AREA 5 • PHYTOREMEDIATION • REVIEW ARTICLE

Implications of metal accumulation mechanismsto phytoremediation

Abdul R. Memon & Peter Schröder

Received: 17 September 2008 /Accepted: 11 November 2008 / Published online: 6 December 2008# Springer-Verlag 2008

AbstractBackground, aim, and scope Trace elements (heavy metalsand metalloids) are important environmental pollutants, andmany of them are toxic even at very low concentrations.Pollution of the biosphere with trace elements has acceler-ated dramatically since the Industrial Revolution. Primarysources are the burning of fossil fuels, mining and smeltingof metalliferous ores, municipal wastes, agrochemicals, andsewage. In addition, natural mineral deposits containingparticularly large quantities of heavy metals are found inmany regions. These areas often support characteristic plantspecies thriving in metal-enriched environments. Whereasmany species avoid the uptake of heavy metals from thesesoils, some of them can accumulate significantly highconcentrations of toxic metals, to levels which by far exceedthe soil levels. The natural phenomenon of heavy metaltolerance has enhanced the interest of plant ecologists, plantphysiologists, and plant biologists to investigate the physi-ology and genetics of metal tolerance in specialized hyper-accumulator plants such as Arabidopsis halleri and Thlaspicaerulescens. In this review, we describe recent advances inunderstanding the genetic and molecular basis of metaltolerance in plants with special reference to transcriptomics

of heavy metal accumulator plants and the identification offunctional genes implied in tolerance and detoxification.Results Plants are susceptible to heavy metal toxicity andrespond to avoid detrimental effects in a variety of differentways. The toxic dose depends on the type of ion, ionconcentration, plant species, and stage of plant growth.Tolerance to metals is based on multiple mechanisms such ascell wall binding, active transport of ions into the vacuole,and formation of complexes with organic acids or peptides.One of the most important mechanisms for metal detoxifi-cation in plants appears to be chelation of metals by low-molecular-weight proteins such as metallothioneins andpeptide ligands, the phytochelatins. For example, glutathione(GSH), a precursor of phytochelatin synthesis, plays a keyrole not only in metal detoxification but also in protectingplant cells from other environmental stresses includingintrinsic oxidative stress reactions. In the last decade,tremendous developments in molecular biology and successof genomics have highly encouraged studies in moleculargenetics, mainly transcriptomics, to identify functional genesimplied in metal tolerance in plants, largely belonging to themetal homeostasis network.Discussion Analyzing the genetics of metal accumulation inthese accumulator plants has been greatly enhanced through thewealth of tools and the resources developed for the study of themodel plant Arabidopsis thaliana such as transcript profilingplatforms, protein and metabolite profiling, tools dependingon RNA interference (RNAi), and collections of insertion linemutants. To understand the genetics of metal accumulationand adaptation, the vast arsenal of resources developed in A.thaliana could be extended to one of its closest relatives thatdisplay the highest level of adaptation to high metal environ-ments such as A. halleri and T. caerulescens.Conclusions This review paper deals with the mechanismsof heavy metal accumulation and tolerance in plants.

Environ Sci Pollut Res (2009) 16:162–175DOI 10.1007/s11356-008-0079-z

Responsible editor: Lee Young

A. R. Memon (*)TÜBİTAK, Marmara Research Center,Institute for Genetic Engineering and Biotechnology,P.O. Box 21, 41470 Gebze, Kocaeli, Turkeye-mail: Abdulrezzak.Memon@mam.gov.tr

P. SchröderHelmholtz-Zentrum München,German Research Center for Environmental Health,Ingolstaedter Landstrasse 1,85758 Neuherberg, Germany

Detailed information has been provided for metal trans-porters, metal chelation, and oxidative stress in metal-tolerantplants. Advances in phytoremediation technologies and theimportance of metal accumulator plants and strategies forexploring these immense and valuable genetic and biologicalresources for phytoremediation are discussed.Recommendations and perspectives A number of specieswithin the Brassicaceae family have been identified asmetal accumulators. To understand fully the genetics ofmetal accumulation, the vast genetic resources developed inA. thaliana must be extended to other metal accumulatorspecies that display traits absent in this model species. A.thaliana microarray chips could be used to identifydifferentially expressed genes in metal accumulator plantsin Brassicaceae. The integration of resources obtained frommodel and wild species of the Brassicaceae family will beof utmost importance, bringing most of the diverse fields ofplant biology together such as functional genomics,population genetics, phylogenetics, and ecology. Furtherdevelopment of phytoremediation requires an integratedmultidisciplinary research effort that combines plant biolo-gy, genetic engineering, soil chemistry, soil microbiology,as well as agricultural and environmental engineering.

Keywords Accumulator plants . Heavy metals .

Metal transporters . Metallothioneins . Phytochelatins .

Phytoremediation

1 Background, aim, and scope

Trace elements (heavy metals and metalloids) are importantenvironmental pollutants, and many of them are toxic evenat very low concentrations. Pollution of the biosphere withtrace elements has accelerated dramatically since thebeginning of the Industrial Revolution (Padmavathiammaand Li 2007). The primary sources of this pollution are theburning of fossil fuels, mining and smelting of metallifer-ous ores, municipal wastes, fertilizers, pesticides, andsewage (Wei and Zhou 2008).

In addition to sites contaminated by human activity,natural mineral deposits containing particularly largequantities of heavy metals are present in many regions ofthe globe. These areas often support characteristic plantspecies that thrive in these metal-enriched environments.Whereas many species avoid the uptake of heavy metalsform these soils, some of these species can accumulatesignificantly high concentrations of toxic metals, to levelswhich by far exceed the soil levels (Baker and Brooks1989). It is known that the essential metals Fe, Mn, Zn, Cu,Mo, and Ni are taken up and accumulated by plants(Williams et al. 2000). Certain plants are also able toaccumulate heavy metals, which have no known biological

function. These include Cd, Cr, Pb, Co, Ag, and Hg (Bakerand Brooks 1989). However, excessive accumulation ofthese heavy metals can be toxic to most plants. The abilityto acquire a tolerance both against heavy metals and anaccumulation to very high concentrations have evolvedboth independently and together in a number of differentplant species (Baker and Walker 1990; Stearns et al. 2007).

In this review, we summarize current knowledgeconcerning metal accumulation and detoxification mecha-nisms in plants and the potential commercial application ofthis phenomenon in phytoremediation.

2 Results

2.1 Plant survival strategies to increasing metalconcentrations

Generally spoken, there are three different types of plantsthat have developed three basic strategies for growing oncontaminated and metalliferous soils (Baker and Walker1990). Metal excluders—effectively prevent metal fromentering their aerial parts over a broad range of metalconcentrations in the soil; however, they can still containlarge amounts of metals in their roots. Metal indicators—accumulate metals in their above-ground tissues and themetal levels in the tissues of these plants generally reflectmetal levels in the soil. Metal accumulators are usuallyreferred to as hyperaccumulators that concentrate metals intheir above-ground tissues to levels far exceeding thosepresent in the soil or in nonaccumulating species growingnearby. It has been proposed that a plant containing morethan 0.1% of Ni, Co, Cu, Cr, and Pb or 1% of Zn on a dryweight basis is called a hyperaccumulator, irrespective of themetal concentration in the soil (Baker and Walker 1990).There are around 400 plant species known worldwide toaccumulate metals in large amounts and these species are ofinterest for potential use in phytoremediation of metal-contaminated soils (Brooks 1983; Memon and Yatazawa1984; Baker et al. 2000; Pilson-Smits 2005). Informationrelated to accumulator plants is most needed in four areas:First, the metal-accumulating ability of various species as afunction of soil metal concentrations, physicochemical soilproperties, and physiological state of the plant; second, thespecificity of metal uptake, transport, and accumulation;third, the physiological, biochemical, and molecular mecha-nisms of accumulation; and fourth, the biological andevolutionary significance of metal accumulation.

2.2 Mechanisms of metal accumulation

In plant species growing on metal-rich soils, the internaldistribution may differ significantly. Metal ions may be

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localized in roots and shoots, or they may accumulate andbe stored in nontoxic forms. One distinct mechanism oftolerance or accumulation appears to involve binding ofpotentially toxic metals in cell walls of roots and leaves,before they can get in contact with sensitive sites within thecell. Others allow cellular uptake, but then sequester metalsin the vacuoles (Memon and Yatazawa 1984; Cosio et al.2004). A pressing question about heavy metals in theenvironment concerns the amounts that plants can tolerateand accumulate without adverse effects. A further questionrelates more to the amount and speciation and their roles forplant performance and metal transfer to the food chain.Several types of heavy metal resistance and tolerancemechanisms are being suggested in plants growing onmetalliferous soils (Hall 2002; Milner and Kochian 2008).Several heavy metal accumulator plants, for example, Mn(Acanthopanax sciadophylloides, Maytenus founieri) Ni(Sasa borealis, Alyssum sps.) Co (Clethra barbinervis),Cd, Zn (Clethra barbinervis, Ilex crenata, Thlaspi caer-ulescens, Arabidopsis halleri), Pb (Thlaspi rotundifoliumssp. Cepaeifolium, T. caerulescens, Sesbania drummondii),and Se (Brassica juncea Czern L.) have been reported(Banuelos and Meek 1990; Baker et al. 2000; Memon et al.2000; Reeves et al. 2001; Sahi et al. 2002; Assunçao et al.2003; McGrath et al. 2006; Fernando et al. 2007). Some ofthese accumulator plants have very unique ecophysiologicalbehavior and have the capacity to accumulate significantamounts of metals and compartmentalize them efficiently inthe cell wall, vacuole, and to the specific subcompartmentand/or compartments of the cytosol in order to render theminnoxious or nontoxic and keep them away from activemetabolic sites in plant cells (Memon et al. 1981; Memonand Yatazawa 1982, 1984). While surveying the flora of Cumining areas of Southeastern Anatolia, we discoveredseveral endemic metal accumulator plants and, interesting-ly, a Brassica nigra ecotype found from a Diyarbakir sitecontained a very high amount of Cu in their shoots. Whenplants from this ecotype were regenerated from callusculture and grown in soil culture containing 200 ppm Cu,the shoots accumulated three times more Cu (700 μg/g dryweight) than roots (Memon et al. 2006). The γ-glutamyl-cysteine (γ-EC) expression in the shoots of Cu-treatedplants was around 3.5 times that of control plants (Memonet al. 2008a). This ecotype could be considered a goodcandidate for Cu phytoremediation. Several mechanismsmay contribute to heavy metal tolerance, depending on thetype of metal and plant species (Memon and Yatazawa1982, 1984). The recent development of transcriptomics(microarray analysis), proteomics, and metabolomicsallows a deeper exploration of the function and regulationof the cell when it encounters high metal concentration inthe environment. The current research data on transcrip-tome analysis of transporters and the participation of

multiple gene families in response to metal stress in twomodel accumulator plant species such as T. caerulescensand A. halleri have also been summarized. For example,P1B-type ATPase of the divalent transport group (HMA4)has been implicated in Zn homeostasis and Cd detoxifica-tion and in the translocation of these metals from the root tothe shoots in Arabidopsis (Hussain et al. 2004; Verret et al.2004; Courbot et al. 2007; Hanikenne et al. 2008). RNAi ofHMA4 in A. halleri downregulated its expression andreduced the Zn hyperaccumulation and full tolerance to Cdand Zn in the plants (Hanikenne et al. 2008). Several metal-binding proteins in plants have been reported which includemetallothioneins, metalloenzymes, metal-activatedenzymes, and various metal storage, carrier, and channelproteins (Zhou and Goldsbrough 1994; Zhu et al. 1999a,1999b; Hirschi et al. 2000). In addition, phytochelatins,enzymatically synthesized low-molecular-weight (LMW)γ-Glu-Cys peptide ligands with a high affinity for transitionmetals, are widely distributed in yeast as well as in lowerand higher plants (Cobbett 2000).

2.3 Genetics of metal accumulation in plants

Over the past decade, significant progress has been made inelucidating the molecular basis of metal uptake, accumula-tion, and tolerance into plant cells (Cobbett 2000; Cobbettand Meagher 2002; Clemens 2006). Until now, most of thegenetic studies on species with a metalliferous populationhave dealt with the genetic determinism of heavy metaltolerance (Schat and Ten Bookum 1992; Macnair 1993).Such studies have yielded valuable insight into the mecha-nisms that might underlie plant adaptation to metal avail-ability. Despite recent advances, the mechanism underlyinghyperaccumulation is still not well-defined. Therefore, thereis a need to develop a model system for molecular geneticstudies of metal hyperaccumulation in plants. Arabidopsisthaliana, although it is not a metal accumulator, will besuitable because around 25% of the reported hyperaccumu-lator species are members of Brassicaceae. Therefore,microarray gene chips of A. thaliana to analyze thetranscriptome of model metal accumulator plants like A.halleri and T. caerulescens could provide a platform fromwhich plant geneticists can relate the phenotypic and geneticvariations in physiological traits of underlying genes or genenetworks related to metal tolerance and/or accumulation(Plaza et al. 2007; Willems et al. 2007; Milner and Kochian2008; Roosens et al. 2008). Few groups, however, havetapped the full potential of gene expression profiles,especially as they relate to understanding how geneticchange translates to phenotypic variation and the resultantadaptive physiological traits (Maathuis et al. 2003; Weber etal. 2004). Hammond et al. (2006) have developed a robustmethod to profile and compare the transcriptome of two

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plant species from Brassicasae, T. caerulescens J & C Presl.,a Zn hyperaccumulator, and T. arvense L., a nonhyperaccu-mulator, using Affymetrix A. thaliana ATH-121501 (ATH1)GeneChip® arrays (Affymetrix, Santa Clara, CA, USA).Approximately 5,000 genes were differentially expressed inthe shoots of T. caerulescens compared with T. arvense,including genes involved in Zn transport and compartmen-talization. Partial sequencing of cDNA clones (e.g., ESTs-expressed sequence tags) is another efficient method toobtain metal-related gene expression for metalliferousspecies of which no detailed genomic information isavailable. Comparing EST sequences of the target specieswith the appropriate reference model species such as A.thaliana, O. sativa, P. trichocarpa, M. truncatula, C.reinhardtii, and with additional public databases for annota-tions of their putative functions, provides an important toolfor the identification of unique genes, their function, andtheir role in metal tolerance. This approach could be usefulfor species whose complete genome sequence information isnot available, as in the case of Brassica sps. (A. halleri, T.caerulescens, T. goesingense, B. juncea, B. nigra, Thellun-giella, etc.) (Rigola et al. 2006; Van de Mortel 2006;Freeman and Salt, 2007; Milner and Kochian 2008; Roosenset al. 2008). The fully sequenced genome of species, such asArabidopsis, rice, poplar, Chlamydomonas, together with alarge-scale “omics” and systems biology approach will allowone to address many of the outstanding questions related tometal accumulation and tolerance in plant cells through aglobal analysis of gene expression. It is possible to usesynteny between the genomes of the test and model speciesto deduce or identify genetic regions that contain quantitativetrait loci (QTLs) involved in metal tolerance or accumula-tion. In several studies, these techniques have been used todeduce chromosomal regions probably containing QTLs formetal accumulation and identifying genes involved in Zn andCd hyperaccumulation in A. halleri and T. caerulescens(Deniau and Pieper 2006; Filatov et al. 2006; Filatov et al.2007; Willems et al. 2007; Roosens et al. 2008). Duringrecent years, important information has been produced byQTL analysis and has been used to investigate the geneticbase of adaptation of metallophytes to metal-polluted soils.These studies have specified genes underlying metal toler-ance as well as indications on epistatic relationships betweenthose genes (Bratteler et al. 2006; Deniau and Pieper 2006).A wide range of genomic technologies are now available tohelp biologists and ecologists to understand the phenomenonof hyperaccumulation.

2.4 Metal transporters

Plants have developed flexible strategies to cope withfluctuations in their environment in order to minimize theadverse effects of metal deficiency or toxicity. Adaptive

responses can include a significant alteration in geneexpression, particularly of membrane transporters that areresponsible for the uptake, efflux, translocation, and seques-tration of essential and nonessential mineral nutrients. Metalhyperaccumulator plants possess several unique character-istics, such as the ability to take up and translocateexceedingly large amounts of metals to their shoots andhypertolerate the toxic metals (Stearns et al. 2007; Freemanand Salt 2007). Of major concern with respect to plantexposure as well as food chain accumulation are themetalloids, arsenic (As) and selenium (Se), and the metals,cadmium (Cd), mercury (Hg), and lead (Pb) (Welch andGraham 2003; Prasad 2008). Generally, there are vastdifferences in the bioavailability of metals to plants. Somemetals (for example, Cr, Ag, Hg, or Sn) are practically notavailable for plant uptake because of their low solubility insoil. Other metals such as Pb can be major pollutants andcould be present in huge amounts along roadsides in heavilypopulated large cities, yet are hardly taken up into plantsbecause of low solubility and strong interaction with soilparticles. Toxic metal ions that do enter plant roots aretransported to shoots and accumulate in edible plant parts,which represent the principal route of toxic metal entry intothe food chain (Welch and Graham 2003; Marmiroli andMaestri 2008; Memon et al. 2008b). Over the past decade,significant progress has been made in elucidating themolecular basis of metal uptake into plant cells. A numberof important membrane transporter families have beendiscovered by heterologous complementation screens andsequencing of both plant ESTs and the Arabidopsis and ricegenomes. The completion of Arabidopsis, rice, poplar, andChlamydomonas genomes now give us impetus to analyzecomplete sets of transporter gene families in plant and algaspecies. Powerful genetic approaches have been developedthat allow high throughput selection of point mutations thatreduce or block transport of toxic ions, while maintainingnutrient transport (Rogers et al. 2000; Rogers and Guerinot2002). Several toxic metal transporters have been analyzedand some of them are shown to transport Cd. Amongst themare members of Zn-regulated transporter (ZRT), Fe-regulatedtransporter (IRT)-like proteins (ZIP), natural resistance-associated macrophage proteins (NRAMP), and cationdiffusion facilitator (CDF) (Thomine et al. 2000; Maser etal. 2001; Hall and Williams 2003). The overview of themetal transporters and their tissue-specific expression inplants is summarized in Table 1. Microarray analysis ofArabidopsis has demonstrated that a number of genesencoding ZIP proteins are induced in plants under Zn-deficient conditions (Wintz et al. 2003). Plant ZIP proteinsthat mediate uptake of Zn into yeast have also been identifiedin rice (Oryza sativa), soybean (Glycine max), Medicagotruncatula, and the Zn/Cd hyperaccumulator, T. caerulescens(Pence et al. 2000; Moreau et al. 2002; Lopez-Millan et al.

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2004; Ishimaru et al. 2005). Eighteen genes are predicted toencode ZIPs in Arabidopsis (Gaither and Eide 2001) and,although physiological functions of Zn-transporting ZIPs areyet to be described in plants, they are predicted to beinvolved in cellular uptake and in mobilization of stored Zn.A number of ZIP genes have been found to be highlyexpressed in Zn/Cd accumulators like T. caerulescens(Hammond et al. 2006) and A. halleri (Weber et al. 2004;Talke et al. 2006), compared to nonaccumulator species.Differences in expression of ZIP transporter genes not onlyprevail among species, but are also reported to be present inecotypes of some species. Detailed studies with twocontrasting ecotypes of the hyperaccumulator T. caerulescensshowed differences in expression patterns of ZIP transportergenes among ecotypes (Plaza et al. 2007). The four ZIPgenes studied (TcIRT1, TcIRT2, TcZNT1, and TcZNT5)were expressed only in roots and not in leaves, indicatingtheir specificity in metal uptake and transport in roots. Theecotypic difference in ZIP gene expression in the roots of T.caerulescence in the presence and absence of Cd wereclearly identified. TcZNT1 expression was not affected byCd and expression levels were similar for both ecotypes(Ganges and Prayon), but the expression level of TcZNT5 inGanges was below that for Prayon and not affected by Cdtreatment. The expression level of TcIRT1 was increased inroots of both ecotypes when treated with Cd but transcriptlevels of TcIRT2 were not detectable in either ecotype, eitherin the presence or absence of Cd (Plaza et al. 2007). Thesignificance in terms of metal ion uptake needs furtherinvestigation through functional analysis. A number ofpotential metal ligands are highly conserved among ZIP

family members. For example, the replacement of glutamicacid residues at position 103 in wild-type IRT1 with alanineand by heterologous expression in yeast increases thesubstrate specificity of the transporter by selectively elimi-nating its ability to transport Zn (Rogers and Guerinot 2002).A number of other conserved residues in or near transmem-brane domains appear to be essential for transport function.For example, replacing aspartic acid residues at position 100or 136 with alanine also increases IRT1 metal selectivity byeliminating transport of both iron and manganese. Inaddition, the ATP-binding cassette (ABC) family transportersrepresent one of the largest protein families in livingorganisms ranging from bacteria to humans (Hall andWilliams 2003). A. thaliana and rice (O. sativa) containapproximately 130 ABC transporters, the precise functionsof which still remain obscure for the most part (Sanchez-Fernandez et al. 2001; Rea 2007). They can be assigneddepending on their nucleotide-binding folds (NBFs) ornucleotide-binding domains (NBDs), which share 30–40%sequence identity between family members (Higgins 1992).In addition to NBDs, ABC transporters also containtransmembrane domains (TMDs), each composed of severalhydrophobic α-helices (Verrier et al. 2008). There are threeprominent features of these transporters in plants. First of all,it is energized directly by MgATP, but not by free ATP ornonhydrolyzable ATP analogs. Secondly, transport is insen-sitive to the transmembrane H+ electrochemical potentialdifference, and thirdly, transport is exquisitely sensitive tovanadate (Rea 2007). ABC transporters have been classifiedinto three major subfamilies: (1) full-sized transporterscontaining at least two NBFs and two TMDs, (2) half-sized

Table 1 Overview of some of the identified metal transporters and their tissue-specific expression in plants

Plant name Proteinfamilies

Gene name Metals Tissueexpression

References

A. thaliana, A. halleri,L. esculentum

P-TypeATPase

AtHMA1-8,AhHMA3-4,TcHMA4,GmHMA8,OsHMA9

Cu, Zn,Cd, Co,Pb

Shootsand roots

Becher et al. 2004; Bernard et al. 2004;Bernal et al. 2007; Courbot et al. 2007;Hussain et al. 2004; Lee et al. 2007;Papoyan and Kochian 2004; Roosenset al. 2008; Talke et al. 2006; Verretet al. 2004; Willems et al. 2007; Xinget al. 2008

A. thaliana, A. halleri,T. caerulescens, G. max,O. sativa

Nramp AtNRAMP1-6,LeNRAMP1-3,AhNRAMP3

Fe, Cd Shootsand roots

Bereczky et al. 2003; Lanquar et al. 2005;Thomine et al. 2000

A. thaliana, O. sativa ZIP AtZIP1-12, OsZIP4 Zn Shootsand roots

Filatov et al. 2006; Ishimaru et al. 2005;Roosens et al. 2008; Weber et al. 2004

A. thaliana, T. caerulescens,L. esculentum, O. sativa,N. tabacum

IRT AtIRT1, OsIRT1-2,LeIRT1-2, TcIRT1-2,NtIRT1

Cd, Zn Shootsand roots

Hodoshima et al. 2007; Kerkeb et al. 2008;Plaza et al. 2007

A. thaliana, A. halleri,T. goesingense, N. tabacum,P. trichocarpa, P. deltoids

CDF AtMTP1, TgMTP1,AhMTP1, PtdMTP1,NtMTP1

Zn Roots Blaudez et al. 2003; Kawachi et al. 2008;Kim et al. 2004; Shingu et al. 2005;Willems et al. 2007

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ABC transporters with one NBF and one TMD, and (3)soluble ABC transporters with one or two NBFs (Sanchez-Fernandez et al. 2001). Recently, some Arabidopsis ABCtransporters were found to participate in detoxificationprocesses as well as in plant growth and development (Nohet al. 2001; Campbell et al. 2003; Geisler et al. 2005).AtATM3, an ATP-binding cassette transporter of Arabidopsisis shown to be a mitochondrial protein involved in thebiogenesis of iron–sulfur clusters and iron homeostasis inplants and is shown to be upregulated in roots of plants whenexposed with Cd (II) or Pb (II) (Kim et al. 2006).Additionally AtATM3 overexpressed in Arabidopsis plantsshowed enhanced Cd and Pb resistance compared to wild-type, whereas AtATM3 knockout plants showed Cd-sensitivephenotypes. This Cd sensitivity of the mutants was rescuedby overexpressing wild-type AtATM3. AtMHX is anothermetal transporter reported to be located in tonoplastmembrane and involved in Zn and Fe homeostasis (David-Assael et al. 2006). The expression AtMHX is induced byplant hormones, auxin and ABA, and exhibits two distin-guished regulatory properties. Its leader intron is absolutelyessential for expression and a repetitive genomic element of530 bp (or part of it) functions as an enhancer. Recent studiesshowed the common QTL for Zn and Cd tolerance in A.halleri (Courbot et al. 2007; Willems et al. 2007). In A.thaliana, the QTL common to Zn and Cd tolerance covers aregion comprising 739 genes and, among them, 11 genes aredifferentially expressed in A. hallari and A. thaliana(Roosens et al. 2008). From these 11 genes, only one hasbeen depicted as heavy metal-transporting ATPase4 (HMA4)and belongs to a P-type ATPase family involved in thetransport of transition metals (Chiang et al. 2006; Talke et al.2006). The Zn hyperaccumulation and full hypertolerance toZn and Cd in A. halleri was considerably reduced whenHMA4 expression was reduced through RNAi (Hanikenne etal. 2008). It has been demonstrated that AhHMA4 plays amajor role in Zn hyperaccumulation and associated Cd andZn hypertolerance, and its high expression in A. halleri isspecified in cis-regulatory factors and amplified by genecopy number expansion. Increased expression of HMA4 inA. halleri enhanced the Zn transport from the root symplasminto the xylem vessels necessary for shoot Zn hyper-accumulation (Hanikenne et al. 2008). These findingsindicate the importance of these transporters in phytoreme-diation. Manipulation of these transporters to achieveremoval of metal ions from the cell holds great potential inthe plant species for hyperaccumulation (Tong et al. 2004;Roosens et al. 2008).

2.5 Metal chelation and detoxification

Induction of metal-chelating proteins related to metal-lothioneins (MTs) (Robinson et al. 1993; Rauser 1999)

and/or phytochelatins (PCs) (γ-glutmylcysteinyl-isopepti-des) (Clemens et al. 1999; Cobbett 2000; Cobbett andGoldsbrough 2002; Sarry et al. 2006) increases the level ofcell tolerance to an excess of metal ions. These cysteine-rich polypeptides sequester heavy metals efficiently andcontribute to detoxification and accumulation in plant cells.While MTs are independent gene products, PCs aresynthesized enzymatically from GSH or related peptides.The tripeptide GSH is not only involved in PC synthesis,after heavy metal exposure, but is also an important productin countering oxidative stress and may be conjugated todiverse xenobiotic compounds as a prerequisite for theirvacuolar sequestration (Grill et al. 1989; Cobbett 2000;Guo et al. 2008).

2.5.1 Metallothioneins

Metallothioneins (MT) are products of mRNA translationand characterized as LMW cysteine-rich metal-bindingproteins. They are found throughout the animal and plantkingdoms. In animals and fungi, MTs have been shown toplay a role in the detoxification of heavy metals (Robinsonet al. 1993; Hall 2002), although their exact function is notcompletely understood. In plants, a correlation has beenreported between MT RNA levels and naturally observeddifferences in the tolerance to heavy metals in Arabidopsisecotypes, suggesting a role in metal homeostasis (Murphyand Taiz 1995; Murphy et al. 1997; Guo et al. 2008).

Some functions proposed for MTs in plants includemetal detoxification (Roosens et al. 2005; Domenech et al.2006), a role during development (Ledger and Gardner1994), in senescence (Coupe et al. 1995; Hsieh et al. 1995),and in protection against abiotic stress (Zhou et al. 2005).The relationship between MT expression and metal con-centration in different organisms suggests that MTs can beeffective reporters of environmental conditions (Morris etal. 1999). Isolation and characterization of MTs in modelbioindicator organisms could also contribute to our under-standing of the biological response to biomonitoringpollutants in the environment (Morris et al. 1999).

2.5.2 Phytochelatins

Phytochelatins form a family of peptides that consists ofrepetitions of the γ-Glu-Cys dipeptide followed by aterminal Gly with the basic structure (γ-Glu-Cys)n-Gly[(PC)n]) where n is in the range of two to five.Phytochelatins are synthesized enzymatically from GSHin response to many metals (Rauser 1990; Cobbett 2000).They are structurally related to GSH and not directlyencoded by genes, but the products of a biosyntheticpa t hway (G ly+Cys→GCS γ -G lu -Cys+Glu→GS

GSH→PCS+Cd PC→PC–Cd→HMTI vacuole where γ-glu-

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tamylcysteine synthetase (GCS), GSH synthetase (GS),phytochelatin synthase (PCS), heavy metal tolerance 1(HMT1), ABC type vacuolar membrane transporter ofPC–Cd complexes). A number of other structural variantsof PCs, such as (γ-Glu-Cys)n-β-Ala, (γ-Glu-Cys)n-Ser,and (γ-Glu-Cys)n-Glu, have been identified in plants(Rauser 1990, Cobbett 2000). Phytochelatins (PCs) arerapidly induced in vivo by a wide range of heavy metalions. The enzyme-synthesizing PCs from GSH is a γ-Glu-Cys dipeptididyl transpeptidase (EC 2.3.2.15), commonlyreferred to as phytochelatin synthase (PC synthase) (Grillet al. 1989). Mutants of Arabidopsis lacking PC synthaseare unable to synthesize PCs and hypersensitive to Cd andHg. The cad 1 mutant of A. thaliana L. Heynh iscadmium-sensitive and its GSH level is similar to that ofthe wild-type but both are deficient in PC and lack PCsynthase activity in vitro. It is predicted that cad1 is thestructural gene for PC synthase (Howden et al. 1995). TheArabidopsis cad 1 gene (referred to as AtPCS1) (Howdenet al. 1995; Ha et al. 1999; Vatamanuik et al. 1999) and asimilar gene in wheat (TaPCS1) (Clemens et al. 1999)have been shown to confer resistance to Cd whenexpressed in the yeast S. cerevisiae (Clemens et al. 1999;Cobbett and Meagher 2002). However, these mutants haveessentially wild-type levels of tolerance to Cu and Zn(Howden et al. 1995). The occurrence of PC synthase indifferent higher plants has been confirmed (Clemens et al.1999; Cobbett 2000) and gel filtration analysis show Cd–PC interactions as LMW and high-molecular-weight(HMW) complexes (Howden et al. 1995; Rauser 2000).The LMW Cd-binding complex is cytosolic and containsshorter PCs. The HMW complex is vacuolar, containslonger PCs, acid-labile sulfide, and usually dominatesover that of the LMW complex when plants are exposed toCd for longer time (Ortiz et al. 1995; Rauser 2000).Accumulation of the HMW Cd–PC complex is essentialfor Cd detoxification in A. thaliana (Howden et al. 1995;Ha et al. 1999). Aside from detoxification, PC plays a rolein homeostasis of heavy metals in plants, and this is themechanism that regulates the metal ions availability inplant cells (Guo et al. 2008).

2.6 Oxidative stress and metal tolerance in plants

GSH, the tripeptide γ-glutamylcysteinylglycine (γ-glu-cys-gly), is a precursor of phytochelatin synthesis and the majorsource of nonprotein thiols in most plant cells (Noctor et al.1998; Maughan and Foyer 2006). GSH biosynthesis is atwo-step ATP-dependent reaction (Meister and Anderson1983), including the synthesis of γ-EC from glutamate andcysteine, followed by the formation of GSH through theaddition of glycine to the C-terminal end of γ-EC. The firstreaction is catalyzed by γ-glutamylcysteine synthetase (γ-

EC synthetase) and the second one by GS. Both enzymesare encoded by gsh1 and gsh2 genes, respectively (May andLeaver 1995; Wang and Oliver 1996).

γ-EC is feedback-inhibited by GSH, and obviously actsas the rate-limiting step in the pathway. Numerousphysiological functions have been attributed to GSHlevels in plants and its role as a regulator of geneexpression (Foyer et al. 1997) and in the regulation ofenzyme activity after glutathionation of proteins has beenreported (Dixon et al. 2005). Moreover, it is also involvedin the redox regulation of the cell cycle (Shaul et al. 1996;Sanchez-Fernandez et al. 1997). GSH, due to its redox-active thiol group, has often been considered important indefense of plants and yeast against oxidative stress(Alscher 1989; Grant et al. 1996). However, as yet, anabsolute dependence on GSH for stress tolerance has onlybeen demonstrated for H2O2, but direct induction of GSHsynthesis in response to other oxidative stimuli is lacking(May and Leaver 1995). Furthermore, research has failedto demonstrate that such an elevation has physiologicalsignificance in plants. On the other hand, studies in animalsystems have shown that depletion of GSH renders cellssusceptible to oxidative processes (Arrick et al. 1982).Recent data suggest that the cytosolic antioxidant capacityis sufficient to maintain cell viability even in the absenceof catalase, and that under such conditions, a strongincrease in the level of reduced GSH can be measured.The fact that a transient accumulation of hydrogenperoxide led to a significant increase in the level ofreduced GSH suggests that the mechanism of hydrogenperoxide-induced GSH synthesis is much more complexthan previously proposed. However, exposure of maizeseedlings, tomato, parsley, and tobacco cell cultures toheavy metals accelerates GSH synthesis (Rüegsegger andBrunold 1992; Schneider and Bergmann 1995). Thesestudies clearly indicate the importance of GSH in protect-ing plants against various forms of stress. Manipulatingthe expression of enzymes involved in GSH synthesisappears to be a promising strategy for the production ofplants with superior capacity for heavy metal phytoreme-diation (Ducruix et al. 2006; Vestergaard et al. 2008).

3 Discussion

3.1 Phytoremediation: a versatile technology with manypotential applications

Phytoremediation defines the use of plants and theirassociated microbes to extract, sequester, and/or detoxifyvarious kinds of environmental pollutants from water,sediment, soils, and air. Phytoremediation of heavy metalsis an emerging technology and several subsets of this

168 Environ Sci Pollut Res (2009) 16:162–175

technology are being developed (Salt et al. 1995, 1998;Pilson-Smits 2005).

3.2 Use of metal accumulator plants for phytoremediation

A variety of naturally occurring and specially selected plantspecies are used in phytoremediation. Many metallophyteplants are used in prospecting for mineral deposit (Bakerand Brooks 1989) but only recently has the value of metal-accumulating terrestrial plants for environmental remedia-tion been fully realized (Padmavathiamma and Li 2007;Stearns et al. 2007). A number of terrestrial and aquaticplants are known to be natural hyperaccumulators ofmetals, but since these tend to be slow growers, researchershave turned to other species, more recently identified orselected, as more promising commercial candidates. Deep-rooted trees such as poplar, willow, and cottonwood aremost commonly used for applications requiring withdrawalof large amounts of water from the subsurface, while anumber of different plants, trees, and grasses are used tostimulate microbial degradation of organic contaminants insoil. Among plants at earlier stages of research are plantsand trees expressing biodegradative enzymes, halophytic(salt-loving) plants, and transgenic (genetically engineered)plants created to meet specific marketplace needs.

3.3 Current aspects of phytoremediation

Metallophyte species that occur naturally on metal-enrichedsoils represent major biological resources for the improve-ment of phytoremediation, a benign and cost-effectivetechnology that uses plants to clean up metal-polluted soils.Within the last decade, molecular genetic studies carried outon several model organisms (including A. halleri and T.caerulescens) have considerably enhanced our understand-ing of metal tolerance and hyperaccumulation in plants, butthe identification of genes of interest for phytoremediationpurposes remains a challenge. In the past decade, extensiveresearch efforts have been made to dissect the basicmolecular mechanisms of metal tolerance in metallophytes(Clemens 2001; Clemens 2006). Physiological approaches,transcriptomics in particular, have allowed the identificationof several candidates that seemed to result from modifica-tions of the sequence or expression of genes belonging tothe ubiquitous metal homeostasis network (Bernard et al.2004; Drager et al. 2004; Roosens et al. 2004; Mirouze etal. 2006; Talke et al. 2006).

A better understanding of the biochemical processesinvolved in plant heavy metal uptake, transport, accumula-tion, and resistance will help to systematically improvephytoremediation using molecular genetic approaches. Agrowing knowledge of factors important to phytoremedia-tion can provide a basis for genetic modification of plants

for improved performance. For example, to improve thehigh potential of plants for phytoremediation is to introducegenes responsible for accumulation and resistance fromwild slow-growing plants or from bacterial or animalsources into fast-growing, high-biomass plant species.However, long-term efforts should be directed toward thedevelopment of a “gene bank” composed of genes valuablefor phytoremediation. Systematic screening of plant speciesand genotypes for metal accumulation and resistance willbroaden the spectra of genetic material available foroptimization and transfer. Mutagenesis of selected high-biomass plant species may also produce improved phytor-emediating cultivars. Another approach would be togenetically engineer crop species with improved metaltolerance and accumulation capacities (Wangeline et al.2004; Whiting et al. 2004). Phytoremediation engineeringcould then involve either classical breeding or transgenicapproaches (Zhu et al. 1999a, b; Wangeline et al. 2004;Singla-Pareek et al. 2006) or the exploitation of availablegenetic variation for metal tolerance in crops throughmarker-assisted selection (Ghandilyan et al. 2006).

In summary, merging molecular and ecological geneticsto the study of plant metal tolerance greatly improves theoverall knowledge of metal tolerance mechanisms andprovides a currently unreleased context for the exploitationof metal tolerance genes. This approach should quicklyallow scientists to identify the metal tolerance genes that, inreturn, could be investigated in other pseudometallophyteor crop species and finally be used in phytoremediationengineering.

4 Conclusions

Phytoremediation holds great potential as an environmentalclean up technology and has been investigated substantiallysince the last two decades. Considerable interest inphytoremediation exists by both government and industry.The biggest advantage of phytoremediation is its low cost.Phytoremediation can be up to 1,000-fold cheaper com-pared with conventional remediation methods such asexcavation and reburial. Moreover, it offers permanent, insitu remediation rather than simply moving the pollution toa different site (Salt et al. 1998).

In each of the industrial countries, several tens ofthousands of sites are possible targets for remediation, e.g., the USA has more than 50,000 metal-contaminated sites(Ensley 2000), Germany 80,000 sites (Franzius 1994), andin other countries, similar numbers are found. Whentechnical remediation methods like soil washing, excava-tion, or pump and treat systems fail, phytoremediation willbe a welcome and environmentally sound alternative, whichcould probably be cheaper than the estimated U.S.

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remediation costs of $7 to $8 billion per year, approxi-mately 35% of which involves remediation of metals(Bennett et al. 2003). While the process of phytoremediationas such is well-understood, the time frame of such an actionis frequently underestimated. Remediation times of decadesare realistic in many scenarios, and this reduces theattractiveness of this technology. A combination of biomassproduction with other land use functions may be interestingto generate an economic benefit for farmers (Schröder 2007;Schröder et al. 2007). A recent study estimated the economicvalue of the phytoremediation function to farmers asassessed by the substitution cost and hedonic price analysisto about 14,600 and 14,850 € ha−1, respectively, over aperiod of 20 years (Lewandowski et al. 2006). Thewillingness of farming communities to adopt this methodwill strongly rely on governmental help, and the insight intothe soundness and added value of phytoremediation. Large-scale remediation has to be designed to address localproblems, gain more scientific knowledge, and meet cleanup standards for the future use of a site. Phytoremediationusually fails if fast results and total removal of plumes isrequired. But long-term remediation to parks, nature areas,and resorts may be combined with an attractive design,yielding areas to be used by the public during and after theremediation process at low to zero risk (Pilson-Smits 2005).The progress in developing and understanding transgenic ormutagenized plants in their potential to hyperaccumulatetrace elements will also be beneficial to the field. It is likelythat such plants will be utilized in the near future to solvespecific problems of pollution removal, under thoroughobservation of safety regulations, and that their biomass willbe used to fuel our energy demands (Schröder et al. 2008).

In general, fast-growing, high-biomass, competitive,hardy, and metal-tolerant plant species could either beselected or could be generated by genetic manipulation andbe used for remediation of different polluted sites. The A.thaliana, rice, poplar, and Chlamydomonas reinhardtiigenomes have already been published and it appears thata large number of phytoremediation-related genes are beingencoded to act directly on environmental pollutants. Thepresence of several hundreds of catabolic enzymes andtransporter sequences suggest that plants may have richpotential to mobilize and detoxify toxic contaminantsincluding organic and inorganic in their environment withintheir tissues and organs. Genomic and proteomic informa-tion gained from these sequenced plant species will greatlyaccelerate the phytoremediation process in situ. Furtherdevelopment of phytoremediation requires an integratedmultidisciplinary research effort that combines plant biolo-gy, genetic engineering, soil chemistry, soil microbiology,ecology, as well as agricultural and environmental engi-neering. In this regard, considerable efforts have been takenby the European Science Foundation, and under this

context, a COST 859 Action entitled “Phytotechnologiesto promote sustainable land use and improve food safety”has been launched since 2004. The main objective of thisaction is to provide a sound understanding of theabsorption/exclusion, translocation, storage, or detoxifica-tion mechanisms of essential or toxic mineral elements, aswell as organic contaminants, and to prepare the best use ofplants for sustainable land use management and improvefood safety. Promotion of cooperation and of data exchangebetween working groups in this action have been encour-aged and the present work is a part of such cooperation.

5 Recommendations and perspectives

The worldwide phytoremediation industry consists of severaldozens of companies falling within discrete categories. Mostvisible are the dedicated phytoremediation companies, whosesole or primary remediation technology is phytoremediation,but a related category includes other specialty companies,diversifying into hazardous waste or wastewater phytoreme-diation from areas such as constructed wetlands. The nextmost active segment includes a number of the large tomidsize consulting/engineering firms that have developed anexpertise in phytoremediation. The number of these firmswith credible phytoremediation expertise has grown encour-agingly in the last few years. Also part of the “industry,”although generally not conducting commercial remediation,are several industrial companies, which conduct research orfield remediation for internal needs, and a large number ofacademic, government, and other nonprofit research groupsconducting research and developing new technologies(Pilson-Smits 2005).

A significant number of companies with phytoremedia-tion experience have emerged inside and outside the US andhave created growing industries in North America and inseveral European countries. There are at least ten companiesin Canada and may be as many as 20 companies in Europewhich are conducting research or which have carried outcommercial remediation using phytoremediation or relatedtechnologies. Most of these companies would be considered“diversifying specialty companies,” but they include anumber of consulting/engineering firms, particularly inCanada, and some dedicated phytoremediation firms. Forthe most part, many of these companies are pursuingphytoremediation at the research stage only, but numerouscommercial projects have been undertaken in severalcountries around the world (Glass 2000). Smaller, butemerging, markets exist in developing nations, particularlyin portions of Asia. The total world remediation market isreported to be approximately US $15–18 billion per year.

An estimated 52 million hectares, which is more than16% of the total land area, are affected by some level of soil

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contamination. The most heavily contaminated areas arefound near industrialized regions in northwestern Europe,but many contaminated areas also exit around major citiesof Europe (EEA 2003). There could be between 300,000and 1.5 million of these sites in the EU (EC 2002) presentfor phytoremediation. While not all are priorities forremediation, it is estimated that EU nations may spendbetween 59 and 109 billion € (EC 2002) to clean these sitesover the next 20–25 years. There is an urgent need forcheap and efficient methods to clean up heavily contami-nated industrial areas and this could be achieved by usingwild or genetically modified metal accumulator plants. Theuse of plants provides several striking advantages, com-pared with conventional methods of soil remediation. It ischeap and, after planting, only marginal costs apply forharvesting and field management. After harvesting biomassis burned, no additional carbon dioxide will be released intoatmosphere beyond that which was originally assimilatedby plants during growth. As mentioned above, the biomasscould be also used for bioenergy production (Schröder et al.2008) and, in addition, the potential of phytoremediationwith energy crops together with the production of biodieselwould be one of the economically feasible methods in thenear future.

Acknowledgements This work is part of the cooperation betweengroups working in a COST589 program. We extend our thanks to Dr.Jean-Paul Schwitzguebel for his encouragement and support forcooperation among COST 859 groups. We appreciate the commentsof Dr. Oktay Külen and Gülten Güneş for some parts of the review.

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