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Phytoremediation of Heavy Metals: Physiological and Molecular Mechanisms

December 2009
The Botanical Review 75(4):339-364

DOI:10.1007/s12229-009-9036-x

Authors:

Ruqaya Jabeen

Jazan University

Altaf Ahmad

Aligarh Muslim University

Muhammad Iqbal

Jamia Hamdard

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.

mportant Plants Used for Phytoremediation of Heavy Metals

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arious Examples of Transgenic Plants Used for Phytoremediation

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Phytoremediation of Heavy Metals: Physiological

and Molecular Mechanisms

Ruqaya Jabeen

&Altaf Ahmad

Muhammad Iqbal

1,2,4

Molecular Ecology Laboratory, Department of Botany, Faculty of Science, Hamdard University,

New Delhi 110062, India

Department of Plant Production, College of Food & Agricultural Sciences, King Saud University,

Post Box # 2460, Riyadh 11451, Saudi Arabia

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 plant’s 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) 75:339–364

DOI 10.1007/s12229-009-9036-x

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 150–5,000 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 treat”and “dig and dump”techniques, 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

$60,000–1,000,000, 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 plant’sability(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, nutrient–recycling 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(4–500 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 20–40% lower in

342 R. Jabeen et al.

shoots and 50–70% lower in roots than in wild type when supplied with selenite (Van

Huysen et al., 2003). Phytovolatization has been successful also in removing Tritium

(

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 3–7 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

(Cohen et al., 1998; Hart et al., 1998), Cu

(Thornton, 1991) and Zn

(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

) (Li et al., 1997), Ag-GS

(Ghosh et al., 1996) and Hg-GS

(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

thiol–rich peptides of a general structure (γ-Glu-Cys) n-Gly, where n is normally in

the range of 2–5 (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 (1.3–2.1 fold at 85µM CdCl

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

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

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

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

tolerance (Vatamaniuk et al., 1999). The S. cerevisiae cells

expressing these genes display a Cd

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

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 9–16 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 10–500 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 4–17

fold greater fresh shoot weight and accumulated 2–3 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

(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

erior

toremediation ca

abilit

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

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 2–3 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 wild–type plants,

when grown on a soil supplemented with selenite (SeO

−

) (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 “ionomics”screens 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

/Cu

-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

and Cu

. 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 encapsulation”strategies

will help in popularizing the transgenic phytoremediation. “Biological encapsula-

tion”denotes 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 10–20 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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Phytoremediation and Biofortification: Strategies for Sustainable Environmental and Health Management. Plant-Assisted Bioremediation of Heavy Metals in Soil and Water

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Genome-Wide Identification and Characterization of the Homeodomain Leucine Zipper Protein (HD-Zip) Gene Family in Sea Buckthorn (Hippophae rhamnoides) Under Lead Stress

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Exploring the phytoremediation potential of plant species in soils impacted by gold mining in Northern Colombia

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ENVIRON SCI POLLUT R

Contamination of soils with toxic metals poses significant threats to human health and ecosystems. Plant-based remediation strategies can play a vital role in mitigating these risks, and the use of plants as a remediation strategy can help reduce these risks. In this study, we investigate the remediation potential of native plants in accumulating and translocating metal(loid)s at a Colombian site impacted by gold mining. The remediation capacity is evaluated using the translocation factor (TF) from roots to shoots and the bioconcentration factor (BCF) from soil to roots. Metal(loid) concentrations in the soil followed the order: Fe > As > Hg > Cd > Pb > Zn > Mn > Cu. In plant tissues, Hg showed higher accumulation in leaves (3.5 mg/kg) compared to roots (2.8 mg/kg). Pb (17.7 mg/kg), As (3.8 mg/kg), Fe (2.5 mg/kg) and Cd (1.2 mg/kg) concentracions were also higher in roots. Metal concentrations in the stems, were generally below 1.0 mg/kg, except for Pb (15.0 mg/kg) and Hg (1.0 mg/kg). The highest BCF values for Hg were observed in Spondias mombin L. (18.7), Cecropia peltata L. (8.3) and Gliricidia sepium (Jacq.) Walp (4.4). On the other hand, Senna alata (L.) Roxb., Psidium guajava L. and Morinda citrifolia L. exhibited notable BFC values for As with 44.7, 6.3 and 5.9, respectively. Musa x paradisiaca L. had the highest BCF for Cd (1.8). M. citrifolia (4.3) and Annona muricata L. (3.2) exhibited the highest TF for Hg, while Tabebuia rosea (Bertol.) Bertero ex A.DC. (4.9) and Paspalum fasciculatum Willd. ex (3.1) demonstrated elevated TF values for Pb. In conclusion, plants such as P. fasciculatum, A. muricata, M. citrifolia, G. sepium and T. rosea exhibit great potential for application in phytoremediation strategies in tropical regions impacted by gold mining activities.

Physiological responses of plants to heavy metals and the quantification of tolerance and toxicity

Article

Full-text available

Mar 1989
CHEM SPEC BIOAVAILAB

Techniques available for assessing the tolerance of plants to heavy metal toxins are reviewed. All are based on physiological responses and range from long-term growth trials in metal-contaminated substrates, to rapid cytological tests. Problems associated with the ecophysiological interpretation of in vitro measurements of tolerance are considered. The implications of multiple tolerance, co-tolerance, constitutional tolerance, inducible tolerance and possible stimulatory effects of metals on plant responses are discussed.

Engineered Phytoremediation of Mercury Pollution in Soil and Water Using Bacterial Genes

Chapter

Nov 2020

Toxic Mercury Reduction and Remediation Using Transgenic Plants with a Modified Bacterial Gene

Article

Jul 1998

Stabilization of toxic mine wastes by the use of tolerant plant populations

Article

Jan 1992

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.

Phytoremediation of Toxic Metals: Using Plants to Clean Up the Environment (ed. I. Raskin)

Article

Cretan bake (Pteris cretica L.): an arsenic-accumulating plant

Article

Jan 2002

Phytodetoxification of TNT by transgenic plants expressing bacterial nitroreductase

Article

Jan 2001
NAT BIOTECHNOL

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.

Reactions of Metal Chelates in Soils and Nutrient Solutions

Chapter