Environmental Science and Pollution Research

, Volume 16, Issue 2, pp 162–175

Implications of metal accumulation mechanisms to phytoremediation

Authors

    • TÜBİTAK, Marmara Research CenterInstitute for Genetic Engineering and Biotechnology
  • Peter Schröder
    • Helmholtz-Zentrum München, German Research Center for Environmental Health
AREA 5 • PHYTOREMEDIATION • REVIEW ARTICLE

DOI: 10.1007/s11356-008-0079-z

Cite this article as:
Memon, A.R. & Schröder, P. Environ Sci Pollut Res (2009) 16: 162. doi:10.1007/s11356-008-0079-z

Abstract

Background, aim, and scope

Trace elements (heavy metals and metalloids) are important environmental pollutants, and many of them are toxic even at very low concentrations. Pollution of the biosphere with trace elements has accelerated dramatically since the Industrial Revolution. Primary sources are the burning of fossil fuels, mining and smelting of metalliferous ores, municipal wastes, agrochemicals, and sewage. In addition, natural mineral deposits containing particularly large quantities of heavy metals are found in many regions. These areas often support characteristic plant species thriving in metal-enriched environments. Whereas many species avoid the uptake of heavy metals from these soils, some of them can accumulate significantly high concentrations of toxic metals, to levels which by far exceed the soil levels. The natural phenomenon of heavy metal tolerance has enhanced the interest of plant ecologists, plant physiologists, and plant biologists to investigate the physiology and genetics of metal tolerance in specialized hyperaccumulator plants such as Arabidopsis halleri and Thlaspi caerulescens. In this review, we describe recent advances in understanding the genetic and molecular basis of metal tolerance in plants with special reference to transcriptomics of heavy metal accumulator plants and the identification of functional genes implied in tolerance and detoxification.

Results

Plants are susceptible to heavy metal toxicity and respond to avoid detrimental effects in a variety of different ways. The toxic dose depends on the type of ion, ion concentration, plant species, and stage of plant growth. Tolerance to metals is based on multiple mechanisms such as cell 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 detoxification in plants appears to be chelation of metals by low-molecular-weight proteins such as metallothioneins and peptide ligands, the phytochelatins. For example, glutathione (GSH), a precursor of phytochelatin synthesis, plays a key role not only in metal detoxification but also in protecting plant cells from other environmental stresses including intrinsic oxidative stress reactions. In the last decade, tremendous developments in molecular biology and success of genomics have highly encouraged studies in molecular genetics, mainly transcriptomics, to identify functional genes implied in metal tolerance in plants, largely belonging to the metal homeostasis network.

Discussion

Analyzing the genetics of metal accumulation in these accumulator plants has been greatly enhanced through the wealth of tools and the resources developed for the study of the model plant Arabidopsis thaliana such as transcript profiling platforms, protein and metabolite profiling, tools depending on RNA interference (RNAi), and collections of insertion line mutants. To understand the genetics of metal accumulation and adaptation, the vast arsenal of resources developed in A. thaliana could be extended to one of its closest relatives that display the highest level of adaptation to high metal environments such as A. halleri and T. caerulescens.

Conclusions

This review paper deals with the mechanisms of heavy metal accumulation and tolerance in plants. Detailed information has been provided for metal transporters, metal chelation, and oxidative stress in metal-tolerant plants. Advances in phytoremediation technologies and the importance of metal accumulator plants and strategies for exploring these immense and valuable genetic and biological resources for phytoremediation are discussed.

Recommendations and perspectives

A number of species within the Brassicaceae family have been identified as metal accumulators. To understand fully the genetics of metal accumulation, the vast genetic resources developed in A. thaliana must be extended to other metal accumulator species that display traits absent in this model species. A. thaliana microarray chips could be used to identify differentially expressed genes in metal accumulator plants in Brassicaceae. The integration of resources obtained from model and wild species of the Brassicaceae family will be of utmost importance, bringing most of the diverse fields of plant biology together such as functional genomics, population genetics, phylogenetics, and ecology. Further development of phytoremediation requires an integrated multidisciplinary research effort that combines plant biology, genetic engineering, soil chemistry, soil microbiology, as well as agricultural and environmental engineering.

Keywords

Accumulator plantsHeavy metalsMetal transportersMetallothioneinsPhytochelatinsPhytoremediation

1 Background, aim, and scope

Trace elements (heavy metals and metalloids) are important environmental pollutants, and many of them are toxic even at very low concentrations. Pollution of the biosphere with trace elements has accelerated dramatically since the beginning of the Industrial Revolution (Padmavathiamma and Li 2007). The primary sources of this pollution are the burning of fossil fuels, mining and smelting of metalliferous ores, municipal wastes, fertilizers, pesticides, and sewage (Wei and Zhou 2008).

In addition to sites contaminated by human activity, natural mineral deposits containing particularly large quantities of heavy metals are present in many regions of the globe. These areas often support characteristic plant species that thrive in these metal-enriched environments. Whereas many species avoid the uptake of heavy metals form these soils, some of these species can accumulate significantly high concentrations of toxic metals, to levels which by far exceed the soil levels (Baker and Brooks 1989). 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 to accumulate heavy metals, which have no known biological function. These include Cd, Cr, Pb, Co, Ag, and Hg (Baker and Brooks 1989). However, excessive accumulation of these heavy metals can be toxic to most plants. The ability to acquire a tolerance both against heavy metals and an accumulation to very high concentrations have evolved both independently and together in a number of different plant species (Baker and Walker 1990; Stearns et al. 2007).

In this review, we summarize current knowledge concerning metal accumulation and detoxification mechanisms in plants and the potential commercial application of this phenomenon in phytoremediation.

2 Results

2.1 Plant survival strategies to increasing metal concentrations

Generally spoken, there are three different types of plants that have developed three basic strategies for growing on contaminated and metalliferous soils (Baker and Walker 1990). Metal excluders—effectively prevent metal from entering their aerial parts over a broad range of metal concentrations in the soil; however, they can still contain large amounts of metals in their roots. Metal indicators—accumulate metals in their above-ground tissues and the metal levels in the tissues of these plants generally reflect metal levels in the soil. Metal accumulators are usually referred to as hyperaccumulators that concentrate metals in their above-ground tissues to levels far exceeding those present in the soil or in nonaccumulating species growing nearby. It has been proposed that a plant containing more than 0.1% of Ni, Co, Cu, Cr, and Pb or 1% of Zn on a dry weight basis is called a hyperaccumulator, irrespective of the metal concentration in the soil (Baker and Walker 1990). There are around 400 plant species known worldwide to accumulate metals in large amounts and these species are of interest for potential use in phytoremediation of metal-contaminated soils (Brooks 1983; Memon and Yatazawa 1984; Baker et al. 2000; Pilson-Smits 2005). Information related to accumulator plants is most needed in four areas: First, the metal-accumulating ability of various species as a function of soil metal concentrations, physicochemical soil properties, and physiological state of the plant; second, the specificity of metal uptake, transport, and accumulation; third, the physiological, biochemical, and molecular mechanisms of accumulation; and fourth, the biological and evolutionary significance of metal accumulation.

2.2 Mechanisms of metal accumulation

In plant species growing on metal-rich soils, the internal distribution may differ significantly. Metal ions may be localized in roots and shoots, or they may accumulate and be stored in nontoxic forms. One distinct mechanism of tolerance or accumulation appears to involve binding of potentially toxic metals in cell walls of roots and leaves, before they can get in contact with sensitive sites within the cell. Others allow cellular uptake, but then sequester metals in the vacuoles (Memon and Yatazawa 1984; Cosio et al. 2004). A pressing question about heavy metals in the environment concerns the amounts that plants can tolerate and accumulate without adverse effects. A further question relates more to the amount and speciation and their roles for plant performance and metal transfer to the food chain. Several types of heavy metal resistance and tolerance mechanisms are being suggested in plants growing on metalliferous 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 caerulescens, Arabidopsis halleri), Pb (Thlaspi rotundifolium ssp. 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 of these accumulator plants have very unique ecophysiological behavior and have the capacity to accumulate significant amounts of metals and compartmentalize them efficiently in the cell wall, vacuole, and to the specific subcompartment and/or compartments of the cytosol in order to render them innoxious or nontoxic and keep them away from active metabolic sites in plant cells (Memon et al. 1981; Memon and Yatazawa 1982, 1984). While surveying the flora of Cu mining areas of Southeastern Anatolia, we discovered several endemic metal accumulator plants and, interestingly, a Brassica nigra ecotype found from a Diyarbakir site contained a very high amount of Cu in their shoots. When plants from this ecotype were regenerated from callus culture and grown in soil culture containing 200 ppm Cu, the shoots accumulated three times more Cu (700 μg/g dry weight) than roots (Memon et al. 2006). The γ-glutamylcysteine (γ-EC) expression in the shoots of Cu-treated plants was around 3.5 times that of control plants (Memon et al. 2008a). This ecotype could be considered a good candidate for Cu phytoremediation. Several mechanisms may contribute to heavy metal tolerance, depending on the type of metal and plant species (Memon and Yatazawa 1982, 1984). The recent development of transcriptomics (microarray analysis), proteomics, and metabolomics allows a deeper exploration of the function and regulation of the cell when it encounters high metal concentration in the environment. The current research data on transcriptome analysis of transporters and the participation of multiple gene families in response to metal stress in two model accumulator plant species such as T. caerulescens and 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 detoxification and in the translocation of these metals from the root to the shoots in Arabidopsis (Hussain et al. 2004; Verret et al. 2004; Courbot et al. 2007; Hanikenne et al. 2008). RNAi of HMA4 in A. halleri downregulated its expression and reduced the Zn hyperaccumulation and full tolerance to Cd and Zn in the plants (Hanikenne et al. 2008). Several metal-binding proteins in plants have been reported which include metallothioneins, metalloenzymes, metal-activated enzymes, and various metal storage, carrier, and channel proteins (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 transition metals, are widely distributed in yeast as well as in lower and higher plants (Cobbett 2000).

2.3 Genetics of metal accumulation in plants

Over the past decade, significant progress has been made in elucidating the molecular basis of metal uptake, accumulation, and tolerance into plant cells (Cobbett 2000; Cobbett and Meagher 2002; Clemens 2006). Until now, most of the genetic studies on species with a metalliferous population have dealt with the genetic determinism of heavy metal tolerance (Schat and Ten Bookum 1992; Macnair 1993). Such studies have yielded valuable insight into the mechanisms that might underlie plant adaptation to metal availability. Despite recent advances, the mechanism underlying hyperaccumulation is still not well-defined. Therefore, there is a need to develop a model system for molecular genetic studies of metal hyperaccumulation in plants. Arabidopsis thaliana, although it is not a metal accumulator, will be suitable because around 25% of the reported hyperaccumulator species are members of Brassicaceae. Therefore, microarray gene chips of A. thaliana to analyze the transcriptome of model metal accumulator plants like A. halleri and T. caerulescens could provide a platform from which plant geneticists can relate the phenotypic and genetic variations in physiological traits of underlying genes or gene networks related to metal tolerance and/or accumulation (Plaza et al. 2007; Willems et al. 2007; Milner and Kochian 2008; Roosens et al. 2008). Few groups, however, have tapped the full potential of gene expression profiles, especially as they relate to understanding how genetic change translates to phenotypic variation and the resultant adaptive physiological traits (Maathuis et al. 2003; Weber et al. 2004). Hammond et al. (2006) have developed a robust method to profile and compare the transcriptome of two plant species from Brassicasae, T. caerulescens J & C Presl., a Zn hyperaccumulator, and T. arvense L., a nonhyperaccumulator, using Affymetrix A. thaliana ATH-121501 (ATH1) GeneChip® arrays (Affymetrix, Santa Clara, CA, USA). Approximately 5,000 genes were differentially expressed in the shoots of T. caerulescens compared with T. arvense, including genes involved in Zn transport and compartmentalization. Partial sequencing of cDNA clones (e.g., ESTs-expressed sequence tags) is another efficient method to obtain metal-related gene expression for metalliferous species of which no detailed genomic information is available. Comparing EST sequences of the target species with the appropriate reference model species such as A. thaliana, O. sativa, P. trichocarpa, M. truncatula, C. reinhardtii, and with additional public databases for annotations of their putative functions, provides an important tool for the identification of unique genes, their function, and their role in metal tolerance. This approach could be useful for species whose complete genome sequence information is not available, as in the case of Brassica sps. (A. halleri, T. caerulescens, T. goesingense, B. juncea, B. nigra, Thellungiella, etc.) (Rigola et al. 2006; Van de Mortel 2006; Freeman and Salt, 2007; Milner and Kochian 2008; Roosens et al. 2008). The fully sequenced genome of species, such as Arabidopsis, rice, poplar, Chlamydomonas, together with a large-scale “omics” and systems biology approach will allow one to address many of the outstanding questions related to metal accumulation and tolerance in plant cells through a global analysis of gene expression. It is possible to use synteny between the genomes of the test and model species to deduce or identify genetic regions that contain quantitative trait loci (QTLs) involved in metal tolerance or accumulation. In several studies, these techniques have been used to deduce chromosomal regions probably containing QTLs for metal accumulation and identifying genes involved in Zn and Cd 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). During recent years, important information has been produced by QTL analysis and has been used to investigate the genetic base of adaptation of metallophytes to metal-polluted soils. These studies have specified genes underlying metal tolerance as well as indications on epistatic relationships between those genes (Bratteler et al. 2006; Deniau and Pieper 2006). A wide range of genomic technologies are now available to help biologists and ecologists to understand the phenomenon of hyperaccumulation.

2.4 Metal transporters

Plants have developed flexible strategies to cope with fluctuations in their environment in order to minimize the adverse effects of metal deficiency or toxicity. Adaptive responses can include a significant alteration in gene expression, particularly of membrane transporters that are responsible for the uptake, efflux, translocation, and sequestration of essential and nonessential mineral nutrients. Metal hyperaccumulator plants possess several unique characteristics, such as the ability to take up and translocate exceedingly large amounts of metals to their shoots and hypertolerate the toxic metals (Stearns et al. 2007; Freeman and Salt 2007). Of major concern with respect to plant exposure as well as food chain accumulation are the metalloids, arsenic (As) and selenium (Se), and the metals, cadmium (Cd), mercury (Hg), and lead (Pb) (Welch and Graham 2003; Prasad 2008). Generally, there are vast differences in the bioavailability of metals to plants. Some metals (for example, Cr, Ag, Hg, or Sn) are practically not available for plant uptake because of their low solubility in soil. Other metals such as Pb can be major pollutants and could be present in huge amounts along roadsides in heavily populated large cities, yet are hardly taken up into plants because of low solubility and strong interaction with soil particles. Toxic metal ions that do enter plant roots are transported to shoots and accumulate in edible plant parts, which represent the principal route of toxic metal entry into the food chain (Welch and Graham 2003; Marmiroli and Maestri 2008; Memon et al. 2008b). Over the past decade, significant progress has been made in elucidating the molecular basis of metal uptake into plant cells. A number of important membrane transporter families have been discovered by heterologous complementation screens and sequencing of both plant ESTs and the Arabidopsis and rice genomes. The completion of Arabidopsis, rice, poplar, and Chlamydomonas genomes now give us impetus to analyze complete sets of transporter gene families in plant and alga species. Powerful genetic approaches have been developed that allow high throughput selection of point mutations that reduce or block transport of toxic ions, while maintaining nutrient transport (Rogers et al. 2000; Rogers and Guerinot 2002). Several toxic metal transporters have been analyzed and some of them are shown to transport Cd. Amongst them are members of Zn-regulated transporter (ZRT), Fe-regulated transporter (IRT)-like proteins (ZIP), natural resistance-associated macrophage proteins (NRAMP), and cation diffusion facilitator (CDF) (Thomine et al. 2000; Maser et al. 2001; Hall and Williams 2003). The overview of the metal transporters and their tissue-specific expression in plants is summarized in Table 1. Microarray analysis of Arabidopsis has demonstrated that a number of genes encoding ZIP proteins are induced in plants under Zn-deficient conditions (Wintz et al. 2003). Plant ZIP proteins that mediate uptake of Zn into yeast have also been identified in rice (Oryza sativa), soybean (Glycine max), Medicago truncatula, and the Zn/Cd hyperaccumulator, T. caerulescens (Pence et al. 2000; Moreau et al. 2002; Lopez-Millan et al. 2004; Ishimaru et al. 2005). Eighteen genes are predicted to encode ZIPs in Arabidopsis (Gaither and Eide 2001) and, although physiological functions of Zn-transporting ZIPs are yet to be described in plants, they are predicted to be involved in cellular uptake and in mobilization of stored Zn. A number of ZIP genes have been found to be highly expressed 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 only prevail among species, but are also reported to be present in ecotypes of some species. Detailed studies with two contrasting ecotypes of the hyperaccumulator T. caerulescens showed differences in expression patterns of ZIP transporter genes among ecotypes (Plaza et al. 2007). The four ZIP genes studied (TcIRT1, TcIRT2, TcZNT1, and TcZNT5) were expressed only in roots and not in leaves, indicating their specificity in metal uptake and transport in roots. The ecotypic difference in ZIP gene expression in the roots of T. caerulescence in the presence and absence of Cd were clearly identified. TcZNT1 expression was not affected by Cd and expression levels were similar for both ecotypes (Ganges and Prayon), but the expression level of TcZNT5 in Ganges was below that for Prayon and not affected by Cd treatment. The expression level of TcIRT1 was increased in roots of both ecotypes when treated with Cd but transcript levels of TcIRT2 were not detectable in either ecotype, either in the presence or absence of Cd (Plaza et al. 2007). The significance in terms of metal ion uptake needs further investigation through functional analysis. A number of potential metal ligands are highly conserved among ZIP family members. For example, the replacement of glutamic acid residues at position 103 in wild-type IRT1 with alanine and by heterologous expression in yeast increases the substrate specificity of the transporter by selectively eliminating its ability to transport Zn (Rogers and Guerinot 2002). A number of other conserved residues in or near transmembrane domains appear to be essential for transport function. For example, replacing aspartic acid residues at position 100 or 136 with alanine also increases IRT1 metal selectivity by eliminating transport of both iron and manganese. In addition, the ATP-binding cassette (ABC) family transporters represent one of the largest protein families in living organisms ranging from bacteria to humans (Hall and Williams 2003). A. thaliana and rice (O. sativa) contain approximately 130 ABC transporters, the precise functions of which still remain obscure for the most part (Sanchez-Fernandez et al. 2001; Rea 2007). They can be assigned depending on their nucleotide-binding folds (NBFs) or nucleotide-binding domains (NBDs), which share 30–40% sequence identity between family members (Higgins 1992). In addition to NBDs, ABC transporters also contain transmembrane domains (TMDs), each composed of several hydrophobic α-helices (Verrier et al. 2008). There are three prominent features of these transporters in plants. First of all, it is energized directly by MgATP, but not by free ATP or nonhydrolyzable ATP analogs. Secondly, transport is insensitive to the transmembrane H+ electrochemical potential difference, and thirdly, transport is exquisitely sensitive to vanadate (Rea 2007). ABC transporters have been classified into three major subfamilies: (1) full-sized transporters containing at least two NBFs and two TMDs, (2) half-sized 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 ABC transporters were found to participate in detoxification processes as well as in plant growth and development (Noh et al. 2001; Campbell et al. 2003; Geisler et al. 2005). AtATM3, an ATP-binding cassette transporter of Arabidopsis is shown to be a mitochondrial protein involved in the biogenesis of iron–sulfur clusters and iron homeostasis in plants and is shown to be upregulated in roots of plants when exposed with Cd (II) or Pb (II) (Kim et al. 2006). Additionally AtATM3 overexpressed in Arabidopsis plants showed enhanced Cd and Pb resistance compared to wild-type, whereas AtATM3 knockout plants showed Cd-sensitive phenotypes. This Cd sensitivity of the mutants was rescued by overexpressing wild-type AtATM3. AtMHX is another metal transporter reported to be located in tonoplast membrane and involved in Zn and Fe homeostasis (David-Assael et al. 2006). The expression AtMHX is induced by plant hormones, auxin and ABA, and exhibits two distinguished regulatory properties. Its leader intron is absolutely essential for expression and a repetitive genomic element of 530 bp (or part of it) functions as an enhancer. Recent studies showed 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 a region comprising 739 genes and, among them, 11 genes are differentially expressed in A. hallari and A. thaliana (Roosens et al. 2008). From these 11 genes, only one has been depicted as heavy metal-transporting ATPase4 (HMA4) and belongs to a P-type ATPase family involved in the transport of transition metals (Chiang et al. 2006; Talke et al. 2006). The Zn hyperaccumulation and full hypertolerance to Zn and Cd in A. halleri was considerably reduced when HMA4 expression was reduced through RNAi (Hanikenne et al. 2008). It has been demonstrated that AhHMA4 plays a major role in Zn hyperaccumulation and associated Cd and Zn hypertolerance, and its high expression in A. halleri is specified in cis-regulatory factors and amplified by gene copy number expansion. Increased expression of HMA4 in A. halleri enhanced the Zn transport from the root symplasm into the xylem vessels necessary for shoot Zn hyperaccumulation (Hanikenne et al. 2008). These findings indicate the importance of these transporters in phytoremediation. Manipulation of these transporters to achieve removal of metal ions from the cell holds great potential in the plant species for hyperaccumulation (Tong et al. 2004; Roosens et al. 2008).
Table 1

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

Plant name

Protein families

Gene name

Metals

Tissue expression

References

A. thaliana, A. halleri, L. esculentum

P-Type ATPase

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

Cu, Zn, Cd, Co, Pb

Shoots and 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; Roosens et al. 2008; Talke et al. 2006; Verret et al. 2004; Willems et al. 2007; Xing et al. 2008

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

Nramp

AtNRAMP1-6, LeNRAMP1-3, AhNRAMP3

Fe, Cd

Shoots and roots

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

A. thaliana, O. sativa

ZIP

AtZIP1-12, OsZIP4

Zn

Shoots and 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

Shoots and 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

2.5 Metal chelation and detoxification

Induction of metal-chelating proteins related to metallothioneins (MTs) (Robinson et al. 1993; Rauser 1999) and/or phytochelatins (PCs) (γ-glutmylcysteinyl-isopeptides) (Clemens et al. 1999; Cobbett 2000; Cobbett and Goldsbrough 2002; Sarry et al. 2006) increases the level of cell tolerance to an excess of metal ions. These cysteine-rich polypeptides sequester heavy metals efficiently and contribute to detoxification and accumulation in plant cells. While MTs are independent gene products, PCs are synthesized 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 product in countering oxidative stress and may be conjugated to diverse xenobiotic compounds as a prerequisite for their vacuolar sequestration (Grill et al. 1989; Cobbett 2000; Guo et al. 2008).

2.5.1 Metallothioneins

Metallothioneins (MT) are products of mRNA translation and characterized as LMW cysteine-rich metal-binding proteins. They are found throughout the animal and plant kingdoms. In animals and fungi, MTs have been shown to play a role in the detoxification of heavy metals (Robinson et al. 1993; Hall 2002), although their exact function is not completely understood. In plants, a correlation has been reported between MT RNA levels and naturally observed differences in the tolerance to heavy metals in Arabidopsis ecotypes, suggesting a role in metal homeostasis (Murphy and Taiz 1995; Murphy et al. 1997; Guo et al. 2008).

Some functions proposed for MTs in plants include metal detoxification (Roosens et al. 2005; Domenech et al. 2006), a role during development (Ledger and Gardner 1994), 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 concentration in different organisms suggests that MTs can be effective reporters of environmental conditions (Morris et al. 1999). Isolation and characterization of MTs in model bioindicator organisms could also contribute to our understanding of the biological response to biomonitoring pollutants in the environment (Morris et al. 1999).

2.5.2 Phytochelatins

Phytochelatins form a family of peptides that consists of repetitions of the γ-Glu-Cys dipeptide followed by a terminal 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 GSH in response to many metals (Rauser 1990; Cobbett 2000). They are structurally related to GSH and not directly encoded by genes, but the products of a biosynthetic pathway (Gly+Cys→GCS γ-Glu-Cys+Glu→GS GSH→PCS+Cd PC→PC–Cd→HMTI vacuole where γ-glutamylcysteine synthetase (GCS), GSH synthetase (GS), phytochelatin synthase (PCS), heavy metal tolerance 1 (HMT1), ABC type vacuolar membrane transporter of PC–Cd complexes). A number of other structural variants of 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) are rapidly induced in vivo by a wide range of heavy metal ions. The enzyme-synthesizing PCs from GSH is a γ-Glu-Cys dipeptididyl transpeptidase (EC 2.3.2.15), commonly referred to as phytochelatin synthase (PC synthase) (Grill et al. 1989). Mutants of Arabidopsis lacking PC synthase are unable to synthesize PCs and hypersensitive to Cd and Hg. The cad 1 mutant of A. thaliana L. Heynh is cadmium-sensitive and its GSH level is similar to that of the wild-type but both are deficient in PC and lack PC synthase activity in vitro. It is predicted that cad1 is the structural gene for PC synthase (Howden et al. 1995). The Arabidopsis cad 1 gene (referred to as AtPCS1) (Howden et al. 1995; Ha et al. 1999; Vatamanuik et al. 1999) and a similar gene in wheat (TaPCS1) (Clemens et al. 1999) have been shown to confer resistance to Cd when expressed in the yeast S. cerevisiae (Clemens et al. 1999; Cobbett and Meagher 2002). However, these mutants have essentially wild-type levels of tolerance to Cu and Zn (Howden et al. 1995). The occurrence of PC synthase in different 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 contains shorter PCs. The HMW complex is vacuolar, contains longer PCs, acid-labile sulfide, and usually dominates over that of the LMW complex when plants are exposed to Cd for longer time (Ortiz et al. 1995; Rauser 2000). Accumulation of the HMW Cd–PC complex is essential for Cd detoxification in A. thaliana (Howden et al. 1995; Ha et al. 1999). Aside from detoxification, PC plays a role in homeostasis of heavy metals in plants, and this is the mechanism that regulates the metal ions availability in plant 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 major source of nonprotein thiols in most plant cells (Noctor et al. 1998; Maughan and Foyer 2006). GSH biosynthesis is a two-step ATP-dependent reaction (Meister and Anderson 1983), including the synthesis of γ-EC from glutamate and cysteine, followed by the formation of GSH through the addition of glycine to the C-terminal end of γ-EC. The first reaction is catalyzed by γ-glutamylcysteine synthetase (γ-EC synthetase) and the second one by GS. Both enzymes are encoded by gsh1 and gsh2 genes, respectively (May and Leaver 1995; Wang and Oliver 1996).

γ-EC is feedback-inhibited by GSH, and obviously acts as the rate-limiting step in the pathway. Numerous physiological functions have been attributed to GSH levels in plants and its role as a regulator of gene expression (Foyer et al. 1997) and in the regulation of enzyme activity after glutathionation of proteins has been reported (Dixon et al. 2005). Moreover, it is also involved in 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 in defense of plants and yeast against oxidative stress (Alscher 1989; Grant et al. 1996). However, as yet, an absolute dependence on GSH for stress tolerance has only been demonstrated for H2O2, but direct induction of GSH synthesis in response to other oxidative stimuli is lacking (May and Leaver 1995). Furthermore, research has failed to demonstrate that such an elevation has physiological significance in plants. On the other hand, studies in animal systems have shown that depletion of GSH renders cells susceptible to oxidative processes (Arrick et al. 1982). Recent data suggest that the cytosolic antioxidant capacity is sufficient to maintain cell viability even in the absence of catalase, and that under such conditions, a strong increase in the level of reduced GSH can be measured. The fact that a transient accumulation of hydrogen peroxide led to a significant increase in the level of reduced GSH suggests that the mechanism of hydrogen peroxide-induced GSH synthesis is much more complex than previously proposed. However, exposure of maize seedlings, tomato, parsley, and tobacco cell cultures to heavy metals accelerates GSH synthesis (Rüegsegger and Brunold 1992; Schneider and Bergmann 1995). These studies clearly indicate the importance of GSH in protecting plants against various forms of stress. Manipulating the expression of enzymes involved in GSH synthesis appears to be a promising strategy for the production of plants with superior capacity for heavy metal phytoremediation (Ducruix et al. 2006; Vestergaard et al. 2008).

3 Discussion

3.1 Phytoremediation: a versatile technology with many potential applications

Phytoremediation defines the use of plants and their associated microbes to extract, sequester, and/or detoxify various kinds of environmental pollutants from water, sediment, soils, and air. Phytoremediation of heavy metals is an emerging technology and several subsets of this 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 plant species are used in phytoremediation. Many metallophyte plants are used in prospecting for mineral deposit (Baker and Brooks 1989) but only recently has the value of metal-accumulating terrestrial plants for environmental remediation been fully realized (Padmavathiamma and Li 2007; Stearns et al. 2007). A number of terrestrial and aquatic plants are known to be natural hyperaccumulators of metals, but since these tend to be slow growers, researchers have turned to other species, more recently identified or selected, as more promising commercial candidates. Deep-rooted trees such as poplar, willow, and cottonwood are most commonly used for applications requiring withdrawal of large amounts of water from the subsurface, while a number of different plants, trees, and grasses are used to stimulate microbial degradation of organic contaminants in soil. Among plants at earlier stages of research are plants and 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-enriched soils represent major biological resources for the improvement of phytoremediation, a benign and cost-effective technology that uses plants to clean up metal-polluted soils. Within the last decade, molecular genetic studies carried out on several model organisms (including A. halleri and T. caerulescens) have considerably enhanced our understanding of metal tolerance and hyperaccumulation in plants, but the identification of genes of interest for phytoremediation purposes remains a challenge. In the past decade, extensive research efforts have been made to dissect the basic molecular mechanisms of metal tolerance in metallophytes (Clemens 2001; Clemens 2006). Physiological approaches, transcriptomics in particular, have allowed the identification of several candidates that seemed to result from modifications of the sequence or expression of genes belonging to the ubiquitous metal homeostasis network (Bernard et al. 2004; Drager et al. 2004; Roosens et al. 2004; Mirouze et al. 2006; Talke et al. 2006).

A better understanding of the biochemical processes involved in plant heavy metal uptake, transport, accumulation, and resistance will help to systematically improve phytoremediation using molecular genetic approaches. A growing knowledge of factors important to phytoremediation can provide a basis for genetic modification of plants for improved performance. For example, to improve the high potential of plants for phytoremediation is to introduce genes responsible for accumulation and resistance from wild slow-growing plants or from bacterial or animal sources into fast-growing, high-biomass plant species. However, long-term efforts should be directed toward the development of a “gene bank” composed of genes valuable for phytoremediation. Systematic screening of plant species and genotypes for metal accumulation and resistance will broaden the spectra of genetic material available for optimization and transfer. Mutagenesis of selected high-biomass plant species may also produce improved phytoremediating cultivars. Another approach would be to genetically engineer crop species with improved metal tolerance and accumulation capacities (Wangeline et al. 2004; Whiting et al. 2004). Phytoremediation engineering could then involve either classical breeding or transgenic approaches (Zhu et al. 1999a, b; Wangeline et al. 2004; Singla-Pareek et al. 2006) or the exploitation of available genetic variation for metal tolerance in crops through marker-assisted selection (Ghandilyan et al. 2006).

In summary, merging molecular and ecological genetics to the study of plant metal tolerance greatly improves the overall knowledge of metal tolerance mechanisms and provides a currently unreleased context for the exploitation of metal tolerance genes. This approach should quickly allow scientists to identify the metal tolerance genes that, in return, could be investigated in other pseudometallophyte or crop species and finally be used in phytoremediation engineering.

4 Conclusions

Phytoremediation holds great potential as an environmental clean up technology and has been investigated substantially since the last two decades. Considerable interest in phytoremediation exists by both government and industry. The biggest advantage of phytoremediation is its low cost. Phytoremediation can be up to 1,000-fold cheaper compared with conventional remediation methods such as excavation and reburial. Moreover, it offers permanent, in situ remediation rather than simply moving the pollution to a different site (Salt et al. 1998).

In each of the industrial countries, several tens of thousands 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), and in other countries, similar numbers are found. When technical remediation methods like soil washing, excavation, or pump and treat systems fail, phytoremediation will be a welcome and environmentally sound alternative, which could probably be cheaper than the estimated U.S. remediation costs of $7 to $8 billion per year, approximately 35% of which involves remediation of metals (Bennett et al. 2003). While the process of phytoremediation as such is well-understood, the time frame of such an action is frequently underestimated. Remediation times of decades are realistic in many scenarios, and this reduces the attractiveness of this technology. A combination of biomass production with other land use functions may be interesting to generate an economic benefit for farmers (Schröder 2007; Schröder et al. 2007). A recent study estimated the economic value of the phytoremediation function to farmers as assessed by the substitution cost and hedonic price analysis to about 14,600 and 14,850 € ha−1, respectively, over a period of 20 years (Lewandowski et al. 2006). The willingness of farming communities to adopt this method will strongly rely on governmental help, and the insight into the soundness and added value of phytoremediation. Large-scale remediation has to be designed to address local problems, gain more scientific knowledge, and meet clean up standards for the future use of a site. Phytoremediation usually fails if fast results and total removal of plumes is required. 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 the remediation process at low to zero risk (Pilson-Smits 2005). The progress in developing and understanding transgenic or mutagenized plants in their potential to hyperaccumulate trace elements will also be beneficial to the field. It is likely that such plants will be utilized in the near future to solve specific problems of pollution removal, under thorough observation of safety regulations, and that their biomass will be 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 be selected or could be generated by genetic manipulation and be used for remediation of different polluted sites. The A. thaliana, rice, poplar, and Chlamydomonas reinhardtii genomes have already been published and it appears that a large number of phytoremediation-related genes are being encoded to act directly on environmental pollutants. The presence of several hundreds of catabolic enzymes and transporter sequences suggest that plants may have rich potential to mobilize and detoxify toxic contaminants including organic and inorganic in their environment within their tissues and organs. Genomic and proteomic information gained from these sequenced plant species will greatly accelerate the phytoremediation process in situ. Further development of phytoremediation requires an integrated multidisciplinary research effort that combines plant biology, genetic engineering, soil chemistry, soil microbiology, ecology, as well as agricultural and environmental engineering. In this regard, considerable efforts have been taken by the European Science Foundation, and under this context, a COST 859 Action entitled “Phytotechnologies to promote sustainable land use and improve food safety” has been launched since 2004. The main objective of this action is to provide a sound understanding of the absorption/exclusion, translocation, storage, or detoxification mechanisms of essential or toxic mineral elements, as well as organic contaminants, and to prepare the best use of plants for sustainable land use management and improve food safety. Promotion of cooperation and of data exchange between working groups in this action have been encouraged and the present work is a part of such cooperation.

5 Recommendations and perspectives

The worldwide phytoremediation industry consists of several dozens of companies falling within discrete categories. Most visible are the dedicated phytoremediation companies, whose sole or primary remediation technology is phytoremediation, but a related category includes other specialty companies, diversifying into hazardous waste or wastewater phytoremediation from areas such as constructed wetlands. The next most active segment includes a number of the large to midsize consulting/engineering firms that have developed an expertise in phytoremediation. The number of these firms with credible phytoremediation expertise has grown encouragingly in the last few years. Also part of the “industry,” although generally not conducting commercial remediation, are several industrial companies, which conduct research or field remediation for internal needs, and a large number of academic, government, and other nonprofit research groups conducting research and developing new technologies (Pilson-Smits 2005).

A significant number of companies with phytoremediation experience have emerged inside and outside the US and have created growing industries in North America and in several European countries. There are at least ten companies in Canada and may be as many as 20 companies in Europe which are conducting research or which have carried out commercial remediation using phytoremediation or related technologies. Most of these companies would be considered “diversifying specialty companies,” but they include a number of consulting/engineering firms, particularly in Canada, and some dedicated phytoremediation firms. For the most part, many of these companies are pursuing phytoremediation at the research stage only, but numerous commercial projects have been undertaken in several countries around the world (Glass 2000). Smaller, but emerging, markets exist in developing nations, particularly in portions of Asia. The total world remediation market is reported to be approximately US $15–18 billion per year.

An estimated 52 million hectares, which is more than 16% of the total land area, are affected by some level of soil contamination. The most heavily contaminated areas are found near industrialized regions in northwestern Europe, but many contaminated areas also exit around major cities of Europe (EEA 2003). There could be between 300,000 and 1.5 million of these sites in the EU (EC 2002) present for phytoremediation. While not all are priorities for remediation, it is estimated that EU nations may spend between 59 and 109 billion € (EC 2002) to clean these sites over the next 20–25 years. There is an urgent need for cheap and efficient methods to clean up heavily contaminated industrial areas and this could be achieved by using wild or genetically modified metal accumulator plants. The use of plants provides several striking advantages, compared with conventional methods of soil remediation. It is cheap and, after planting, only marginal costs apply for harvesting and field management. After harvesting biomass is burned, no additional carbon dioxide will be released into atmosphere beyond that which was originally assimilated by plants during growth. As mentioned above, the biomass could be also used for bioenergy production (Schröder et al. 2008) and, in addition, the potential of phytoremediation with energy crops together with the production of biodiesel would be one of the economically feasible methods in the near future.

Acknowledgements

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

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© Springer-Verlag 2008