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Heavy Metal Toxicity in Plants and Phytoremediation

R.C. Setia, Navjyot Kaur, Neelam Setia and Harsh Nayyar1
Department of Botany, Punjab Agricultural University, Ludhiana-141004, India
1
Department of Botany, Panjab University, Chandigarh-160014
email: rc_setia@yahoo.co.in

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 z Editors: R.C. Setia, Harsh Nayyar and Neelam Setia
© 2008 I.K. International Publishing House Pvt. Ltd., New Delhi, pp 206-218
 Heavy Metal Toxicity in Plants and Phytoremediation 207

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).
208 Crop Improvement: Strategies and Applications

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 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-
 Heavy Metal Toxicity in Plants and Phytoremediation 209

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., 2000), and
ZIP family (Guerinot, 2000). Further, heavy metal ions such as Cd enters the plant cell by transporters
for essential cations such as Fe2+(Thomine et al., 2000). AtNramp genes in Arabidopsis encode the
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.
210 Crop Improvement: Strategies and Applications

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 (Vmax) 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-glu-
cys-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).
 Heavy Metal Toxicity in Plants and Phytoremediation 211

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
212 Crop Improvement: Strategies and Applications

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
concentrations in their tissues. For example, Brassica juncea, while having one-third the concentration
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 metal-
tolerant 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
 Heavy Metal Toxicity in Plants and Phytoremediation 213

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 gene-
encoded, 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
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, metal-
induced, 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 ATP-
dependent 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 SeO3 (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. 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
214 Crop Improvement: Strategies and Applications

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,
is biosynthesized in Hg-contaminated soils, and to detoxify this compound, A. thaliana plants were
genetically manipulated to express bacterial genes mer A (Catalyzes proteolysis of the carbon-
mercury bond with the release of Hg2+) 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
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, cad1 and 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 E. coli gsh 1 gene encoding ³-glutamylcysteine synthetase (³-ECS) 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,
 Heavy Metal Toxicity in Plants and Phytoremediation 215

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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