gene 1996; 179, 9-19.
Silver S, Ji G. Newer systems for bacterial resistances to toxic heavy metals. Environmental Health Perspectives 1994; 102, 107-113.
Solioz M, Vulpe C. CPx-type ATPases: a class of P-type ATPases that pump heavy metals. Trends in Biochemical Sciences 1996; 21, 237-241.
Steffens JC. The heavy-metal binding peptides of plants. Annu. Rev. Plant Physiol. Plant. Mol. Biol., Jan 1990; 41: 553 - 575.
Strandberg GW, Shumate SE and Parrot JR. Accumulation of uranium by Saccharomyces cerevisiae and Pseudomonas aeruginosa. Appl. Environ. Microbiol. 1981; 41, 237- 245.
Strange J, Macnair MR. Evidence for a role for the cell membrane in copper tolerance of Mimulus guttatus Fischer ex DC. New Phytologist 1991; 119, 383-388.
Stumm W, Morgan JJ; 1996. Aquatic Chemistry. 3rd Ed. John Wiley & Sons, New York, NY, USA.
Thomine S, Wang R, Ward JM, Crawford NM, Schroeder JI. Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes. PNAS, Apr 2000; 97: 4991 - 4996.
Tobin JM, Copper DG, Neufeld RJ. Uptake of metal ions by Rhizopus arrhizus biomass. Appl. Environ. Microbiol. 1984; 47, 821- 824.
Tomsett AB, Thurman DA. Molecular biology of metal tolerances of plants. Plant Cell Environ. 1988; 11, 383–394.
Tseng TS, Tzeng SS, Yeh CH, Chang FC, Chen YM, Lin CY. The heat-shock response in rice seedlings isolation and expression of cDNAs that encode class-I low-molecular-weight heat-shock proteins. Plant and Cell Physiology 1993; 34, 165-168.
Van der Zaal BJ, Neuteboom LW, Pinas JE, Chardonnens AN, Schat H, Verkleij JAC, Hooykaas PJJ. Overexpression of a novel Arabidopsis gene related to putative zinc-transporter genes from animals can lead to enhanced zinc resistance and accumulation. Plant Physiology, Mar 1999; 119: 1047 - 1056.
Van Vliet C, Andersen CR, Cobbett CS. Copper-sensitive mutant of Arabidopsis thaliana. Plant Physiol. 1995; 109: 871-878.
Vatamaniuk OK, Mari S, Lu Y-P, Rea PA: AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proc Natl Acad Sci USA 1999, 96:7110-7115.
Verkleij JAC, Koevoets PLM, Mechteld MA, Blake-Kalff MMA, Chardonnens AN. Evidence for an important role of the tonoplast in the mechanism of naturally selected zinc tolerance in Silene vulgaris. Journal of Plant Physiology 1998; 153, 188-191.
Vierling E. The roles of heat shock proteins in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42, 579–620.
Vílchez C, Garbayo I, Lobato MV, Vega JM. Microalgae-mediated chemicals production and wastes removal, Enzyme Microb. Technol. 1997; 20, 562–572.
Visviki I, Rachlin JW. Acute and chronic exposure of Dunaliella salina and Chlamydomonas bullosa to copper and cadmium: effects of ultrastructure. Arch Environ Contam Toxicol 1994 Feb; 26(2): 154-62.
Volesky B. (1990) Biosorption of Heavy Metals. CRC Press, Boca Raton, U.S.A.
Volesky B. Advances in biosorption of metals: selection of biomass types. FEMS Microbiol. Rev. 1994; 14, 291-302.
Volesky B, May H, Holan ZR. Cadmium biosorption by Saccharomyces cerevisiae. Biotechnol. Bioeng. 1993; 41, 826-829.
Wainwright SJ, Woolhouse HW. Some physiological aspects of copper and zinc tolerance in Agrosis tenuis Sibth. : cell elongation and membrane damage. Journal of Experimental Botany 1997; 28, 1029-1036.
Wilde EW, Benemann JR. Bioremoval of heavy metals by the use of microalgae, Biotechnol. Adv. 1993; 11, 781–812.
Williams LE, Pittman JK, Hall JL. Emerging mechanisms for heavy metal transport in plants. Biochimica et Biophysica Acta 2000; 77803, 1-23.
Wollgiehn R, Neumann D. Metal stress response and tolerance of cultured cells from Silene vulgaris and Lycopersicon peruvianum: role of heat stress proteins. Journal of Plant Physiology 1999; 154, 547-553.
Wong MY, Sauser KR, Chung KT, Wong TY, Liu JK. Response of the ascorbate-peroxidase of Selenastrum capricornutum to copper and lead in stormwaters. Environ Monit Assess 2001 Mar; 67(3): 361-78.
Xiang C, Oliver DJ: Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell 1998, 10:1539-1550.
Xiang C, Werner BL, Christensen EM, Oliver DJ. The biological functions of glutathione revisited in Arabidopsis transgenic plants with altered glutathione levels. Plant Physiol. 2001; 126: 564-574.
Yong LZ, Pilon-Smits EAH, Tarun AS, Weber SU, Jouanin L, Terry N: Cadmium tolerance and accumulation in Indian Mustard is enhanced by overexpressing -glutamylcysteine synthetase. Plant Physiol 1999, 121:1169-1177.
Zenk MH: Heavy metal detoxification in higher plants – a review. Gene 1996, 179:21-30.
Zhou J, Goldsbrough PB. Functional homologs of fungal metallothionein genes from Arabidopsis. Plant Cell, Jun 1994; 6: 875 - 884.
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