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Phytoremediation of Heavy Metals: Physiological and Molecular Mechanisms

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Phytoremediation of Heavy Metals: Physiological and Molecular Mechanisms

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Heavy metals (HM) are a unique class of toxicants since they cannot be broken down to non-toxic forms. Concentration of these heavy metals has increased drastically, posing problems to health and environment, since the onset of the industrial revolution. Once the heavy metals contaminate the ecosystem, they remain a potential threat for many years. Some technologies have long been in use to remove, destroy and sequester these hazardous elements. Even though effective techniques for cleaning the contaminated soils and waters are usually expensive, labour intensive, and often disturbing. Phytoremediation, a fast-emerging new technology for removal of toxic heavy metals, is cost-effective, non-intrusive and aesthetically pleasing. It exploits the ability of selected plants to remediate pollutants from contaminated sites. Plants have inter-linked physiological and molecular mechanisms of tolerance to heavy metals. High tolerance to HM toxicity is based on a reduced metal uptake or increased internal sequestration, which is manifested by interaction between a genotype and its environment. The growing interest in molecular genetics has increased our understanding of mechanisms of HM tolerance in plants and many transgenic plants have displayed increased HM tolerance. Improvement of plants by genetic engineering, i.e., by modifying characteristics like metal uptake, transport and accumulation and plant’s tolerance to metals, opens up new possibilities of phytoremediation. This paper presents an overview of the molecular and physiological mechanisms involved in the phytoremediation process, and discusses strategies for engineering plants genetically for this purpose.
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Phytoremediation of Heavy Metals: Physiological
and Molecular Mechanisms
Ruqaya Jabeen
1
&Altaf Ahmad
1
&
Muhammad Iqbal
1,2,4
1
Molecular Ecology Laboratory, Department of Botany, Faculty of Science, Hamdard University,
New Delhi 110062, India
2
Department of Plant Production, College of Food & Agricultural Sciences, King Saud University,
Post Box # 2460, Riyadh 11451, Saudi Arabia
3
Author for Correspondence; e-mail: iqbalg5@yahoo.co.in
Published online: 7 November 2009
#The New York Botanical Garden 2009
Abstract Heavy metals (HM) are a unique class of toxicants since they cannot be broken
down to non-toxic forms. Concentration of these heavy metals has increased drastically,
posing problems to health and environment, since the onset of the industrial revolution.
Once the heavy metals contaminate the ecosystem, they remain a potential threat for many
years. Some technologies have long been in use to remove, destroy and sequester these
hazardous elements. Even though effective techniques for cleaning the contaminated soils
and waters are usually expensive, labour intensive, and often disturbing. Phytoremedia-
tion, a fast-emerging new technology for removal of toxic heavy metals, is cost-effective,
non-intrusive and aesthetically pleasing. It exploits the ability of selected plants to
remediate pollutants from contaminated sites. Plants have inter-linked physiological and
molecular mechanisms of tolerance to heavy metals. High tolerance to HM toxicity is
based on a reduced metal uptake or increased internal sequestration, which is manifested
by interaction between a genotype and its environment. The growing interest in molecular
genetics has increased our understanding of mechanisms of HM tolerance in plants and
many transgenic plants have displayed increased HM tolerance. Improvement of plants by
genetic engineering, i.e., by modifying characteristics like metal uptake, transport and
accumulation and plants tolerance to metals, opens up new possibilities of phytoreme-
diation. This paper presents an overview of the molecular and physiological mechanisms
involved in the phytoremediation process, and discusses strategies for engineering plants
genetically for this purpose.
Introduction
Global industrialization has resulted in a widespread contamination of environment
with persistent addition of organic and inorganic wastes. The contaminants enter the
environment either by natural processes or through human activity. The natural
Bot. Rev. (2009) 75:339364
DOI 10.1007/s12229-009-9036-x
3
contamination originates from excessive withering of minerals from rocks or
displacement from the groundwater or subsurface layers of the soil. Disposal of
industrial effluents, sewage sludges, deposition of air-borne industrial wastes,
military operations, mining, land-fill operations, industrial solid-waste disposal and
the growing use of agricultural chemicals such as pesticides, herbicides and
fertilizers are sources of human-assisted contamination of the environment.
Heavy metals (HMs) are among the major environmental contaminants and pose a
severe threat to human and animal health by their long-term persistence in the
environment (Gisbert et al., 2003; Halim et al., 2003). For instance, lead (Pb) may have
a soil-retention time of 1505,000 years and was reported to maintain a high
concentration for as long as 150 years after application of sludge to the soil
(Nandakumar et al., 1995). The biological half-life of cadmium (Cd) is about 18 years
(Forstner, 1995). Given this, a long-term plan of pollution remediation measures that
may lower the rate of pollution increase has become indispensable. The commonly used
technologies for in situ and ex situ remediation of HM-contaminated soil are pneumatic
fracturing, soil flushing, solidification, vitrification, electrophoresis, chemical reduc-
tion, soil washing and excavation. All these conventional methods, colloquially termed
as pump and treatand dig and dumptechniques, are limited in their applicability to
small areas and have their own limitations.
Over the last one decade, a fast emerging, low-cost and eco-friendly alternative to
the conventional remediation techniques has gained ground both in public and private
sectors. Termed as phytoremediation, this technique engages plants to cleanse the
nature, as plants can absorb, accumulate and detoxify contaminants of their substrates
(soil, water and air) through physical, chemical or biological processes. Various soil
and plant factors such as the physical and chemical properties of the soil, the plant and
microbial exudates, bioavailability of metals, and the ability of plants to uptake,
accumulate, translocate, sequester and detoxify metals account for phytoremediation
efficiency (Hooda, 2007). Several microbes including the mycorrhizal and non-
mycorrhizal fungi, and the cultivated and wild metal-hyperaccumulating plants are
being tested in labs and fields for decontaminating the metalliferrous substrates
present in the environment. According to Salt et al. (1995), to clean up one acre of
soil to a depth of 50 cm, phytoextraction (a type of phytoremediation) costs US
$60,0001,000,000, whereas soil excavation (a type of physical remediation)
requires at least US $4,000,000. Understanding of the mechanisms of plant tolerance
to a particular metal is important for developing plants that are suitable for
phytoremediation of the contaminated sites. This review focuses on the physiological
and molecular aspects of phytoremediation of the HM-infested soil and water.
Types of Phytoremediation
Plants utilize several methods to remediate the polluted sites. Phytoremediation technology
can be subdivided, on the basis of its underlying process and applicability, as follows:
Phytoextraction
Phytoextraction, a common process of phytoremediation, involves uptake of the
contaminant by plant roots with subsequent accumulation in the aerial plant parts,
340 R. Jabeen et al.
followed generally by harvest and then disposal of plant biomass. The metal-
accumulating plants are seeded or transplanted into the metal-contaminated soil and
then cultivated with established agricultural practices. The roots of these plants absorb
metal elements from the soil and translocate them to the aerial shoots, where they
accumulate. After a sufficient plant growth and metal accumulation, the aerial plant
parts are harvested and removed, thus ensuring a permanent removal of metals, such as
Pb, Cd, Ni, Cu, Cr, and V, from the contaminated soils. However, it is applicable only to
those sites containing low to moderate levels of metal pollution, because plant growth
does not sustain in heavily pollutedsites (Padmavathiamma & Loretta, 2007). Chelating
agents are added to solubilize metals that have a low solubility in the soil solution
(Prasad, 2003). EDDS, a chelating agent, increased Cu accumulation in Cannabis
sativa (Meers et al., 2005). Plants to be used for phytoextraction should have: (a)
tolerance to high concentrations metals, (b) high metal-accumulation capability, (c)
heavy biomass, (d) ability to grow fast and a (e) profuse root system. The success of
phytoextraction depends especially on the plantsability(a)toaccumulatebiomass
rapidly, and (b) to store large quantities of the uptaken metals in the shoot tissue
(Blaylock et al., 1997;McGrath,1998; Blaylock & Huang, 2000). Ability of plants to
withstand difficult soil conditions (i.e. soil pH, salinity, soil structure, water content)
and produce a dense root system are also important.
The strategies used in developing a phytoremediation plan are (a) screening of
hyperaccumulator candidate plants, (b) plant breeding, and (c) development of improved
hyperaccumulators using genetic tools. Applicability of phytoextraction technology in
terms of cost and required time must shift the current paradigm of remedial targets,
which is based on total metal concentrations, towards the bioavailable contaminant
stripping (BCS), as proposed initially by Hamon and McLaughlin (1999). With this
strategy, the clean up time can be shortened by targeting extraction of only the most
liable and bioavailable metal pools (Schnept et al., 2002; Sommer et al., 2002).
Rhizofiltration
This technique relies on the ability of plant roots to take up and sequester metal
contaminants or excess nutrients from the aqueous growth substrates (waste-water
streams, nutrientrecycling systems). It remediates metals like Pb, Cd, Ni, Cu, Cr, Vand
radionucliides (U, Cs, Sr). The ideal plants should produce significant amounts of root
biomass or root surface area, be able to accumulate and tolerate significant amounts of
target metals, involve easy handling and a low maintenance cost, and have a minimum of
secondary waste that requires disposal (Dushenkov & Kapulnik, 2000). Terrestrial plants
more suitable for rhizofiltration because they produce longer, more substantial and often
fibrous root systems with large surface areas for metal sorption. Indian mustard
(Brassica juncea) and sunflower (Helianthus annuus) are most promising for metal
removal from water. Indian mustard effectively removes Cd, Cr, Cu, Ni, Pb, and Zn
(Dushenkov et al., 1995) whereas sunflower absorbs Pb (Dushenkov et al., 1995)andU
(Dushenkov et al., 1997) from hydroponic solutions. Indian mustard could effectively
removeawiderange(4500 mg/l) of Pb concentration (Raskin & Ensley, 2000).
Rhizofiltration can be conducted in situ to remediate the contaminated surface-
water bodies, or ex-situ where an engineered system of tanks is used to hold the
introduced plants and the contaminated water. Commercialization of this technology is
Phytoremediation of Heavy Metals 341
driven by economics as well as by technical advantages like applicability to many
problem metals, ability to treat high volumes of water, minimum requirement of
chemicals and likelihood of regulatory and a public acceptance (Dushenkov et al., 1995).
Phytostabilization
In this technique, plants are used to transform toxic soil metals to less toxic forms
(Eapen & Dsouza, 2005), which are not removed from the soil. Phytostabilization
stabilizes wastes, prevents exposure pathways via wind and water erosion, provides a
hydraulic control that suppresses vertical migration of contaminants into ground water
and immobilizes the contaminants physically and chemically by root sorption and
chemical fixation with various soil amendments (Cunningham et al., 1995;Berti&
Cunningham, 2000). It requires plants that are able to grow in contaminated soil with
their roots growing into the contamination zone, and alter the biological, chemical or
physical conditions in the soil that convert the toxic forms of metal to less toxic ones.
Immobilization of toxic metals by plants may be enhanced by soil amendments that
increase the soil organic matter and pH (using lime), or by binding certain constituents
with phosphate or carbonate without using soil amendments (Schnoor, 2000).
Smith and Bradshaw (1992) developed two cultivars of Agrostis tenius and one of
Festuca rubra, which are now commonly available for phytoremediation of the Pb-,
Zn- and Cu-contaminated soils. Phytostabilization, though most effective at sites
having fine-textured soils with high organic matter content, can treat a wide range of
surface contamination (Cunningham et al., 1995; Berti & Cunningham, 2000). Deep-
rooting plants could reduce the highly toxic Cr VI to Cr III, which is much less
soluble and therefore, less bioavailable (James, 2001). Soil amendments should fix
metals rapidly, followed by their incorporation, whereas chemical alterations should
be long lasting if not permanent. The most important soil amendments are phosphate
fertilizers, organic matter or bio-solids, iron or manganese oxyhydroxides, natural or
artificial clay minerals or mixtures of these amendments. Phytostabilization does not
require soil removal and/or disposal of the hazardous material or the biomass.
Phytovolatization
Use of plants to absorb HM contaminants and make convert them to volatile, less
toxic chemical species through transpiration is called phytovolatization. Some
metals, like As, Hg and Se, may exist as gaseous species in the environment. Some
naturally occurring or genetically modified plants, like Chara canescens (musk-
grass), Brassica juncea (Indian mustard) and Arabidopsis thaliana, are reported to
possess capability to absorb heavy metals and convert them to gaseous species
within the plant and subsequently release them into the atmosphere (Ghosh & Singh,
2005). Phytovolatization has been used primarily for the removal of mercury
wherein mercuric ion is transformed into the less toxic gaseous elemental mercury
(Ghosh & Singh, 2005). Some plants growing in high Se media e.g, Arabidopsis
thaliana and Brassica juncea, produce volatile Se in the form of dimethylselenide
and dimethyldiselenide (Banuelos, 2000). Overexpression of CGS in Brassica
promoted selenium volatilization and the CGS seedlings were more tolerant to
selenite than wild type. The CGS plants contained Se levels that were 2040% lower in
342 R. Jabeen et al.
shoots and 5070% lower in roots than in wild type when supplied with selenite (Van
Huysen et al., 2003). Phytovolatization has been successful also in removing Tritium
(
3
H), a radioactive isotope of hydrogen, which is decayed to stable helium with a half-
life of about 12 years (Dushenkov, 2003). Effort has been made to insert the bacterial
Hg ion reductase genes into plants for purpose of Hg phytovolatization (Rugh et al.,
1996;Rughetal.,1998). Nicotiana tabacum and Arabidopsis thaliana, which include
a gene for mercuric reductase to convert ionic mercury (Hg II) to less toxic metallic
mercury (Hg 0) and volatilize it, have been genetically modified (Meagher et al.,
2000). Volatile Se compounds, such as dimethylselenide are 500 to 600 times less
toxic than the inorganic forms of Se found in the soil (Deesouza et al., 2000).
Furthermore, phytovolatization involves minimal site disturbance, little erosion and no
disposal of contaminated plant material (Rugh et al., 2000). A gene, encoding the
enzyme SMT, has been cloned from the Se-hyperaccumulator, Astragulus bisculatus
(Neuhieral et al., 1999). When overexpressed in Arabidopsis and Indian mustard, it
increased selenium tolerance, accumulation and volatilization. The SMT transgenic
seedlings tolerated Se, particularly selenite, significantly better than the wild type,
producing a 37 fold higher biomass and 3-fold longer roots (Le Duc et al., 2004).
However, unlike other remediation techniques, once the contaminants have been
removed via volatilization, one has no control over their migration to other areas.
Phytodegradation (Phytotransformation)
In this method, plants degrade organic pollutants by metabolic processes and using the
rhizospheric associations between plants and soil microorganisms. Plant enzymes that
metabolize contaminants may be released into the rhizosphere, where they may play active
role in transformation of contaminants. Enzymes, like dehalogenase, nitroreductase,
peroxidase, laccase and nitrilase, have been discovered in plant sediments and soils.
Organic compounds such as munitions, chlorinated solvents, herbicides and insecticides
and the inorganic nutrients can be degraded by this technology (Schnoor et al., 1995). The
dissolved TNT (trinitrotoluene) concentrations in flooded soil decreased from 128 ppm
to 10 ppm within one weak in the presence of the aquatic plant, Myriophyllum aquaticum,
which produces nitroreductase enzyme that can partially degrade TNT (Schnoor et al.,
1995). To engineer plant tolerance to TNT, two bacterial enzymes (PETN reductase and
nitroreductase), able to reduce TNT into less harmful compounds, were overexpressed in
tobacco plants. The two genes onr and nfs, under the control of a constitutive promotor,
provided the transgenic plants with increased tolerance to TNT at a concentration that
severely affected the development of wild type plants (Hannink et al., 2001).
Various plants tested for different phytoremediation technologies are enlisted in
Table 1.
Mechanisms of Metal Sequestration
Metal Uptake Mechanism
Metal uptake depends primarily on metal bioavailability. In soils, metals exist as a
variety of chemical species in a dynamic equilibrium governed by the physical,
Phytoremediation of Heavy Metals 343
chemical and biological processes of the soil (Chaney, 1988). Bioavailability of soil
pollutants, a primary basis of remediation efficacy, refers to a fraction of the total
pollutant mass in the soil and sediment available to plants. Uptake of metals by
plants involves root interception of metal ions, entry of metal ions into roots and
Table 1 Important Plants Used for Phytoremediation of Heavy Metals
Contaminant Medium Process Plant References
Arsenic Soil Phytoextraction Pteris vittata L. Gonzaga et al. (2006)
Phytostabilization Piricum sativum L. Jonnalagadda and
Nenzou (1997)
Boron Soil Phytoextraction Gypophila sphaerocephala
Fenzel
Babaoglu et al. (2004)
Cadmium Soil Phytoextraction Oryza sativa L. Murakami et al. (2007)
Phytostabilization Vettiveria zizanioides L. Lai and Chen (2004)
Water Rhizofiltration Lemna minor L. Hou et al. (2007)
Chromium Soil Phytoextraction Brassica juncea L. Zhang et al. (2007)
Phytostabilization Brassica juncea L. Salt et al. (1995)
Water Rhizofiltration Brassica juncea L. Diwan et al. (2008)
Cobalt Soil Phytoextraction Berkheya coddii Roessler Keeling et al. (2003)
Copper Soil Phytoextraction Elsholtzia splendens
Nakai ex Maekawa
Jiang et al. (2004)
Phytostabilization Festuca rubra L. Smith and Bradshaw (1979)
Water Rhizofiltration Lemna minor L. Hou et al. (2007)
Lead Soil Phytoextraction Chenopodium album L. Celestino et al. (2006)
Phytostabilization Vetiveria zizanioides L. Rotkittikhun et al. (2007)
Water Rhizofiltration Hemidesmus indicus L. Chandrashekhar et al.
(2004)
Manganese Soil Phytoextraction Phytolacca americana L. Min et al. (2007)
Mercury Soil Phytoextraction Marrubium vulgare L. Jimenez et al. (2006)
Water Rhizofiltration Pistia stratiotes L. Skinner et al. (2007)
Nickel Soil Phytoextraction Alyssum lesbiacum
(Candargy) Rech. f.
Singer et al. (2007)
Phytostabilization Agropyron elongatum
(Host.)P. Beauv.
Chen and Wong (2006)
Water Rhizofiltration Lemna minor L. Axtell et al. (2003)
Selenium Soil Phytoextraction Brassica rapa L. Moreno et al. (2005)
Phytovolatization Brassica spp (Wild type) Banuelos et al. (2005)
Water Rhizofiltration Lemna minor L. Zayed et al. (1998)
Uranium Soil Phytoextraction Lolium perenne L. Vadenhov and Heese
(2004)
Water Rhizofiltration Chenopodium amaranticolor
H.J.Coste & Reyn
Eapen et al. (2003)
Zinc Soil Phytoextraction Cynodon dactylon (L.)Pers. Celestino et al. (2006)
Phytostabilization Cynodon dactylon (L.) Pers. Pierzynski et al. (1994)
Water Rhizofiltration Brassica juncea L. Dushenkov et al. (1995)
344 R. Jabeen et al.
their translocation to the shoot through mass flow and diffusion. The uptake is achieved
by mobilizing metals bound to soil particles through the metal-chelating molecules
(mugenic and aveic acids) secreted into the rhizosphere, specific plasma-membrane-
bound metal reductase and the proton extrusion from roots (Salt et al., 1995).
Another type of exudate produced by grasses are phytosiderophores, which bind Fe
and facilitate its uptake. Phytosiderophores are biosynthesized from nicotinamide,
which is composed of three methionines coupled via non-peptide bonds (Higuchi et al.,
1999). Chelation of phytosiderophores can help in the transport of metal ions across
the plasma membrane as a metal-siderophore complex via specialized transporters. By
reducing the chelated Fe (III) with a root ferric chelate reductase, plants are able to
release soluble Fe (II) for uptake by roots (Salt et al., 1994). Plants can also solubilize
iron and other metals by exuding protons from roots to acidify the rhizosphere (Salt et
al., 1994). It is therefore possible to enhance the bioavailability of metal pollutants by
manipulating the root processes. Once the metal is bioavailable to the plant, the entry of
metal ions inside the plant, either through symplast (intercellular) or apoplast
(extracellular), depends on the type of metal and the plant species. The apoplast
continuum of root epidermis and cortex is readily permeable for solutes. Apoplastic
pathway is relatively unregulated, because water and dissolved substance can flow and
diffuse without crossing the membrane. The cell walls of the endodermal layer act as a
barrier for apoplastic diffusion into the vascular system (Ghosh & Singh, 2005).
Apoplastic transport is limited by high cation exchange capacity of the cell wall (Raskin
et al., 1997). In the symplastic transport, metal ions move across the plasma membrane,
which usually has a large negative resting potential of approximately 170 mV (negative
inside the membrane). This membrane potential provides a strong electrochemical
gradient for the inward movement of the metal ions (Ghosh & Singh, 2005). Most metal
ions enter plant cells by an energy-dependent process via specific or generic metal-ion
carriers or channels (Bubb & Lester, 1991). Cutler and Rains (1974) found that a large
fractionofCdwastakenupbybarleytissuesthrough exchange absorption, and through
diffusion coupled with sequestration, without any concomitant active metabolic uptake.
Although there is no direct evidence for a role of plasma membrane efflux
transporters in the HM tolerance in plants, recent research has revealed that plants
possess several classes of metal transporters such as heavy metal (or CPX-type)
ATPases that are involved in the overall metal-ion homeostasis and tolerance in
plants (Williams et al., 2000), the natural resistance-associated macrophage-protein
(Nramp) family, cation-diffusion facilitator (CDF) proteins family and the zinc-iron
permease (ZIP) family (Guerinot, 2000). Yang et al. (2005) found a correlation
between uptake capacity and hyperaccumulation of ZIP family members in the plant,
Thlaspi caerulescens. Under Zn-replete conditions, two ZIP cDNA (ZNT1 and
ZNT2) are expressed at significantly higher levels in the roots of different
T. caerulescens accessions than those of the non-hyperaccumulating, T. arvense
(Pence et al., 2000; Assuncao et al., 2001). Thus, overexpression of the uptake
systems may result in enhanced accumulation of the metals.
Metal Translocation to Shoots
Once the metal ions have entered the roots, they can either be stored in the root or
forwarded to the shoot, primarily through the xylem. The rate of metal translocation
Phytoremediation of Heavy Metals 345
to the shoot may depend on metal concentration in the root (Hardiman et al., 1984).
Prezemeck and Haase (1991) suggested a phytochelatin-mediated metal binding in
the xylem sap as a possible mechanism for metal translocation. Low molecular
weight chelators such as citrate (Lee et al., 1977) and free histidine as in Alyssum
lesbiacum (Kramer et al., 1996) were associated with this process. Other chelating
compounds like malate, citrate, and histidine may also have a role in the metal-ion-
mobility in plants (Von Wiren et al., 1999). Membrane transport systems are likely to
play a central role in the translocation process. Many gene families that are involved
in metal transport have been identified. Some of them are heavy-metal ATPases,
natural resistance-associated macrophage proteins (NRAMPs), cation diffusion
facilitators, the Zrt- and Irt- like proteins family, and the cation antiporters (Hall &
Williams, 2003).
Tolerance Mechanism
Plant tolerance to a particular metal is governed by an inter-related network of
physiological and molecular mechanisms, to be understood essentially for
developing plants suitable for phytoremediation of the contaminated sites. The
apparent tolerance to increasing levels of toxic elements can result from the
exclusion of toxic elements or the metabolic tolerance of plants to specific elements.
(Singh et al., 2003).
Exclusion
Transport across the root-cell membrane initiates the process of metal absorption by
plant tissues. The electrical charge prevents metal ions from diffusing freely across
the lipophilic cellular membranes into the cytosol (Horst et al., 2002). Therefore, ion
transport into cells must be mediated by membrane proteins with transport functions.
Root-uptake kinetics has been investigated for a variety of metal ions including Cd
2+
(Cohen et al., 1998; Hart et al., 1998), Cu
2+
(Thornton, 1991) and Zn
2+
(Santa Maria
& Cogliatti, 1988; Vazquez et al., 1994). Entry of Cd in the cells of radish seeds
through Ca channels was proposed by Rivetta et al. (1997).
Vacuolar Compartmentalization
Metal compartmentalization in the vacuole is well documented (Vazquez et al.,
1994; Kupper et al., 1999). Significant for metal detoxification and plant tolerance,
this process prevents free concentration of metal ions in the cytosol and forces them
into a limited space (Tong et al., 2004). Several transporters have been shown to
mediate Zn fluxes across the cellular membranes (Paulsen and Saier, 1997) including
the tonoplast (Macdiarmid et al., 2000). In yeast, over-expression of membrane
MTPS (metal tolerance proteins) from Thlaspi goesingense confers resistance to Cd,
Co, Ni and Zn possibly due to their transport into the vacuole (Persans et al., 2000).
Cd-phytochelatin complexes as well as apo-phytochelatins are transported through
specific carriers against concentration gradient across the tonoplast. They accumulate
inside the tonoplast vesicles (Salt & Rauser, 1995). Intact vacuoles isolated from the
tobacco and barley leaves exposed to Zn contained Zn ions (Brune et al., 1994;
346 R. Jabeen et al.
Krotz et al., 1989). High-level expression of a vacuolar metal-ion transporter
TgMTP1 in T. goesingense was proposed to account for the enhanced ability of
plants to accumulate metal ions within shoot vacuoles (Persans et al., 2001). Within
plant cells, PC-metal complexes bound by GSH or PCs are shuttled to the vacuole
by an AB-type transporter protein in the tonoplast (Lu et al., 1997). Anthocyanins
can also bind metals (Pilon-Smits & Pilon, 2002) and have a role in metal
sequestration. Other molecules involved in metal complexation in the vacuole are the
organic acids (Kramer et al., 2000). Vacuolar Zn accumulation has been confirmed
in roots and shoots of Thlaspi caerulescens (Vazquez et al., 1992,1994). Increase in
the vacuolar volume fraction of meristematic cells in Festuca rubra during zinc
exposure also confirms Zn accumulation within the vacuole as a detoxification
mechanism (Davies et al., 1991). To date, the best characterized vacuolar transporter
and channel involved in metal tolerance is YCF1 from Saccharomyces cerevisiae.
This is an MgATP-energized glutathione-S-conjugate transporter responsible for
vacuolar sequestration of organic compounds after their S-conjugation with
glutathione and GSH-metal complexes. It catalyses transport of bis (glutathione)
cadmium (Cd-GS
2
) (Li et al., 1997), Ag-GS
3
(Ghosh et al., 1996) and Hg-GS
2
(Gueldry, 2003) into vacuoles. Overexpression of such vacuolar transporters can be
used to engineer advanced phytoremediators with increased ability to pump heavy
metals into a safe compartment.
Phytochelatins
HM accumulation in plants induces production of phytochelatins (PCs), a family of
thiolrich peptides of a general structure (γ-Glu-Cys) n-Gly, where n is normally in
the range of 25 (Steffens, 1990; Rauser, 1995). Cd and As effectively induce
phytochelatin synthesis, while Zn and Ni hardly do so (Grill et al., 1989).
Glutathione, the substrate for PC synthase, is synthesized from its constituent amino
acids in two steps; the first step is catalyzed by γ-glutamyl-cys synthase (γ-ECS)
and the second one by glutathione synthase (GS). The γ-glutamyl-Cys-synthase
activity is controlled through feedback regulation by glutathione and is dependent on
the availability of cysteine (Mejare & Billow, 2001). The gene encoding this enzyme
has been identified in Arabidopsis thaliana (Clemens et al., 1999; Ha et al., 1999;
Vatamaniuk et al., 1999), Triticum aestivum (Clemens et al., 1999)andSchizo-
saccharomyces pombe (Clemens et al., 1999; Ha et al., 1999).
Although many earlier studies have suggested a role of PCs in metal
detoxification, it was clearly demonstrated by the PC-deficient Cad1-3 mutant of
Arabidopsis and the PC-synthase targeted deletion mutant of S. pombe. Both the
mutants are highly sensitive to Cd and As but with a little or no sensitivity to metals
like Cu, Hg, and Ni, showing that PCs are essential for the detoxification of Cd and
As but have little involvement in that of Cu, Hg and Ni. (Ha et al., 1999). In such
cases there may be a more effective alternative like metallothioneins and histidine.
Lee et al. (2003) overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic
Arabidopsis with the goal of increasing transgenic plants showing increased
production of PCs (1.32.1 fold at 85µM CdCl
2
stress for 3 days) as compared
with wild type plants. However, PCs lines paradoxically showed hypersensitivity to
cadmium and zinc when grown on agar medium containing a 50 or 85 µM CdCl
2
.
Phytoremediation of Heavy Metals 347
GSH-dependent PC-synthase activity was identified in cultured cells of Silense
cucubalis (Grill et al., 1989). The enzyme was active only in the presence of Cd, Cu,
Zn, Ag, Hg and Pb. Similar activities have been identified, inter acia, in tomato
(Howden et al., 1995) and pea (Klapheck et al., 1995). Despite the detection of PC-
synthase activity a decade ago, identification of a corresponding gene remained
elusive until Vatamaniuk et al. (1999) identified an Arabidopsis cDNA; named
AtPCS1. Expression of AtPCS1 protein mediated increase in Cd accumulation,
suggesting a possible role of AtPCS1 in Cd chelation or sequestration. Clemens et al.
(1999) identified a wheat cDNA, TaPCS1, that increased Cd resistance in wild-type
yeast. The resistance, associated with increase in Cd accumulation, was GSH-
dependent. Occurrence of the AtPCS1 and TaPCS1 mediated tolerance in vacuole-
deficient mutants suggests that PCs are localized in cytosol and play important role
in tolerance mechanism. The significance of PC-Cd complex formation for
detoxification of Cd
2+
in plants was supported by the isolation of the Arabidopsis
cad 1 mutant, which contains wild-type levels of GSH, but is PC-deficient and Cd
2+
hypersensitive (Howden et al., 1995). The CAD1 gene, also called AtPCS1, encodes
a PC-synthase as evident by the detection of GSH- and metal-dependent PC
synthesis in Escherichia coli cells expressing AtPCS1 (Ha et al., 1999).
Independently, AtPCS1 and TaPCS1 from wheat were isolated in screens for plant
cDNAs conferring Cd
2+
tolerance (Vatamaniuk et al., 1999). The S. cerevisiae cells
expressing these genes display a Cd
2+
tolerance phenotype that is GSH-dependent
and correlates with PC synthesis. Purified recombinant PCS proteins from
Arabidopsis and S. pombe catalyze the formation of PCs from GSH (Clemens et
al., 1999; Vatamaniuk et al., 1999). The Arabidopsis cad1-3 mutant is highly
sensitive to Cd
2+
and AsO
42-
, compared with the wild type, and displays slightly
elevated sensitivities towards Cu, Hg and Ag (Ha et al., 1999). Gisbert et al. (2003)
reported that overexpression of wheat TaPCS1 gene encoding PC synthase in
tobacco greatly increased its tolerance to Pb and Cd, causing the seedling roots to
grow 160% longer than in the wild type. The transgenic seedlings grown in the
mining soils containing 1,572 mg/Kg Pb accumulated double the amount of Pb and
Cd accumulated by the wild type. Raab et al. (2004) have developed a method to
ascertain the nature of As-PC complex in extracts of the As-tolerant grass (Holeus
lanatuss) and As-hyperaccumulator fern (Pteris cretica), using the metal-specific
(inductively coupled plasma-mass spectroscopy) and organ-specific (electro spray
ionization-mass spectroscopy) detection systems.
The HM-detoxification process is not limited to the chelation of the metal ions.
After the activation of PC synthase by the metal ions and metal chelation by the PCs
synthesized, the metal ion complex is transported to the vacuole and stabilized there
by forming a complex with sulfides or organic acids (Rauser, 1990). The tonoplast
transport and vacuolar compartmentation of PC-Cd complexes increase the metal-
binding capacity of phytochelatins (Vogeli-Lange & Wagner, 1989; Salt & Rauser,
1995; Bae & Mehra, 1998).
Metallothioneins
Metallothioneins (MTs) is a group of low molecular weight, cysteine-rich, metal-
binding proteins, which provide thiols for metal chelation. Overexpression of genes
348 R. Jabeen et al.
involved in the synthesis of metal chelators may lead to enhanced or reduced metal
uptake and enhanced metal translocation and or sequestration (Cherian & Oliveira,
2005). An MT- gene (CUP1), when overexpressd in cauliflower, resulted in a 16-
fold higher cadmium tolerance (Hesegawa et al., 1997). Similarly, high Cu
accumulation has been reported in Arabidopsis thaliana by the overexpression of
a pea MT gene (Pan et al., 1994). Although PCs formally belong to this group of
compounds (Class III MTs), the enzymatic synthesis of PCs distinguishes them from
MT proteins. Since their discovery as the Cd-binding proteins in the equine kidney
(Margoshes & Valte, 1957), MT proteins and genomes have been reported in
animals as well as in eukaryotic microorganisms and plants. Plants have a family of
metallothionein (MT) genes encoding peptides that generally consist of 60-80 amino
acids and contain 916 cysteine residues (Chatthai et al., 1997). Metallothionein-
metal complex can be glutathioned (Brouwer et al., 1993), suggesting that they may
be transported into vacuoles for long-time sequestration. The MT-gene structure and
expression are described and discussed extensively in some recent reviews (Rauser,
1999; Cobbett & Golsbrough, 2002). Although metals, including copper, either do
not affect or repress MT-gene expression in some species, copper induces expression
of a Type 1 MT gene (Murphy & Taiz, 1995). Since other stresses, like heat shock
and aluminium, also induce this type of expression, it was suggested that these MTs
might express as part of a general stress response (Cobbett & Golsbrough, 2002).
There is, however, some evidence to suggest that MTs are involved in copper
homeostasis and detoxification (Zhou & Goldsbrough, 1995). Given the wide range
of factors that induce MT synthesis, Karin (1985) has suggested that metal
detoxification is not the primary role of MTs. They are known to sequester metals
in the cell and pass metal ions to the apoenzymes that require them. Reactivation of
theZn-requiringapoenzymesbytransferofzincionsfromMTshasbeen
demonstrated (Udom & Brady, 1980); similar is the case of the transfer of copper
from Nuerospora crassa MT to copper-requiring apoenzymes (Beltramini & Lerch,
1982).
Expression of plant MT genes is well studied. A detailed investigation of the
Arabidopsis MT1a and MT2a expression using reverse transcription polymerase
chain reaction (RT-PCR) and in situ hybridization has revealed distinct patterns for
the two genes (Garcia-Hernandez et al., 1998). While both mRNA species were
detected in root maturation zones and leaf trichomes, only MT1a expressed in the
vascular tissue and the mesophyll cells. The MT2a induction by Cu treatment is
correlated with Cu tolerance of the Arabidopsis ecotypes (Murphy & Taiz, 1995). A
significant amount of evidence suggests that metal tolerance, an important feature of
hyperaccumulator plants, is regulated by few major genes. This provides hope that
biotechnology may be used to engineer more efficient hyperaccumulator plants.
Transcriptional regulation of genes is affected by metal exposure and responds to
metal deficiency. For instance, the cDNA-AFLP expression profiling (Bachem et al.,
1996) in the metallophyte Arabidopsis halleri has shown that a large number of
genes are either up-regulated or down-regulated upon metal treatment (Clemens,
2001). However, the respective signal-transduction pathway is not known in plants
(Xiang & Oliver, 1998). The metal-responsive transcription factors (ACE1, MAC1,
ZAP1) described in S. cerevisiae appear to bind directly and specifically to the
respective metal and may function as metal sensors (Winge et al., 1997). The
Phytoremediation of Heavy Metals 349
growing interest in problems related to metal transport, trafficking and tolerance, the
use of model systems such as S. cerevisiae and S. pombe, and the beginning of
molecular analysis of model hyperaccumulators like Arabidopsis halleri and some
Thlaspi species, are likely to elucidate further the molecular mechanism of metal
tolerance and homeostasis in plants.
Improving Plants for Phytoremediation
Plant capability for hyperaccumulation can be improved by genetic manipulations or
the chelator-regulated strategies.
Genetic Strategies
The growing knowledge of factors affecting phytoremediation can form a basis for
genetic modification of plants for improved remediation performance. Identification
of metal hyperaccumulators has shown that plants have a genetic potential to clean
up the contaminated soil and water. According to Haque et al. (2007), a plant is a
hyperaccumulator, it meets the following criteria: (1) concentrations of heavy metals
in plant shoots should reach hyperaccumulating level, which is different for different
heavy metals e.g, it is more than 1,000 mg Kg
-1
for Pb and Cu (Baker & Walker,
1989, Baker et al., 1994), for As (Ma et al., 2001), for Ni and Co (Brooks, 1998) and
for Cr (Lombi et al., 2001) and more than 10,000 mg Kg
1
for Zn (Brown et al.,
1994) (2) concentrations of heavy metals in shoots should be 10500 times greater
than those in normal plant (Shen & Lui, 1998), (3)HM concentration in shoots
should be invariably greater than in roots (Baker et al., 1994) and (4) an enrichment
coefficient(the ratio of metal concentration in shoot to that in soil) should be greater
than one (Brown et al., 1994, Wei et al., 2002). Metal hyperaccumulators are
notorious for their small size and slow growth. These characteristics have an adverse
impact on their potential for metal phytoextraction and severely restrict the
employment of effective agronomic practices such as mechanical harvest (Tong et
al., 2004). To overcome these disadvantages, conventional breeding has been used
whereby slow-growing, low-biomass hyperaccumulator plants are bred into high-
biomass varieties (Li et al., 2004a). Another approach aims at enhancing the
capability of plants to detoxify HM ions in the cytoplasm through their inactivation
via the chelation, or conversion of toxic ions into a less toxic or easier to handle
form, and/or compartmentalization. Modification or overexpression of enzymes
involved in the synthesis of GSH and PCs might be a good approach to enhance the
HM tolerance and consequently the phytoremediation potential in plants. Zhu et al.
(1999) overexpressed the Escherichia coli counterparts of γ-ECS and GSH
synthetase in the Indian mustard plants that accumulate more Cd than the wild-type
plants. Pilon-Smits et al. (1999) overexpressed the ATP-sulfurylase (APS) gene in
Indian mustard; the transgenic plants had a four-fold higher APS activity and
accumulated three times more Se than the wild-type plants.
Recently, Dhankher et al. (2002) have reported a genetics-based strategy to
remediate As from contaminated soils. They overexpressed two bacterial genes in
Arabidopsis, the E. coli AsrC gene encoding arsenate reductase coupled with a light-
350 R. Jabeen et al.
induced soybean rubisco promoter, and the E.coli γ-ECS gene coupled with a strong
constitutive actin promoter. The AsrC protein, which expresses strongly in the stem
and leaves, catalyzes the reduction of arsenate to arsenite, whereas γ-ECS, which is
the first enzyme in the PC-biosynthetic pathway, increases the pool of PCs in the
plant. The transgenic plants expressing both AsrC and γ-ECS proteins have shown
a substantially high As tolerance; when grown on As, these plants gained a 417
fold greater fresh shoot weight and accumulated 23 fold more As than the wild-type
plants. The strategy for designing the metal-accumulating plants, as modified from
Karenlampi et al. (2000), is summarized below:
Search for metal accumulators
Testing the plant species at high and low regimes of metal stress
C
(OR)
omparing tolerant species with close non-tolerant relatives
Isolation and cloning of the tolerance gene
Characterization of the gene product in a yeast/prokaryotic system
Induction of the gene into the target plant
Plant with su
p
erior
p
h
y
toremediation ca
p
abilit
y
Production of transgenic plant, search of microbes and field trials for remediation
of heavy metals will make phytoremediation technology more applicable and
effective. Bacteria can reduce several HM ions to less toxic states (Lovely, 1993).
Mercury resistance in gram-negative bacteria is encoded by an operon, including a
mercuric-ion reductase gene (merA). MerA is an enzyme that converts toxic Hg
2+
to
the less toxic mercury (Hg0) by the reaction:
Hg2þþNADPH mer A !Hgð0ÞþNADPþþHþ
Rugh et al. (1996) constructed a mutagenised merA sequence and transformed it
to the Arabidopsis thaliana transgenic seedlings that evolved 23 times more Hg0
than in the control. Hg (0) can be volatized by the cell.
Transgenic Populus deltoides overexpressing merA9 and mer18 genes evolved 2
4 fold Hg (0) relative to wild plants when exposed to Hg (II) (Che et al., 2003).
These transgenic trees when grown in soil with 40 ppm of Hg (II), developed larger
Phytoremediation of Heavy Metals 351
biomass. Sub-cellular targeting of methylmercury lyase may enhance plant potential
for the organic mercury detoxification (Bizily et al., 2003). The transgenic A.
thaliana plants expressing a selenocysteine methyltransferase (SMTA) isolated from
the Se hyperaccumulator, Astragalus bisculcatus, accumulated methylselenocysteine
and contained up to eight-fold higher Se concentrations than the wildtype plants,
when grown on a soil supplemented with selenite (SeO
3
) (Ellis et al., 2004).
Induction of various proteins by metals is another perspective of genetic strategies
for phytoremediation. The modern proteome and DNA array technologies may be
applied for searching candidate genes/proteins for phytoremediation; some of the
metal-induced proteins may play a role in metal tolerance or accumulation.
However, examples of a process correlation between protein induction and metal
tolerance are not many. Xiang and Oliver (1998) have shown increased transcription
of genes for synthesis of glutathione, γ-Glutamyl cysteine synthetase, glutathione
synthetase and glutathione reductase under the influence of Cd and Cu. Glutathione
S-transferase is known to mediate glutathione conjugation, which is followed by
transport of the resulting complex to the vacuole (Marrs, 1996). Expression of citrate
synthase gene (dela Fuente et al., 1997) resulted in plants with enhanced Al
tolerance. These plants produced 10- fold citrate in their roots and released a greater
amount than the control plants. Transfer of nicotinamide amino-transferase genes
(NAAT) resulted in overproduction of iron chelator-deoxymuginic acid in rice
(Takashi et al., 2001). The transgenic plants released phytosiderophores and grew
bettering the Fe-deficient soils. Transfer of iron binding protein, ferritin, enhanced
the level of iron in tobacco leaves (Goto et al., 1999). A comprehensive knowledge
of the genetic basis for hyperaccumulation is essential for using biotechnology
effectively to design transgenic plants capable of efficient phytoremediation. Critical
analysis of enhanced metal acquisition, translocation, tolerance and accumulation
abilities in natural metal hyperaccumulators will help in identifying genes
responsible for synthesis of metal-binding proteins/peptides. Identification of genes
encoding both MTs and enzymes involved in PC synthesis thus forms the first step
towards elucidation of the molecular mechanism of phytoremediation.
Completion of the Arabidopsis genome project, followed eventually by genome
sequencing for other plants, should lead to identification of a full range of genes that
are potentially involved in HM homeostasis and accumulation (Dhankher et al.,
2002). Recently ionomicsscreens have been initiated in the phytoremediation
related research involving unbiased multi-element profiling in the A. thaliana mutant
populations in order to identify mutants with altered elemental composition of
rosette leaves. (Lahner et al., 2003; Salt, 2004). These and other similar screens will
serve to identify novel genes with a key role in metal accumulation. Table 2
enumerates transgenic plants used for phytoremediation. Comparison of amino acid
sequences of metal transporters from several hyperaccumulator species might be a
starting point in the identification of determinants of the differential metal
specificities (Rogers et al., 2000). Efforts are on to understand the genetics and
biochemistry of metal uptake, transport and storage in hyperaccumulator plants so as
to be able to develop transgenic plants with improved phytoremediation capability
(Salt & Kramer, 2000; Baker et al., 2000). In addition, manipulation of metal
transporters and the vacuolar targeting of metals will find fruitful application in
developing plants for phytoremediation.
352 R. Jabeen et al.
Table 2 Various Examples of Transgenic Plants Used for Phytoremediation
Authors Gene Origin Target Effect
Misra and Gedamu (1989) MT2 gene Human Nicotiana tabacum L. Cd tolerance
Evans et al. (1992) PsMTA Pisum sativum L. Arabidopsis thaliana L. Cu accumulation
Pan et al. (1994) MT-1 gene Mouse Nicotiana tabacum L. Cd tolerance
Rugh et al. (1998) MerAPe9, merA18 Shigella.Liriodendron tulipifera L. Maximum Hg evolution
Samuelson et al. (1998) FRE1, FRE2 Saccharomyces cerevisiae LNicotiana tabacum L. Elevated Fe-III reduction
Zhu et al. (1999)γ-glutamylcysteine Synthase . Escherichia coli LBrassica juncea L. Cd tolerance
Goto et al. (1999) Ferretin Glycine max L. Nicotiana tabacum L. Increased Fe accumulation
Goto et al. (1999) Ferretin Glycine max L. Oryza sativa L. Increased Fe uptake in seeds
Pilon-Smits et al. (1999) APSI Arabidopsis thaliana L. Brassica juncea L. Increased Se uptake
Van der Zaal et al. (1999)ZAT Arabidopsis thaliana L. Arabidopsis thaliana L. Increased Zn accumulation
Ezaki et al. (2000) Glutathione-S-transferase Nicotiana tabacum L. Arabidopsis thaliana L. Al, Cu, Na tolerance
Dhankher et al. (2002) MerA Gram-ve bacteria Arabidopsis thaliana L. Mercury volatization
Dhankher et al. (2003) arsC Escherichia coli (T. Escherich) Nicotiana tabacum L. Increased Cd tolerance
Gisbert et al. (2003) Phytochelatin synthase (TaPCS) Triticum aestivum L. Nicotiana glauca Graham Pb accumulation
Song et al. (2003) YCF1 Saccharomyces cerevisiae L. Arabidopsis thaliana L. Cd and Pb tolerance
Ellis et al. (2004)SMTA Arabidopsis bisculatus L. Brassica juncea L. Increased Se volatization
Van Huysen et al. (2004) Cystathionine-gamma synthase (CGS) Arabidopsis bisculatus L. Brassica juncea L. Se volatization
Phytoremediation of Heavy Metals 353
Characterization of single genes involved in metal homeostasis has yielded
important insights into their functions and potential use in phytoremediation (Song et
al., 2004), the most important being the high affinity iron-uptake system, IRT1, of A.
thaliana (Connolly et al., 2002; Vert et al., 2002; Verret et al., 2003) and the two
P1B-type Zn
2+
/Cu
2+
-ATPases, HMA2 and HMA4 (Eren & Arguello, 2004; Hussein
et al., 2004; Papoyan & Kochian, 2004; Verret et al., 2004), which function in the
root-to-shoot transport of Zn
2+
and Cu
2+
. Somatic cell hybrids, both symmetric and
asymmetric, have been produced between Brassica juncea, a high-biomass Pb
accumulator and Thlaspi caerulescens, a known Zn and Ni hyperaccumulator (Gleba
et al., 1999). The hybrid has shown increased resistance to Pb, Ni and Zn and the
total amount of the phytoextracted Pb was much greater because of a huge amount
of the biomass produced (Gleba et al., 1999; Dushenkov et al., 2002). So, attempting
somatic cell hybridization between high-biomass plants and low-biomass metal
hyperaccumulators can be helpful in obtaining hybrids with high-biomass and
hyperaccumulation capabilities.
The problem of low-biomass phytoremediators can be overcome by increasing
plant yield and metal uptake by engineering common plants with hyperaccumulating
genes. If the non-native transgenic plants are used for phytoremediation, proper
control of their dissemination has to be adopted to avoid introduction of new weed
species. Development and implementation of biological encapsulationstrategies
will help in popularizing the transgenic phytoremediation. Biological encapsula-
tiondenotes procedures that dramatically decrease probability of the spread of a
transgenic from a genetically modified crop to the natural plant populations, as for
example, introduction of transgene into chloroplast genome instead of the nuclear
genome (Ruiz et al., 2003).
Chelate-assisted Strategies
A chelate is a complex chemical compound composed of a central metal ion attached
to a large molecule (ligand), forming a ring structure. A chelating agent, also called a
chelator is a chemical that can form several bonds to a single metal ion. In other
words, a chelating agent is a multi-dentate ligand. EDTA, NTA, citrate, oxalate,
malate, succinate, tartrate, phthalate, salicylate and acetate etc have been used as
chelators for rapid mobility and uptake of metals by plants from contaminated soils.
Use of synthetic chelators significantly increased Pb and Cd uptake and translocation
from roots to shoots, thus facilitating phytoextraction of metals from the low-grade
ores (Raskin et al., 1997; Blaylock et al., 1997). Synthetic cross-linked poly-
acrylates, hydrogels have protected plant roots from HM toxicity and prevented the
entry of toxic metals into roots. The synthetic and natural zeolites are applied to the
soil through irrigation at specific stages of plant growth in a bid to accelerate metal
accumulation in plant tissues (Blaylock et al., 1997). Synthetic organic chelating
agents are being used in agriculture; EDTA, DTPA (diethylene triamino- pentacetic
acid), HEDTA (N- hydroxy ethylene triamine triacetic acid), EGTA (ethylene glycol-
bis (β-amino-ethyl ether)-N, N, N,N-tetraacetic acid) and NTA (nitrilo-triacetic
acid) can enhance uptake of metals by increasing their availability to plants and their
transport to shoots (Blaylock et al., 1997). These chemicals increase the amount of
the bioavailable metal in the soil solution by either liberating or displacing the metal
354 R. Jabeen et al.
from the solid phase of the soil or by making the precipitated metal species more
soluble (Prasad, 2003). Research in this area has been moderately successful.
Addition of chelating materials, such as EDTA, HEDTA and EDDHA, to soils is
most effective in liberating the labile metal-contaminants into the soil solution.
Chelates complex the free metal ions in solution, allowing further dissolution of the
sorbed or precipitated phases until equilibrium is reached between the complexed
metal, free metal and insoluble metal fractions (Norvell, 1999). Chelates are used to
enhance phytoextraction of a number of metal contaminants including Cd, Cu, Ni, Pb
and Zn (Blaylock et al., 1997). The chelate-mediated accumulation of toxic metals in a
non-accumulator species is termed as chelate-assisted hyperaccumulation(Huang et
al., 1997). Metal-accumulation efficiency appears to be directly related to the affinity
of the applied chelating agent (Salt et al., 1998). Thus, synthetic chelating agents with
a high affinity for the metal of interest (e.g., EDTA for PB, EGTA for Cd) are
preferred (Blaylock et al., 1997). When a chelate-induced hyperaccumulation is the
goal, metals on the site are initially immobilized to allow for a rapid establishment and
growth of agronomic crops such as corn. When the crop accumulates sufficient
biomass, chelating materials are applied to the soil so as to liberate large quantities of
metals into the soil solution. After their death, plants with accumulated metals in their
roots are disposed (Prasad, 2003). A chelate-induced hyperaccumulation thus differs
from the normal phytoextraction where plants are given a gradual exposure to non-
toxic quantities of metal in the solution, and accumulation occurs gradually as the
plants grow. The fate of the residual chelate in the soil after the metal absorption has
taken place has caused controversies (Brooks, 1998). Application of synthetic
chelating agents to the soil needs to be coupled with a system capable of containing
leakage of water through the soil to avoid groundwater pollution by the metals that are
mobilized by the chelating agents (Navari-izzo & Quartacci, 2001).
Future Prospects
In order to make phytoremediation a reliable clean-up method, accurate definitions
of the range of applicability and potential profit margins for various applications are
necessary. Furthermore, monitoring and testing for toxicity and bioaccumulation of
transformation products and the pollutants involved will continue to be necessary
until all significant pathways have been defined. This will completely eliminate the
need for costly off-site disposal. Characterization of the functions of proteins (e.g.,
lignin and cellulose) involved in xenobiotic chemical transformation, transfer and
conjugation (i.e. tolerance and detoxification) can be accomplished by classical
isolation methods or reverse genetics, and by genetic transformation (Marmiroli &
McCutcheon, 2003). There exists considerable information about the induction of
different proteins by metals. Proteome and DNA array technology may be used for
searching the suitable candidate genes/proteins for phytoremediation. Such efforts
may lead to a better understanding of metal metabolism in plants, and open up
important plant applications for environmental clean-up. A systemic screening of
plant species and genotypes for metal accumulation and resistance will broaden the
spectra of genetic material available for optimization of phytoremediation technol-
ogy and its transfer to commercial scale.
Phytoremediation of Heavy Metals 355
According to Cherian and Oliveira (2005), research in the following areas appears
to be worth pursuing in the future to gain further in phytoremediation research.
Manipulation of metal transporters and their cellular targeting to specific cell
organelles, such as vacuoles, to allow for safe compartmentation of heavy metals in
locations that do not disturb other cellular functions. Genetic manipulations of the
chloroplast genome, which may be an alternative approach for some plants to
achieve high gene expression while avoiding the risk of transgene escape via pollen
(Ruiz et al., 2003). (3) Identification of candidate plants with substances that may
deter the herbivores from feeding and the subsequent transformation of such plants
with altered or improved metal tolerance capabilities. Such a system will avoid the
transfer of metals to the food chain (Li et al., 2004b). (4) Developing plants with
ability to secrete metal-selective ligands capable of solubilising elements for
phytoremediation (Eapen & Dsouza, 2005). (5) A multigene approach involving a
simultaneous transfer of several genes into suitable candidate plant to remove
contaminants of complex nature. (6) Establishment of data on the field performance
of transgenes developed in phytoremediation studies.
Phytoremediation technology is still in a research and development phase, and
many technical barriers need to be addressed. The complex interactions that take
place under site-specific conditions necessitate a multi-disciplinary approach to
metal phytoextraction. Success will ultimately depend upon employment of a
holistic approach to integrate the efforts of plant biologists, soil microbiologists,
agronomists and environmental engineers. Phytoremediation promises to be an
integral waste management option for the current century.
Conclusions
Phytoremediation technology is specially beneficial in remediating the HM-
contaminated soil and water as plants can grow in large areas, provide aesthetic
value to the landscape of the contaminated sites, and may have potential of
economic returns which would offset the cost involved, which is already low.
Moreover, the process is environment-friendly because plants uptake and accumulate
the environmental contaminants within their tissues.
However, phytoremediation has some technical limitations. Information needed to
consolidate phytoextraction into a cost-effective method is at present deficient.
Expectations from phytoremediation should also be revised and more appropriately
managed. Phytoremediation requires a long time period to be effective. In one
estimate, given the low growth rate and biomass production in hyperaccumulators, a
complete remediation of metals may not be achieved even in 1020 years (Ernest,
1996). Strategies to address this potential difficulty should include identification of
fast-growing plants as hyperaccumulators, and harvesting of vegetation several times
a year.
Since concentrations of the contaminants can be phytotoxic and prevent plant
growth, the preliminary phytotoxicity studies are necessary to screen the candidate
plants. Phytoremediation efficiency is strongly influenced by the ability of plants to
escape deleterious concentrations of toxic form of pollutants and the active oxygen
species that may be generated in the treated tissue. Extensive progress has been
356 R. Jabeen et al.
made in characterizing soil-chemistry management needed for phytoremediation,
and physiology of plants that hyperaccumulate and hypertolerate metals. It is
increasingly clear that hypertolerance is fundamental to hyperaccumulation, and high
rates of uptake and translocation characterize the hyperaccumulating plants.
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... An alternate to the conventional remediation techniques, a fast evolving, economical and eco-friendly substitute called phytoremediation has ground itself in the environmental sectors over the last decade (Jabeen et al., 2009). Phytoremediation is mixture of two words phyto & remediation which means plants that correct/remove an evil (Tangahu et al., 2011) therefore more precisely it can be defined as "the use of plants and their associated microbes for cleaning or safety of environment" (Mani and Kumar, 2014). ...
... In the process, the hyperaccumulating plants are sowed or shifted into the contaminated soil and then cultivated according to approved agricultural methods. Their roots absorb metals from the soil and move them to upper parts of plant shoots, after accumulation of sufficient amount of metal plants are harvested to clean the soil (Jabeen et al., 2009). ...
... It necessitates that plants grow well in contaminated place and their roots modify the conditions of the soil. This process can be improved either by addition of organic matter and change in pH by using lime, or by binding some other matter with phosphate or carbonate (Jabeen et al., 2009). The basis of Phytostabilization is hyperaccumulation and metal sorption (Mani and Kumar, 2014 (Jadia and Fulekar, 2009). ...
... Plants suitable for phytoextraction should possess multiple abilities: first of all absorb (root/soil) and translocate (leaf/root) to aerial parts heavy metals then rusticity, fast growth, high biomass yield and easy harvesting (Jabeen et al., 2009). ...
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... Subsequently, the HM-enriched materials can be disposed of as hazardous materials or they can be utilized again if economically advantageous for the re-extraction of trace elements (Wuana and Okieimen 2011). Phytoextraction activity can be further enhanced by the type of plant species used that is, fast-growing, natural hyper-accumulator of the polluted site, and genetically modified (Jabeen, Ahmad, & Iqbal, 2009). Several plant species have been reported effectively for the HM remediation such as Helianthus annus, Nicotiana tobaccum, Zea mays, and Cannabis sativa (Sharma et al., 2020). ...
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... The technologies of phytoremediation include phytoextraction, phytofiltration, phytostabilization, phytovolatilization, and phytodegradation [117]. To be specific, hyperaccumulators should possess high Cd tolerance and high root-to-shoot translocation [118]. For example, Noccaea caerulescens which has a specific capacity for Cd and Zn, is an extremophile heavy metal hyperaccumulator and has a potential for effective phytoextraction of Cd from Cd-contaminated soil [119][120][121]. ...
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Cadmium (Cd) is a toxic heavy metal with no known biological function in plants and one of the most toxic substances released into the environment. Crops, such as rice, maize, wheat and tobacco are the major sources of Cd for humans. Cd toxicity inhibits crop growth and development by affecting many central physiological and biochemical processes, and finally it affects human health via the food chain. To adapt to Cd toxicity, crops have evolved a series of detoxification mechanisms. Immediate responses include rapid changes at the transcriptional level with simultaneous changes at the physiological and metabolic levels. However, the long-term responses involve genetic modifications and epigenetic changes. During the last decade, many genes involved in Cd uptake and translocation have been identified, and many of them are transporters. To decrease the accumulation of Cd in cereal grains and tobacco leaves, a number of approaches have been proposed, including physical and chemical methods, developing and planting low accumulation genotypes using transgenic strategies or marker–trait association breeding. In this review, we describe the toxicity of Cd to crops and human body, advances in the molecular mechanisms of Cd accumulation in cereal crops and tobacco, and approaches to decrease Cd accumulation.
... Subsequently, the HM-enriched materials can be disposed of as hazardous materials or they can be utilized again if economically advantageous for the re-extraction of trace elements (Wuana and Okieimen 2011). Phytoextraction activity can be further enhanced by the type of plant species used that is, fast-growing, natural hyper-accumulator of the polluted site, and genetically modified (Jabeen, Ahmad, & Iqbal, 2009). Several plant species have been reported effectively for the HM remediation such as Helianthus annus, Nicotiana tobaccum, Zea mays, and Cannabis sativa (Sharma et al., 2020). ...
Chapter
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Chapter
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Chapter
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Chapter
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Chapter
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There is a need to find an economic and permanent method of stabilizing mine wastes containing toxic levels of metals - both to prevent pollution and to improve the visual appearance of mine workings. It should not involve the use of topsoil, since this is expensive and usually not available. Naturally occurring metal-tolerant populations of wild plant species offer a possible technique, since their metal tolerance should overcome the metal toxicity which is the main factor causing mine wastes to be devoid of vegetation. The other factor, lack of plant nutrients, can be overcome by the use of fertilizer.
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There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives.