Heavy Metal Toxicity in Plants and Phytoremediation

12 Heavy Metal Toxicity in Plants and Phytoremediation R.C. Setia, Navjyot Kaur, Neelam Setia and Harsh Nayyar 1 Department of Botany, Punjab Agricultural University, Ludhiana-141004, India 1 Department of Botany, Panjab University, Chandigarh-160014 email: [email protected]

ABSTRACT The increasing heavy metal concentrations in agricultural lands due to various industrial activities adversely affect crop growth and metabolism consequently lowering yields with concomitant quality deteriorations. However, a great deal of research in the past ten years indicates that certain plants have the genetic potential to remove many heavy metals from the soil. Phytoremediation, the use of plants for environmental restoration, consists of four different technologies for the remediation of metal polluted soils, sediments or waters namely phytoextraction, phytostabilization, rhizofiltration and phytovolatilization. These different phytoremediation technologies are reviewed here with their respective advantages and limitations. An attempt has been made to review plant-based mechanisms which allow metal uptake, accumulation and translocation in plants whose better understanding is needed to further enhance the efficiency of phytoremediation. Genetic engineering approaches to improve the potential of phytoremediation are also reviewed and discussed. The future challenge for phytoremediation is to further reduce the cost and increase the spectrum of metals amenable to this technology.

Keywords: Heavy metals, plants, phytoremediation, metal toxicity

INTRODUCTION The heavy metals are important environmental pollutants and also a cause of potential ecological risk. Large areas of agricultural lands, especially near industrialized areas, are contaminated by heavy metals that mainly originate due to burning of fossil fuels, industrial manufacturing and municipal wastes, and application of fertilizers, pesticides and sewage sludge to land. Among an array of heavy metals, Cu, Co, Fe, Mo, Ni and Zn are essential micronutrient mineral elements, whereas Cd, Pb, Hg, As etc. have no known physiological function in plants and are potential toxins. However, elevated levels of both essential and non-essential heavy metals in the plough layers of crop lands pose serious threat for human health and agriculture. The excessive uptake of these metals from the soil can create dual problem: the harvested crops so contaminated serve as a source of heavy metals in our food supply, and yields are reduced due to adverse effect on plant growth (Bala and Setia, 1990; Hall, 2002).

Crop Improvement: Strategies and Applications  Editors: R.C. Setia, Harsh Nayyar and Neelam Setia © 2008 I.K. International Publishing House Pvt. Ltd., New Delhi, pp 206-218

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A large number of studies, though spread over different crop plants, indicate that the excessively absorbed heavy metals interfere with various biochemical, physiological and structural aspects of plant processes that not only lead to inhibited growth but sometimes result in plant death. The toxic levels of heavy metals affect structural and permeability properties of inner membranes and organelles, cause inhibition of enzymatic activities, nutrient imbalances, decreases in rates of photosynthesis and transpiration (Green et al ., ., 2003; Setia et al ., ., 1993; Prasad and Hagemeyer, 1999; Azevado et al., 2005), stimulate formation of free radicals and reactive oxygen species resulting in oxidative stress (Sandalio et al ., ., 2005), suppress seed germination and seedling growth (Beri et al ., ., 1990; Beri and Setia, 1996; Setia et al ., ., 1989b), reproductive development (Setia et al ., ., 1988,1989a), seed yield and seed quality (Beri and Setia, 1995) and induce deleterious anatomical and ultrastructural changes in crop plants (Setia and Beri, 1993; Setia and Bala, 1994; Liu and Kottke, 2004; Maruthi Sridhar et al ., ., 2005). Further, consistently increasing levels of different heavy metals in the soil render the land unsuitable for plant growth and destroy the biodiversity. Remediation of soils contaminated with heavy metals is particularly challenging. The conventional engineered based remediation technologies (other than bioremediation) used for in situ and ex situ remediation of heavy metal contaminated soils include solidification and stabilization, soil flushing, electrokinetics, chemical reduction/oxidation, soil washing, low temperature thermal desorption, incineration, vitrification, pneumatic fracturing, excavation/ retrieval, landfill and disposal (Saxena et al., 1999; Wenzel et al., 1999). But these are prohibitively expensive and often disturb the land scape. Phytoremediation, the use of plants for remediation of soils and waters contaminated with heavy metals, has gained acceptance in the past ten years as a cost effective and non-invasive technology. This approach is emerging as an innovative tool with greater potential that is most useful when contaminants are within the root zone of the plants (top three to six feet). Further, phytoremediation is an energy efficient, cost-effective, aesthetically pleasing method of remediating sites with low to moderate levels of contamination (Schnoor, 1997; Salt et al., 1998). The technique of phytoremediation exploits the use of either naturally occurring metal hyperaccumulator plants or genetically engineered plants (Cunningham et al., 1997; Flathman and Lanza, 1998).The base of phytoremediation is pollutant uptake or bounding by plants under the different processes namely phytoextraction, phytodegradation, phytostabilization and phytovolatilization. This review aims to give a broad overview of processes involved in uptake and transport of heavy metals in plant cells/ tissues and mechanism of phytoremediation of the heavy metal contaminated soils.

PLANT RESPONSES TO HEAVY METALS Plants have evolved several effective mechanisms to deal with the excess of heavy metals in the soil. They can prevent or restrict the uptake of metals through root and/or into protoplast, or minimize the toxic effects of metal ions inside the protoplast, or take up the metals, accumulate and indicate specific symptoms. Accordingly, the plants have been classified as follows: Metal Excluders: These plants prevent metal uptake into roots and/or avoid translocation and accumulation into shoots over a wide range of metal concentrations in the soil (De Vos et al., 1991;Memon et al., 2001). Excluders have a low potential for metal extraction, but they can be used to stabilize the soil, and avoid further contamination spread due to erosion. Such a species is Agrostis tenuis, which avoids Cd, Cu, Pb and Zn uptake by precipitating the metal in the rhizosphere (Lasat, 2002).

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Metal Accumulators: This group of plants can accumulate metals in their above ground tissues in concentrations far exceeding than those present in the soil, and such plant species are termed as hyperaccumulators (Baker and Brooks, 1989). These plants have evolved specific mechanisms for detoxifying heavy metals accumulated in their cells. Such mechanisms allow bioaccumulation of extremely high concentrations of metals. Metal Indicators: These plants show poor control over metal uptake and transport processes, and accumulate metals in their above ground tissues. The extent of metal accumulation in the tissues of these plants reflects metal concentration in the rhizosphere. Indicator species have been used for mine prospecting to find new ore bodies (Raskin et al., 1994).

HEAVY METAL UPTAKE, TRANSLOCATION AND ACCUMULATION The uptake, translocation and accumulation of heavy metals in plants is mediated by integrated network of physiological, biochemical and molecular mechanisms operative at the extracellular (root surface) level as well as inside the cells/tissue of plants growing in contaminated soils. The transfer of heavy metals from soils to plants depends primarily on total amount of potentially available or the bioavailability of the metal (quantity factor), the activity as well as the ionic ratios of elements in soil solution (intensity factor), and rate of element transfer from solid to liquid phases and to plant roots (reaction kinetics) (Brümmer et (Brümmer et al., 1986). Plants distribute metals internally in many different ways. They may localize selected metals mostly in roots and stems, or they may accumulate and store other metals in non-toxic forms for later distribution and use. A mechanism of tolerance or accumulation in some plants apparently involves binding potentially toxic metals at cell walls of roots and leaves, away from sensitive sites within the cell or storing them in a vacuolar compartment (Memon et al., 2001).

Metal Bioavailability The degree to which higher plants are able to take up metal ions depends on their concentration in the soil and bioavailability, modulated by the presence of organic matter, pH, redox potential, temperature and concentration of other elements (Benavides et al., 2005). In soils, metal exist as a variety of chemical species in a dynamic equilibrium governed by soil’s physical, chemical and biological properties (Chaney, 1988). Heavy metals are retained by soils in three ways: by adsorption onto the surface with mineral particles, by complexation with humic substances in organic particles and by precipitation reactions (Walton et al., 1994). In general, only a fraction of soil metal is readily available (bioavailable) for plant uptake. The bulk of soil metals is commonly found as insoluble compounds unavailable for transport into roots (Lasat, 2002). Plants possess highly specialized mechanisms to stimulate metal bioavailability in the rhizosphere, and to enhance uptake into roots (Romheld and Marschner, 1986). Root exudates have an important role in the acquisition of several essential metals. For example, some grass species have been documented to exude from roots a class of organic acids called siderophores (mugineic and avenic acids), which were shown to significantly enhance the bioavailability of soil-bound iron (Kanazawa et al., 1994) and possibly zinc (Cakmak, 1996 a, b). Dicotyledonous species facilitate iron uptake by acidifying the rhizosphere via H+ extrusion from roots. Acidic environment stimulates the reduction of ferric to ferrous iron which is readily taken up by plants (Chaney et al., 1972; Bienfait et al., 1982). Pollutant bioavailability may also be affected by various plant and/or microbial activities. Some bacteria are known to release biosurfactants (e.g., rhamnolipids) that make hydrophobic pollutants more water-


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souble (Volkering et al., 1998). Plant exudates or lysates may also contain lipophilic compounds that increase pollutant solubility or promote biosurfactant producing microbial populations (Siciliano and Germida, 1998). According to Pilon-Smits (2005), bioavailability of metals may be enhanced by metal chelators that are released by plants and bacteria. Some reported plant chelators such as siderophores, organic acids and phenolics can release metal cations from soil particles and make the metals more available for plant uptake.

Metal Uptake by Roots The movement of metals towards the root surface depends on three factors: a) mass flow due to which the soluble metal ions move from soil solids to root surface (driven by transpiration), b) diffusion of elements along the concentration gradient formed due to uptake and thereby depletion of the element in root vicinity, and c) root interception, where soil volume is displaced by root volume due to root growth (Marschner ,1995). The metal uptake by roots may take place at the apical region or from the entire root surface depending upon the type of element under consideration. Further, the uptake depends upon the uptake capacity and growth characteristics of the root system. There are two pathways for solubilized heavy metals to enter a plant. These are apoplastic (extracellular) and symplastic (intracellular). The apoplast continuum of the root epidermis and cortex is readily permeable to solutes. The metals are first taken into the apoplast of the roots where a significant ion fraction is physically adsorbed at the extracellular negatively charged sites (COO-) of the root cell walls (Lasat, 2000). Then, some of the total amount of metal ions associated with root cell walls is translocated into the cell. However, the impermeable suberin layers in the cell wall of the root endodermis (casparian strips) prevent solutes from flowing straight from root apoplast into the root xylem (Taiz and Zeiger, 2002). Therefore, the solutes have to be taken up into the root symplasm before they can enter the xylem apoplast. Metal ions require membrane transporter proteins for their transportation from root endodermis into root xylem (Pilon-Smits, 2005). Some metals are chelated during xylem transport by organic acids (histidine, malate, citrate), nicotianamine, or thiol-rich peptides (Krämer et al., 1996; Pickering et al., 2000). However, for most metal ions it is still unclear via which transporter proteins they are exported to the root xylem and to which chelators they are bound during transport (Pilon-Smits, 2005). Transporters The plant plasma membrane may be regarded as the first living structure that encounters the heavy metal toxicity. Because of their charge, metal ions can not move freely across the cellular membranes, which are lipophilic structures. Therefore, ion transport into cells must be mediated by membrane proteins with transport functions, generally known as transporters (Lasat, 2000). Several classes of metal transporters are reported in plants that are involved in metal uptake and homeostasis in general, and thus could play some role in tolerance (Hall, 2002). These include heavy metal CPx -ATPases, the Nramps, and CDF (cation diffusion facilitators) family (Williams et al., al., 2000), and ZIP family (Guerinot, 2000). Further, heavy metal ions such as Cd enters the plant cell by transporters al., 2000). AtNramp genes in Arabidopsis encode the for essential cations such as Fe 2+(Thomine et al., metal transporter, which transports both the metal nutrient iron and the toxic metal cadmium. Lasat (2000) has summarized the mechanism of transporter function. According to him, “membrane transporters possess an extracellular binding domain to which the ions attach just before the transport, and a transmembrane structure which connects extracellular and intracellular media.

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The binding domain is receptive only to specific ions and is responsible for transporter specificity. The transmembrane structure facilitates the transfer of bound ions from extracellular space through hydrophobic environment of the membrane into the cell. The transporters are characterized by certain kinetic parameters such as transport capacity (V max) and affinity for ion (Km)”. For most elements, multiple transporters occur in plants. For example, Arabidopsis thaliana has been reported to have 150 different cation transporters and 14 transporters for sulphate alone (Axelsen and Palmgren, 2001).

Metal Transport from Root to Shoot The transport of heavy metals from root to shoot primarily takes place through the xylem via specialized membrane transport processes (Salt et al., 1995). For example, the xylem loading of Ni may be facilitated by binding of Ni to free histidine (Krämer et al., 1996). The movement of metal ions in xylem vessels appears to be mainly dependent on transpiration-driven mass flow (Salt et al., 1995). Since xylem cell walls have high cation exchange capacity (CEC), therefore, non-cationic metal-chelate complexers, e.g., Cd-citrate, should be transported more efficiently in the transpiration stream (Senden et al., 1990). Involvement of organic acids for Cd translocation has been observed, and phytochelatins and other thiol-containing ligands play no direct role in Cd transport in the xylem (Salt et al., 1995). Bulk flow in the xylem from root to shoot is driven by transpiration from the shoot, which creates a negative pressure in the xylem that pulls up water and solutes (Taiz and Zeiger, 2002). Import into leaf cells from leaf xylem involves another membrane transport step. Metals are taken up by specific membrane transporter proteins. Once inside the leaf symplast, the pollutant may be compartmentized in certain tissues or cellular locations. In general, toxic pollutants are sequestered in places where they can do the least harm to essential cellular processes. At the cellular level, pollutants are generally accumulated in vacuole or cell wall (Burken, 2003; Cobbett and Goldsbrough, 2000). At the tissue level they may be accumulated in the epidermis or trichomes. When pollutants are sequestered in leaf tissues, they are often bound by chelators or form conjugates. Chelators that are involved in metal sequestration include the tripeptide GSH (³-glucys-gly) and its oligomer, the phytochelatins (see further in text). After chelation by GSH or PCs, an ABC-type transporter activity transports the metal-chelate complex to the vacuole where it is further complexed by the sulphate. Additional metal chelating proteins exist (e.g. MTs) that play a role in sequestration, tolerance, and / or in homeostasis of essential metals (Goldsbrough, 2000).

PHYTOREMEDIATION Phytoremediation, which essentially involves the use of plants for environmental clean up consists of four different technologies for the remediation of metal polluted soils, sediments or waters. These include phytoextraction, phytostabilization, rhizofiltration and phytovolatilization.

Phytoextraction Phytoextraction is the most well known of all phytoremediative technologies involving uptake of metal contaminants from soil through plant roots and thereafter storage of the same in plant stem or leaves. This technology is, however, most suitable for the large areas which are contaminated at shallow depths, and have low to moderate levels of metal contaminants. There are two basic strategies of phytoextraction—induced and continuous phytoextraction (Salt et al., 1998).


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Induced Phytoextraction: Some heavy metals, such as Pb occur as insoluble precipitates of phosphates, carbonates and hydroxy-oxides which are largely unavailable for plant uptake, resulting in binding and immobilization within the soil matrix, and consequently significantly restrict the potential for metal phytoextraction. To overcome this limitation, several chelating agents have been tested to increase phytoextraction of toxic metals including Cd and Pb and the process is known as induced phytoextraction. Induced phytoextraction involves chelate-mediated release of bound metals into soil solution vis-à -vis transport of metals to the harvestable shoot (Salt et al., 1998). Blaylock et al. (1997) reported that addition of EDTA (ethylene-diamine-tetra acetic acid) at a rate of 10mmol kg-1 soil stimulated Pb accumulation in maize to levels as high as 1.6% of shoot dry weight compared to levels only 0.01-0.06% Pb of shoot dry biomass in vegetation growing on heavily lead contaminated soil. The concept of chelate-assisted phytoextraction is applicable to a broad range of heavy metals, e.g., an application of EDTA to heavy metal contaminated soil resulted in the simultaneous accumulation of Pb, Cr, Cu, Ni and Zn in Indian mustard plants. In this way, synthetic chelates having a high affinity for the metal of interest can play a significant role for the reclamation of metal contaminated sites, e.g., EDTA for Pb and Cd, EGTA (Ethylene glycol-[amino ethyl ether] –N, N, N,’ N’, tetra acetic acid) for Cd and NTA (Nitrilotriacetic acid) for Cu and Cd (Hong and Pintauro, 1996 ; Wu et al., 1999). Chelate-mediated transport of metals to shoots appears to occur in the xylem sap via the transpiration stream. The metal is transported within the plant from roots to shoots as a metal-chelate complex where water evaporates and the metal-chlelate complex remains ( Salt et al., 1998). On the negative side, the enhanced solubilization of soil metal contaminants with chemical additives/soil amendments pose a high risk of groundwater contamination because highly soluble Pb-EDTA complex easily percolates through soil profile (Wu et al., 1999). In order to overcome this limitation, an amendment formulation combining lower EDTA doses and surfactants has been suggested as an alternative to higher rates of soil EDTA application for removal of Pb (Elless and Blaylock, 2000). Recent research in this field aims at eliminating the risk of spreading metal contamination due to high solubility of metal-chelate complexes like Pb-EDTA complex by implementing alternative chelate formulations and innovative agronomic practices. Continuous Phytoextraction (The Concept of Hyperaccumulators): An alternative approach to chelate-assisted induced phytoextraction is the continuous phytoextraction that utilizes the unique genetic and physiological capacity of specialized hyperaccumulating plants, which can grow on soils rich in heavy metals, and are able to accumulate, translocate and tolerate high amounts of metals in their tissues. Hyperaccumulators are those plant species which can accumulate one or more inorganic elements to levels 100 fold higher than other species grown under the same conditions, and will concentrate more than 10 mg kg -1 Hg, 100 mg kg-1 Cd, 1000 mg kg-1 Co, Cr, Cu and Pb and 10000 mg kg-1 Zn and Ni (Baker et al., 2000). Thalaspi caerulescens, a member of family brassicaceae, is the best known metal hyperaccumulator that has been reported to accumulate up to 26000 mg kg -1Zn without showing any symptoms of toxicity (Brown et al., 1995). This plant is also able to extract up to 20% of soil exchangeable Cd from a contaminated site (Gerrard et al., 2000). The recently discovered As hyperaccumulating fern, Pteris vittata, has been shown to accumulate as much as 14500 mg kg -1 As in fronds without showing symptoms of toxicity (Ma et al., 2001). Another plant, Alyssum bertolonii, has been found to phytoremediate Ni (Li et al., 2003) and can accumulate Ni at levels as high as 1%, which is over 100-1000 times higher than other plants (Minguzzi and Vergnano, 1948). The main function of metal accumulation in

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hyperaccumulators has been suggested to be protection against fungal and insect attack (Boyd and Martens, 1992). However, most of the hyperaccumulators have limited potential for phytoextraction because most of them are slow growing and attain low biomass. Thus, plants having the ability to produce higher dry biomass even when grown on heavy metal contaminated soils are preferred over well-known hyperaccumulator species that can have considerably higher heavy metal juncea, while having one-third the concentration concentrations in their tissues. For example, Brassica example, Brassica juncea, of Zn in its tissue, is more effective at Zn removal from soil than T. Caerulescens —a known hyperaccumulator of Zn—as the former produces ten times more biomass than the latter. It is expected that further research with classical or molecular genetic methods will produce a range of crops that can be used for the phytoextraction of several heavy metals (Schmidt, 2003).

Phytostabilization Phytostabilization is a plant-based remediative technology that exploits the ability of heavy metaltolerant plants to reduce the mobility of the metal contaminants as the latter are absorbed and accumulated by roots, adsorbed onto the roots or precipitated in the rhizosphere. The goal of phytostabilization is thus not to remove metal contaminants from a site, but rather to stabilize them and reduce the risk to human health and environment by preventing migration of metal contaminants into the groundwater or air (Prasad and Freitas, 2003). Plants chosen for phytostabilization should be poor translocators of metal contaminants to shoots, such as grasses, thus minimizing exposure of wildlife to toxic elements. To further reduce the risk of groundwater contamination by downward leaching, grasses should be grown along with fast transpiring trees, such as poplar, which are deep rooted and transpire at very high rates, creating powerful upward flow (Dawson and Ehleringer, 1991). Rhizofiltration Rhizofiltration is a phytoremediative technology concerned with the removal of metals from the aquatic environments. Plants used for rhizofiltration are first hydroponically grown in clean water until a large root system has developed. This is followed by acclimatization of the plant to the pollutant by substituting the clean water supply for a polluted water supply. Then these acclimatized plants are transplanted into metal-polluted waters where plants absorb and concentrate metals in their roots and shoots (Salt et al., 1995; Zhu et al., 1999 a). In addition to absorption of metal pollutants, latter may also be adsorbed onto the root surfaces triggered by root exudates and changes in rhizosphere pH. Roots or whole plants are harvested for disposal after they become saturated with metal pollutants (Flathman and Lanza, 1998). Several aquatic plant species such as water hyacinth, pennywort and duckweed have the ability to remove heavy metals from water ( Dierberg et al., 1987; Mo et al., 1989; Zhu et al., 1999a). However, these plants have limited potential for rhizofiltration because of their small and slow-growing roots. Phytovolatilization Phytovolatilization is the process where plants absorb water soluble elemental forms of metal contaminants, and biologically convert them to gaseous species within the plant followed by their release into the atmosphere. This process is particularly used to remediate soils contaminated with metals such as As, Hg and Se (Suszcynsky and Shann, 1995). This technology has the added benefits of minimal site disturbance, less erosion and no need to dispose off contaminated plant


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material. The major limitation of phytovolatilization is that there is no control over the migration of metal contaminants that have been removed via volatilization to other areas (Prasad and Freitas, 2003).

MECHANISMS FOR METAL SEQUESTRATION AND DETOXIFICATION When toxic metals are sequestered in plant tissues, they are often bound by heavy metal-binding polypeptides, also known as chelators. The two best-characterized heavy metal binding polypeptides known to exist in plants are metallothioneins and phytochelatins. Metallothioneins (MTs) are geneencoded, low molecular-weight, cysteine-rich polypeptides. MTs were first identified as Cd- binding proteins in mammalian tissues and are classified based on the arrangement of cysteine residues (Robinson et al., 1993). Several classes of MT genes (MT1, MT2, MT3, MT4) have now been Arabidopsis(Goldsbrough, identified in several higher plants, including Arabidopsis (Goldsbrough, 2000). The role of plant metallothioneins in relation to Cu binding and detoxification has been most widely studied. Murphy and Taiz (1995) demonstrated that expression levels of MT2 mRNA in Arabidopsis thaliana strongly correlated with Cu resistance. Earlier, Zhou and Goldsbrough (1994) also reported that MT2 mRNA was strongly induced in Arbabidopsis seedlings by Cu, but only slightly by Cd and Zn, suggesting that metallothioneins are involved in Cu resistance. Phytochelatins (PCs), also known as class III metallothioneins, are short metal-binding, metalinduced, cysteine-rich peptides possessing the general structure: (³-Glu Cys) n-Gly with n=2-11. PCs are synthesized from glutathione (GSH) by a specific transpeptidase named ³-glutamyl cysteine dipeptidyl transpeptidase (EC 2.3.2.15), also known as phytochelatin synthase (PCS) (Vatamaniuk et al., 2004), which is a constitutive enzyme requiring post-translational activation by heavy metals (Klapheck et al., 1995). The best activator for the enzyme phytochelatin synthase (PCS) is Cd, but is also activated in the presence of other heavy metals, such as Ag, Bi, Pb, Zn, Cu, Hg, Au and As, both in vivo and in vitro (Cobbett, 2000). X-ray absorption spectroscopy (XAS) has shown that metals that were complexed by PCs in vivo include Cd and As (Pickering et al., 2000). In vitro studies conducted by Loeffler et al. (1989) had shown that PC biosynthesis continued until the activating metal ions were chelated either by the PCs formed or by the addition of a metal chelators such as EDTA, thus suggesting that activating metal ions autoregulate the biosynthesis of PCs. The final step in heavy metal detoxification involves the sequestration of metal -PC complexes in the vacuole, and transport of the metal-PC complexes through the tonoplast is mediated by an ATPdependent ABC-transporter (Schat et al., 2000). Metal-PC complex in the vacuole is further stabilized by the incorporation of acid-labile sulphide (Cobbett and Goldsbrough, 2000). PCs appear to be important in the detoxification of Cd and As, but have a relatively insignificant role in the detoxification of metal ions such as Cu, Zn, Ni and SeO 3 (Cobbett, 2000). Metal hyperaccumulator species possess additional detoxification mechanisms. For example, studies conducted in a Ni hyperaccumulator- T . goesingense have revealed that the high tolerance of the latter was due to Ni complexation by histidine, which rendered the metal inactive (Krämer et al., 1996).

GENETIC ENGINEERING AND PHYTOREMEDIATION Natural metal hyperaccumulators such as Thalaspi spp spp.. can accumulate and tolerate higher metal concentrations in their tissues than those usually found in non-accumulators without any visible toxicity symptoms. However, most of the metal hyperaccumulators have a limited potential for

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phytoremediation because of their small size and slow growth (Lasat, 2002). Thus, to overcome this limitation and to improve the potential for metal phytoextraction, Brown et al. (1995 ) proposed the transfer of the hyperaccumulator phenotype from small and slow growing hyperaccumulator species to fast growing, high biomass producing nonaccumulator plants. Research data indicates that tolerance to toxic metals is regulated by one or few major genes, thus providing a hope that biotransformation of selected crop species for tolerance and ultimately superior metal extraction potential is feasible. However, the use of conventional breeding programmes to improve plants for metal phytoextraction has limited potential because of anatomical constraints that can severely restrict sexual compatibility between species (Ow, 1996). The most spectacular application of biotechnology for environmental restoration has been the bioengineering of plants capable of removing methyl-mercury from the contaminated soil. Methyl-mercury, a strong neurotoxic agent, thaliana plants were is biosynthesized in Hg-contaminated soils, and to detoxify this compound, A. thaliana plants genetically manipulated to express bacterial genes mer A (Catalyzes proteolysis of the carbonmercury bond with the release of Hg 2+) and mer B (converts Hg2+ taken up by roots to Hgo, a less toxic volatile element which is released into the atmosphere). These transgenic A. thaliana plants thaliana plants were able to grow on media containing 50-fold higher methylmercury concentrations than wild type plants (Rugh et al., 1996). The roles of glutathione and phytochelatins in heavy metal tolerance have been well illustrated in Cd-sensitive mutants of Arabidopsis, of Arabidopsis, cad1 and cad2. cad2. These mutants are deficient in PC production due to mutations in PC synthase and ³-glutamyl-cys synthetase in cad1 and cad 2 mutants, respectively (Howden et al., 1995; Cobbett et al., 1998). To investigate the importance of PC for Cd tolerance, Lee et al., (2003) overexpressed the Arabidopsis PC Synthase gene, AtPCS 1. The results of this study showed that the normal level of Arabidopsis PC synthase expression was sufficient to synthesize the required PCs in response to supplemented levels of Cd and increased capacity of PC synthesis does not lead to Cd tolerance, but paradoxically leads to Cd hypersensitivity. Furthermore, the genes encoding enzymes involved in glutathione synthesis may hold more promise as overexpression of of E. coli gsh 1 gene encoding ³-glutamylcysteine synthetase (³-ECS) or E. or E. coli gshII gene encoding glutathione synthetase (GS) in Brassica juncea enhanced PC synthesis and Cd tolerance (Zhu et al., 1999 b,c).

FUTURE STRATEGIES FOR PHYTOREMEDIATION In order to achieve practical applications of phytoremediation, hyperaccumulators offer an important tool for inexpensive soil decontamination for those elements which these plants hyperaccumulate. But these natural hyperaccumulators have limited potential for phytoremediation, as most of these are slow growing (e.g., mosses, lichens, or the Thalaspi species that take up heavy metals) and/or have low biomass (Salt et al., 1998). Thus, in order to overcome these limitations, attempts have been made to bring heavy metal tolerance from natural hyperaccumulators like Thalaspi species into high biomass crop plant species like Brassica juncea (which has been reported to accumulate Cd as well as other toxic metals) by protoplast fusion technique (Dushenkov et al., 2002). Other new developments in plant genetic engineering are tailored transgenics that overexpress different enzymes in different plant parts or that express a transgene only under certain environmental conditions (Dhankher et al., 2002). But, many oppose introducing transgenic or non-transgenic phytoremediating plant species because they pose a risk of spread to adjacent areas, displacing native or other desirable species or hybridizing with related native species (Gressel and Al-Ahmad,


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2005). To overcome the biological risks of plants used for phytoremediation, Ruiz et al. (2003) have shown that genetic engineering of the chloroplast genome can be used as a novel approach to obtain high expression without the risk of spreading the transgene via pollen. The future challenge for phytoremediation is to further reduce the cost and increase the spectrum of metals amenable to this technology. This goal can be achieved by creating superior plant varieties for phytoextraction by using genetic engineering to introduce valuable traits into plants, optimizing agronomic practices for their cultivation, and designing safer and more effective soil amendments (Gleba et al., 1999). Further, manipulating rhizospheric bacteria and introduction of arbuscular mycorrhizal (AM) fungal inoculums into metal contaminated areas could be used as a strategy for enhancing the establishment of mycorrhizal herbaceous species, as AM fungi play a vital role in metal tolerance and accumulation (Zhu et al., 2001).

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Heavy Metal Toxicity in Plants and Phytoremediation

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