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Critical Reviews in Plant Sciences, 21(5):439–456 (2002) Phytoremediation of Metals Using Transgenic Plants Elizabeth Pilon-Smits and Marinus Pilon Colorado State University, Biology Department, A/Z Building, Fort Collins, CO 80523 [email protected] fax: 970-491-0649 Referee: Dr. Clayton Rugh, Michigan State University, Dept. of Crop and Soil Sciences, 516 Plant and Soil Science Bldg. East Lansing, MI 48824 ABSTRACT: An ideal plant for environmental cleanup can be envisioned as one with high biomass production, combined with superior capacity for pollutant tolerance, accumulation, and/or degradation, depending on the type of pollutant and the phytoremediation technology of choice. With the use of genetic engineering, it is feasible to manipulate a plant’s capacity to tolerate, accumulate, and/or metabolize pollutants, and thus to create the ideal plant for environmental cleanup. In this review, we focus on the design and creation of transgenic plants for phytoremediation of metals. Plant properties important for metal phytoremediation are metal tolerance and accumulation, which are determined by metal uptake, root-shoot translocation, intracellular sequestration, chemical modification, and general stress resistance. If we know which molecular mechanisms are involved in these tolerance and accumulation processes, and which genes control these mechanisms, we can manipulate them to our advantage. This review aims to give a succinct overview of plant metal tolerance and accumulation mechanisms, and to identify possible strategies for genetic engineering of plants for metal phytoremediation. An overview is presented of what has been achieved so far regarding the manipulation of plant metal metabolism. In fact, both enhanced metal tolerance and accumulation have been achieved by overproducing metal chelating molecules (citrate, phytochelatins, metallothioneins, phytosiderophores, ferritin) or by the overexpression of metal transporter proteins. Mercury volatilization and tolerance was achieved by introduction of a bacterial pathway. The typical increase in metal accumulation as the result of these genetic engineering approaches is 2- to 3-fold more metal per plant, which could potentially enhance phytoremediation efficiency by the same factor. As for the applicability of these transgenics for environmental cleanup, results from lab and greenhouse studies look promising for several of these transgenics, but field studies will be the ultimate test to establish their phytoremediation potential, their competitiveness, and risks associated with their use. KEY WORDS: environmental cleanup, genetic engineering, biotechnology, cadmium, copper, iron, zinc, nickel. TABLE OF CONTENTS I. II. III. Introduction .................................................................................................................. 440 A. The Problem with Metals .................................................................................... 440 B. Phytoremediation Approaches for Metals .......................................................... 440 C. How to Make Metal Phytoremediation More Efficient? ................................... 441 Genetic Engineering of Plants — What and How ...................................................... 441 Genetic Engineering of Plants for Metal Phytoremediation: Species to Use, Processes to Target....................................................................................................... 442 0735-2689/02/$.50 © 2002 by CRC Press LLC 439 IV. V. Molecular Mechanisms of Metal Accumulation and Tolerance ................................. 443 Genetic Manipulation of Metal Accumulation and Tolerance — State of the Science .......................................................................................................................... 447 A. Metal-Binding Molecules: Metallothioneins, Phytochelatins, Organic Acids, Phytosiderophores, Ferritin...................................................................... 447 B. Membrane Transporters ...................................................................................... 448 C. Metal Metabolism ............................................................................................... 449 D. General Stress-Resistance Mechanisms .............................................................. 449 VI. Putting Transgenics to the Test — Phytoremediation Case Studies .......................... 450 VII. Risk Assessment Considerations ................................................................................. 450 VIII. Perspectives ................................................................................................................... 451 I. INTRODUCTION A. The Problem with Metals Metals are present naturally in the Earth’s crust at various levels (Angelone and Bini, 1992). Mining, industry, and agriculture lead to accelerated release of metals into ecosystems, causing serious environmental problems and posing a threat to human health (Lantzy and Mackenzie, 1979; Nriagu, 1979; Ross, 1994). Although many metals are essential for cells (e.g. Cu, Fe, Mn, Ni, Zn), all metals are toxic at higher concentrations (Marschner, 1995). One reason metals may become toxic is because they may cause oxidative stress. Especially redox active transition metals, which can take up or give off an electron (e.g. Fe2+/3+, Cu+/2+) can give rise to free radicals that cause damage (Jones et al., 1991; Li and Trush, 1993), but other metals can cause oxidative stress as well (Weckx and Clijsters, 1997; Baccouch et al., 1998; Cho and Park, 2000). Another reason why metals may be toxic is because they can replace other essential metals in pigments or enzymes, disrupting the function of these molecules (Rivetta et al., 1997; van Assche and Clijsters, 1986). Some metal ions (e.g. Hg+ and Cu+) are very reactive to thiol groups and can interfere with protein structure and function. When released into the environment, some metals occur as free cations (e.g. Zn2+), while others form cations that are bound to organics (e.g. Cu2+) and yet others form oxyanions (e.g. CrO43–, MoO42–, WO43–). Some metals occur in the environment as radioactive isotopes, posing an additional health risk (e.g. 238U, 137Cs, 239Pt, 90Sr). 440 More than 50,000 metal-contaminated sites await remediation in the U.S. alone (Ensley, 2000). Approximately 80% of U.S. Superfund sites (designated by the U.S. Environmental Protection Agency as priority sites for cleanup) contain heavy metals, often mixed with organic pollutants (Ensley, 2000). Conventional remediation methods for metal-contaminated substrates include soil washing, excavation, and reburial of soil, and pump and treat systems for water (Glass, 1999). The present costs concerned with U.S. remediation are $7 to 8 billion per year, of which ~35% involves metals remediation (Glass, 1999, 2000). The use of plants for remediation of metals offers an attractive alternative, because it is solar driven and can be carried out in situ, minimizing cost and human exposure (Salt et al., 1995b, 1998). Phytoremediation is also aesthetically pleasing and can be used in conjunction with other cleanup methods. The U.S. phytoremediation market for metals is expected to grow from ~$20 million in 2000 to ~$150 million in 2005 (Glass, 1999, 2000). B. Phytoremediation Approaches for Metals Unlike organic contaminants, metals cannot be degraded. Instead, phytoremediation strategies for metals are based on stabilization, accumulation, and in some cases volatilization (EPA, 1998). Phytostabilization of metals may simply involve the prevention of leaching through the upward water flow created by plant transpiration, reduced runoff due to above-ground vegetation, and reduced soil erosion via stabilization of soil by plant roots (Berti and Cunningham, 2000). In some cases of phytostabilization, metals may be transformed to less bioavailable and therefore less toxic forms. For instance, many wetland plants reduce metals to insoluble precipitates on their root surface (Horne, 2000). Thus, in phytostabilization, mobilization of metals is prevented; although metal concentrations are not reduced, the metal becomes less of a risk to the environment. Accumulation of metals by roots in a hydroponic setup, followed by harvesting of the plant biomass is termed rhizofiltration (Dushenkov and Kapulnik, 2000). The accumulation of metals in shoot tissue, followed by harvesting of shoot biomass, is called phytoextraction (Blaylock and Huang, 2000). After harvesting the root and/or shoot biomass, the plant material may be ashed, followed by recycling of the metals if economically feasible (Chaney et al., 2000), or the disposal of the ashes in a landfill. Alternatively, the plant material may be used for non-food purposes, for example, cardboard or wood products. Certain metal(loid)s can be converted by plants into a gaseous form and emitted into the atmosphere (Hansen et al., 1998). The use of plants for volatilization of contaminants is called phytovolatilization. Each of these metal phytoremediation technologies has already been shown to be effective. To give a few examples: a rhizofiltration system uses sunflowers to remove radioactive U from contaminated wastewater to levels below regulatory limits (95% removal in 24 h, Dushenkov and Kapulnik, 2000); chelator-assisted phytoextraction using Brassica juncea lowered soil Pb levels from 2055 mg.kg–1 to 960 mg.kg–1 in three crops (Blaylock, 2000); constructed wetlands routinely remove over 90% of metals from various wastewater streams (Horne, 2000). Phytoextraction, phytovolatilization, phytostabilization, and rhizofiltration are not exclusive technologies. For instance, in a constructed wetland, phytoextraction, phytostabilization, and phyto-volatilization may be used simultaneously (Hansen et al., 1998). C. How to Make Metal Phytoremediation More Efficient? Several approaches may be used to further enhance the efficiency of metal phytoremediation. First, a screening study may be performed to identify the most suitable plant species or varieties for remediation of a certain metal. Second, agronomic practices may be optimized for a selected species to maximize biomass production and metal uptake. For instance, planting density and fertilization can be optimized to enhance plant productivity (Chaney et al., 2000), and amendments such as organic acids or synthetic chelators may be added to the soil to enhance metal uptake (Salt et al., 1998; Blaylock and Huang, 2000). Different plant species may also be combined, spatially or successively, for maximal phytoremediation efficiency (Horne, 2000). Agronomic practices such fertilization and plant clipping may also affect plant metal uptake by influencing microbial density and composition in the root zone. The selected species or variety can be bred further for the desired property, either through classic breeding or via genetic engineering. The advantage of genetic engineering is that it can be much faster than conventional breeding, and it is possible to introduce genes from other species. Thus, it is possible to introduce properties into plants that could not be introduced via conventional breeding. This review focuses on the second approach: the use of genetic engineering to enhance plants’ phytoremediation potential for metals. II. GENETIC ENGINEERING OF PLANTS — WHAT AND HOW In genetic engineering of plants, a foreign piece of DNA is stably inserted into the genome of a cell, which is regenerated into a mature transgenic plant. The piece of DNA can come from any organism, from bacteria to mammals. The foreign DNA usually contains two genes, one a resistance gene used for selection after transformation, the other the gene of interest. Each gene is coupled to a plant promoter, ensuring the formation of the gene product (usually a protein) in 441 the plant. When the transformed plant is propagated, the foreign gene is inherited by its offspring. The foreign stretch of DNA may be transferred to the plant either via a particle gun, for which the DNA is coated onto metal particles and shot into the plant tissue, or via Agrobacterium, a soil bacterium that makes a living by inserting part of its DNA (called T-DNA) into a plant cell and feeding off of the gene products produced by the plant. The Agrobacterium T-DNA genes can be replaced by genes of interest, which are then inserted into the plant by Agrobacterium infection. (Hoekema et al., 1983). For some plant species (e.g. Arabidopsis thaliana, the model plant for plant molecular biology), transformation simply involves dipping the flowers in an Agrobacterium suspension: some of the resulting seeds will be transgenic (Bechtold et al., 1998). Most plants need to be grown as undifferentiated callus tissue culture in order to be transformed. After the transformation, mature plants are regenerated from the tissue culture using shootinducing plant hormones (Horsch et al., 1985). The gene product can be targeted to certain cellular compartments (e.g. chloroplast, vacuole, mitochondrion, or apoplast) by adding specific targeting information in the gene construct. Often, constitutive promoters such as the 35S-cauliflower mosaic virus promoter are used, that direct expression in all tissues and at all times. However, the expression pattern of the gene may also be programmed to be only in certain tissue types (e.g. roots, vascular tissue, shoot), or under certain environmental conditions (stress-induced, light-induced), by means of different promoters (Kasuga et al., 1999; Su et al., 1998). Besides overexpressing a gene, it is also possible to repress the expression of an endogenous gene, by inserting a copy of that gene in reverse orientation (antisense technology). III. GENETIC ENGINEERING OF PLANTS FOR METAL PHYTOREMEDIATION A. Species to Use The ideal plant species to engineer for phytoremediation purposes is one with a high bio- 442 mass production, sufficiently hardy and competitive in the climate where it is to be used, and with a good phytoremediation capacity to start with. Ideally, there should be an existing transformation protocol for the species, because the development of such a protocol may take a year or more. Some examples of suitable species are Indian mustard (Brassica juncea), poplar (Populus spp.), yellow poplar (Liriodendron tulipifera), and cordgrass (Spartina spp.). In addition to, or prior to, transforming these large biomass phytoremediation species, the same gene construct may be transformed to the model plant species, A. thaliana. This small plant with its short generation time and high seed production is very suitable to test in a short time whether a biotechnological approach works. B. Processes to Target To breed plants with superior phytoremediation potential, one possible strategy is to enhance the biomass productivity of species that are good accumulators; another strategy is to enhance metal tolerance and/or accumulation in high biomass species. General plant productivity is controlled by many genes and difficult to promote by single gene insertion. Therefore, this is difficult to achieve in the short term by means of genetic engineering. To enhance plant metal tolerance and/or accumulation, an existing plant process that is limiting for remediation may be accelerated, for example, a general plant mechanism involved in metal accumulation or tolerance; alternatively, a new pathway may be introduced into the plant from any other organism, for example, a bacterial pathway for metal detoxification. To accelerate existing plant processes that limit phytoremediation of an element (e.g. root uptake, root-shoot translocation, sequestration in specific tissues or cell compartments, biotransformation, plant-microbe interactions), we need to know which pathways and genes are involved. The overexpression of a gene encoding a ratelimiting gene product is expected to lead to a faster overall rate of the pathway and thus to more efficient phytoremediation. Conversely, antisense repression of a rate-limiting gene product will lead to a slower flux through a pathway. Table 1 gives an overview of the factors that are important and may be limiting for different metal phytoremediation applications. Molecules involved in the processes listed above include metal transporter membrane proteins, metal chelating molecules of various sorts, metal-modifying enzymes, enzymes involved in repair of metal damage (e.g. oxidative stress), and regulatory proteins. All of these molecules are produced as the result of the presence and expression of genes. Any (combination) of these genes may be suitable targets for plant genetic engineering for phytoremediation. In the next section, a short overview is given of molecules and genes involved in metal uptake, translocation, sequestration, and tolerance. IV. MOLECULAR MECHANISMS OF METAL ACCUMULATION AND TOLERANCE A. Uptake For metal ions to be taken up by plants, they have to be bioavailable. Roots compete with soil particle cation/anion exchange sites for ions (Figure 1). Especially in soils with high clay and organic matter content metal bioavailability is low (Ross, 1994; Marschner, 1995). Plants have evolved mechanisms to make micronutrient metals more bioavailable. For instance, many plants excrete organic acids (e.g. malate, citrate) that act as metal chelators and decrease the rhizosphere pH, making metal cations more bioavailable (Ross, 1994). Organic acids have been reported to facilitate metal uptake, for example, citrate enhances U uptake in Brassica (Huang et al., 1998). On the other hand, organic acids can also inhibit metal uptake by forming a complex with it outside the root that is not taken up, for example, citrate inhibits Al uptake in various species (de la Fuente et al., 1997; Moffat, 1999; Pineros and Kochian, 2001; Papernik et al., 2001); a similar mechanism appears to be responsible for Cu tolerance in Arabidopsis (Murphy et al., 1999). Plants can also affect their rhizosphere pH via proton pumps in the root cell membrane. For instance, one proton pump, an H+-ATPase en- coded by gene AHA2, is upregulated under iron deficiency, leading to enhanced proton efflux from the root (Fox and Guerinot, 1998). Also, an Arabidopsis mutant was found to have an increased rhizosphere pH, caused by enhanced proton influx (Moffat, 1999). This resulted in the precipitation of Al hydroxides, and therefore reduced Al uptake and higher Al tolerance. Another type of exudate produced by grasses are phytosiderophores, which bind Fe and facilitate its uptake. Phytosiderophores are biosynthesized from nicotianamine, which is composed of three methionines coupled via non-peptide bonds (Higuchi et al., 1994). Rhizosphere microbes can also affect plant uptake of metals: bacteria have been reported to enhance uptake of Se and Hg (de Souza et al., 1999), and mycorrhizae were reported to reduce metal uptake, leading to enhanced tolerance (Frey et al., 2000; Rufyikiri et al., 2000). Likely, certain plant genes are involved in these plant-microbe interactions, for example, in production of signal molecules. The uptake of metals requires transport across the root cell membrane into the symplast. This process involves specific membrane transporter proteins. Membrane transport of cations has been the subject of several recent reviews (Fox and Guerinot, 1988, Williams et al., 2000; Mäser et al., 2001; Axelsen and Palmgren 2001). The genome of the model species A. thaliana encodes for over 150 different cation transporters in at least nine different families. The abundance of genes implied in metal transport in A. thaliana as well as all other organisms underscores the need for metal homeostasis in which organisms must maintain a fine balance between having enough essential metals available for metabolic functions and at the same time avoiding deficiency or toxicity. Membranes serve to separate compartments in which metal concentrations can be regulated with the aid of transporters (Nelson, 1999). The uptake of metal ions into the cell can be driven by the electrochemical gradient (proton gradient) across the plasma membrane, but the energetics of transport are not yet fully understood in all cases (Mäser et al., 2001). Often more than one transport system exists for one metal. For example, A. thaliana has several transporters of the NRAMP family capable of transporting iron into 443 FIGURE 1. Schematic overview of processes and molecules involved in uptake, translocation, and sequestration of metals. Circled arrows represent membrane transporters. Metals are represented by small dots. TABLE 1 Important Factors for Different Metal Phytoremediation Applications Note: the importance of biomass is incorporated in shoot accumulation (=shoot conc. x biomass) and in tolerance (=growth) 444 cells; in addition, the ZIP family member IRT and perhaps several members of the 8-member YSL family are also implied in iron uptake into cells (Mäser et al., 2001; Curie et al., 2001). The presence of several transporters permits having uptake systems with different affinities and capacities. In addition, transporters are present in internal membranes to allow to regulate the storage of metals in organelles such as vacuoles. Transporters may be specific for a certain cell type. Of interest to phytoremediation is the observation that some metal transporters can transport more than one metal ion. For instance, the IRT protein mediates iron uptake in roots but also transports Cd. B. Translocation For root-shoot translocation of metals, metal transporters export metal ions out of the root symplast into the xylem apoplast (Marschner, 1995). Translocation in the xylem is probably transpiration driven (Salt et al., 1995a). Different chelators may be involved in translocation of metal cations through the xylem (Figure 1), such as organic acid chelators (e.g. malate, citrate, histidine, Salt et al., 1995a; von Wiren et al., 1999), or nicotianamine (Stephan et al., 1996; von Wiren et al., 1999). Uptake of metal ions from the xylem apoplast into the shoot symplast is mediated by metal transporters in the shoot cell membrane. For translocation of metals in the phloem, nicotianamine may function as a chelator (von Wiren et al., 1999). C. Sequestration Once inside the shoot cells, essential metals will be translocated to their final destination, which may involve membrane metal transporters, and metal-binding proteins. One class of metal chelating molecules that may play a role in sequestration — they are upregulated under conditions of high metal availability— are the metallothioneins (MTs). Metallothioneins are small (~7 kDa) cysteine-rich metal-binding proteins that occur in all organisms. Although the exact role of MTs is still not clear, they likely play a role in homeostasis of essential metals and perhaps also in tolerance to nonessential metals (Goldsbrough, 2000; Cobbett and Goldsbrough, 2000). Metal chaperones are a different class of proteins that bring metals to specific targets in the cell. An example is the ATX protein, which is upregulated under Cu deficiency (Himelblau et al., 1998). Toxic levels of essential or nonessential metals are stored in a location where the metal can do the least harm to vital cellular processes. This may involve storage in special cellular compartments such as the vacuole by means of specialized transporters such as ZAT1, a CDF-type transporter for zinc (van der Zaal et al., 1999). Sequestration may also be in the apoplast, or in specialized cell types, such as epidermal cells and trichomes (Heath et al., 1997; Küpper et al., 1999; Salt and Krämer, 2000; Hale et al., 2001). For storage in the vacuole, certain metals may be complexed by phytochelatins (PCs). Phytochelatins are small cysteine-rich metal-binding peptides (5 to 23 amino acids) that occur in all plants tested so far (Rauser, 1995; Zenk, 1996; Cobbett, 2000), as well as in some fungi and animals (Vatamaniuk et al., 2001). Phytochelatins are induced only under metal stress and are thought to mainly function in tolerance to toxic metals (Goldsbrough, 2000; Cobbett and Goldsbrough, 2000). They are synthesized enzymatically from glutathione. Complexes of metals bound by glutathione or phytochelatins are shuttled to the vacuole by an ABC-type transporter protein in the tonoplast (Lu et al., 1997). The same type of transporter is involved in shuttling glutathione-conjugated anthocyanins to the vacuole (Marrs, 1996). In fact, anthocyanins can also bind metals (Kondo et al., 1992; Everest and Hall, 1921; Takeda et al., 1985), and recently have been suggested to play a role in metal sequestration (Hale et al., 2001). Other metalbinding molecules that are involved in metal complexation in the vacuole are organic acids (Krämer et al., 2000). Excess iron, in contrast to other metals, is stored in chloroplasts, bound to the protein ferritin (Theil, 1987). D. Chemical Modification Metal-modifying enzymes may be involved in assimilation of metals into organic molecules 445 (e.g. selenate is metabolized to dimethylselenide; de Souza et al., 2000), or in changing the oxidation state of metals (e.g. toxic Cr(VI) is reduced to nontoxic Cr(III), Lytle et al., 1998, and in dicots Fe (and possibly also Cu) is reduced by a reductase at the root cell membrane before uptake; Robinson et al., 1999). E. Hyperaccumulation Mechanisms Metal hyperaccumulators are commonly defined as plant species that accumulate ~100-fold higher metal levels than nonaccumulator species (Brooks, 1998), for example, 1% of DW for Mn and Zn, 0.1% of DW for Cu and Ni, and 0.01% of DW for Cd (Baker et al., 2000). Metal hyperaccumulation has been found in over 500 species from over 75 families (Baker et al., 2000) and has probably evolved independently in these different taxa (Wu, 1990; Pollard et al., 2000). The evolutionary selection pressure for hyperaccumulation may be protection against herbivory (Pollard and Baker, 1997) and pathogens (Boyd et al., 1994). Hyperaccumulators are usually slow growing, low biomass species. They hyperaccumulate metals even from low external metal concentrations, and most of the metal is translocated to the shoot (Salt and Krämer, 2000). For a comprehensive review of hyperaccumulation mechanisms see Salt and Krämer (2000). Following is a summary of findings to date. At the root membrane level, metal uptake is unusually high in hyperaccumulators. This may be due to constitutive high expression of a metal transporter in the plasma membrane, as was found for the Zn and Cd hyperaccumulator Thlaspi caerulescens (Pence et al., 2000; Lombi et al., 2001). The uptake of metals in hyperaccumulators may be enhanced further by the excretion of metal chelators like histidine (Krämer et al., 1996), and/or by rhizosphere microbes capable of mobilizing nonlabile soil metals (Whiting et al., 2001). Thlaspi caerulescens shows reduced metal accumulation in root vacuoles, enhanced root-shoot translocation, enhanced uptake into leaf cells, and higher metal tolerance (Lasat et al., 2000). The high metal tolerance may in part be due to highly efficient intracellular compartmentalization. The Ni hyperaccumulator 446 T. goesingense was shown recently to have a vacuolar metal transporter, TgMTP1, which is constitutively highly expressed compared to orthologues in nonaccumulators (Persans et al., 2001). Efficient chelation is likely to be another key factor for metal tolerance and accumulation in hyperaccumulators. Different chelator molecules may function in different plant parts. In the Ni hyperaccumulator Alyssum lesbiacum, Ni chelation by histidine appears to play an important role in Ni translocation in the xylem (Krämer et al., 1996). In the Ni hyperaccumulator T. goesingense, histidine does not seem to be a key compound for Ni accumulation (Persans et al., 1999); Ni appears to be predominantly localized in the vacuole as a Ni-organic acid complex in this species (Krämer et al., 2000). In the Zn hyperaccumulator T. caerulescens, zinc chelation appears to be mainly by histidine in roots, by organic acids — or no chelator — in the xylem, and by organic acids in shoots (Salt et al., 1999). F. General Stress Resistance Mechanisms One reason why metals are toxic is because they cause oxidative stress. Metal stress activates antioxidative systems, composed of free radical scavenging molecules such as ascorbate and glutathione, and the enzymes involved in their biosynthesis and reduction (Noctor and Foyer, 1998). Other molecules involved in preventing oxidative stress are the superoxide dismutase enzymes, which themselves require either Cu/Zn, Mn, or Fe as cofactors (Bowler et al., 1994). The overproduction of any of these components may lead to higher metal stress tolerance. Alternatively, if regulatory genes can be identified that orchestrate the activation of many metal-induced genes, overexpression of such a regulatory gene may be the most efficient way to enhance metal tolerance. Recently, an irondependent cis-regulatory element was identified in maize that mediates repression of ferritin genes under low iron conditions (Petit et al., 2001). Also, transcription factors that mediate salt, drought, and freezing tolerance have been identified (Su et al., 1998; Kasuga et al., 1999). V. GENETIC MANIPULATION OF METAL ACCUMULATION AND TOLERANCE – STATE OF THE SCIENCE As is clear from the above, many genes are involved in metal uptake, translocation, sequestration, chemical modification, and tolerance. The overexpression of any (combination) of these genes is a possible strategy for genetic engineering. Depending on which phytoremediation application is to be used, the genetic engineering strategy may strive to create plants that accumulate more metals in harvestable plant parts (phytoextraction), or adsorb more metals at their root surface (rhizofiltration, phytostabilization). A plant property essential for all phytoremediation applications is plant tolerance (Table 1), so enhancing plant metal tolerance is an obvious avenue for genetic engineering approaches. Enhanced tolerance to metals may be achieved by reducing metal uptake, by more efficient sequestration of metals in plant storage compartments, overproduction of metal chelating molecules, or increasing activity of enzymes involved in general (oxidative) stress resistance. The overexpression of metal transporter genes may lead to enhanced metal uptake, translocation, and/or sequestration, depending on the tissues where the gene is expressed (root, shoot, vascular tissue, or all), and on the intracellular targeting (e.g. cell membrane, vacuolar membrane). The overexpression of genes involved in synthesis of metal chelators may lead to enhanced or reduced metal uptake, as well as enhanced metal translocation and/or sequestration, depending on the type of chelator and its location. Unless regulatory genes are identified that simultaneously induce many metal-related genes, it is feasible that more than one gene will need to be upregulated in order to substantially enhance metal phytoremediation capacity. To our knowledge, this has not been done so far (in one transformation event) in the context of manipulating plant metal metabolism. Encouraging for transgenic approaches, classic genetic studies indicate that there are usually very few genes (1 to 3) responsible for metal tolerance (MacNair et al., 2000). Also, metal accumulation, tolerance, and plant productivity are not necessarily correlated (Wu, 1990; MacNair et al., 2000). Therefore, it should be possible to breed or genetically engineer a plant with high metal tolerance and metal accumulation as well as high productivity. This would be the ideal plant for metal phytoextraction. In the next section we give an overview of what has been achieved so far with respect to genetic manipulation of plant metal tolerance and accumulation. A. Metal-Binding Molecules: Metallothioneins, Phytochelatins, Organic Acids, Phytosiderophores, Ferritin Overproduction of various metal chelator molecules has been shown to affect plant metal tolerance and accumulation. Several research groups have overexpressed the metal-chelating proteins metallothioneins (MTs). The expression of the human MT2 gene in tobacco or oil seed rape resulted in higher Cd tolerance at the seedling level (Misra and Gedamu, 1989). Similarly, the expression of the mouse MT1 gene in tobacco led to enhanced Cd tolerance at the seedling level (Pan et al., 1994). The overexpression of a pea MT in A. thaliana resulted in a severalfold higher Cu accumulation (Evans et al., 1992). The most pronounced effect of MT overexpression was observed by Hasegawa et al. (1997), who overexpressed the yeast gene CUP1 in cauliflower, leading to a 16-fold higher Cd tolerance, as well as higher Cd accumulation. Thus, it appears that the overexpression of MTs is a promising approach to enhance Cd/Cu tolerance and accumulation. In a different approach to enhance metal tolerance and accumulation, the metal-binding peptides phytochelatins (PCs) were overproduced via expression of enzymes involved in their biosynthesis. Transgenic mustard (Brassica juncea) plants with higher levels of glutathione and phytochelatins were created through the overexpression of either of two glutathione synthesizing enzymes,— γ-glutamylcysteine synthetase (γECS) or glutathione synthetase (GS). Both types of transgenics showed enhanced Cd tolerance and accumulation (Zhu et al., 1999a,b), illustrating the importance of these metal-bind- 447 ing peptides for metal tolerance and accumulation. In a related study, γECS was overexpressed or knocked out (antisense approach) in Arabidopsis, leading to increased or decreased GSH levels (Xiang et al., 2001). Transgenics with decreased GSH levels showed reduced Cd tolerance, confirming the importance of GSH and PCs for Cd tolerance. However, plants with increased GSH levels did not show enhanced Cd tolerance, suggesting that GSH production is not limiting for PC production and Cd tolerance in this species. Harada et al. (2001) also created transgenic plants with enhanced phytochelatin levels, through overexpression of cysteine synthase. The resulting transgenics displayed enhanced Cd tolerance but lower Cd concentrations. To our knowledge, there are no published records concerned with the overexpression of either phytochelatin synthase or a tonoplast PCmetal transporter. However, the overexpression of a tobacco glutathione-S-transferase gene (parB) in Arabidopsis was reported to lead to enhanced Cu, Al, and Na tolerance (Ezaki et al., 2000). Glutathione-S-transferases mediate glutathione conjugation, followed by transport of the resulting complex to the vacuole (Marrs, 1996). Enhanced production of the metal chelator citric acid was achieved by the overexpression of citrate synthase (de la Fuente et al., 1997). The resulting CS transgenics were shown to have enhanced Al tolerance, apparently via extracellular complexation of Al by citrate after excretion from root cells. The same CS transgenics take up more phosphorus (Lopez-Bucio et al., 2000) and are more resistant to iron deficiency (Guerinot, 2001), illustrating that citrate excretion can affect the uptake of different elements in different ways. As citrate amendment has been shown to enhance U uptake (Huang et al., 1998), it would be interesting to test these CS transgenics for U uptake. The overproduction of the iron-chelator deoxymugineic acid (phytosiderophores) was achieved through the overexpression of nicotianamine aminotransferase (NAAT) in rice (Takahashi et al., 2001). The resulting plants released more phytosiderophores and grew better on iron-deficient soils. Iron levels in the plants were not determined. 448 The overexpression of the iron-binding protein ferritin was shown to lead to a 1.3-fold higher iron level in tobacco leaves (Goto et al., 1998) and a three-fold higher level in rice seeds (Goto et al., 1999). B. Membrane Transporters The genetic manipulation of several metal transporters has been shown to result in altered metal tolerance and/or accumulation. The overexpression of the Zn transporter ZAT (also known as AtMTP1) in A. thaliana gave rise to plants with enhanced Zn resistance and two-fold higher root Zn accumulation (van der Zaal et al., 1999). ZAT is a putative vacuolar transporter and of the same gene family as the TgMTP1 isolated from the hyperaccumulator T. goesingense (Persans et al., 2001). The overexpression of the calcium vacuolar transporter CAX2 from A. thaliana in tobacco resulted in enhanced accumulation of Ca, Cd, and Mn, and to higher Mn tolerance (Hirschi et al., 2000). Another vacuolar transporter, AtMHX, was overexpressed in tobacco (Shaul et al., 1999). The resulting plants showed reduced tolerance to Mg and Zn, but it did not show altered accumulation of these elements. Another putative metal transporter gene from tobacco (NtCBP4), encoding a calmodulin-binding protein, when overexpressed resulted in enhanced Ni tolerance and reduced Ni accumulation, as well as reduced Pb tolerance and enhanced Pb accumulation (Arazi et al., 1999). When a truncated form of the protein was overexpressed, however, from which the calmodulin-binding part was removed, the resulting transgenics showed enhanced Pb tolerance and attenuated accumulation (Sunkar et al., 2000). In order to enhance iron uptake by plants, two yeast genes encoding ferric reductase (FRE1 and FRE2, involved in iron uptake) were overexpressed in tobacco (Samuelsen et al., 1998). Iron content in the shoot of the transgenics was 1.5-fold higher compared with wild-type plants. Earlier, enhanced accumulation of various metals (Fe, Cu, Mn, Zn, Mg) was already observed in an Arabidopsis mutant with enhanced ferric-chelate reductase activity (Delhaize, 1996). The affected gene in the Arabidopsis mutant meanwhile has been identified as FRO2 and isolated (Robinson et al., 1999); it will be interesting to see what effect its overexpression has on plant metal uptake. The overexpression of another metal transporter, AtNramp1, resulted in an increase in Fe tolerance (Curie et al., 2000), while the overexpression of AtNramp3 led to reduced Cd tolerance but no difference in Cd accumulation (Thomine et al., 2000). In addition to overexpressing metal transporters, it is also possible to alter their metal specificity. For instance, while IRT1, the Arabidopsis iron transporter, can transport Fe, Zn, Mn, and Cd, the substitution of one amino acid was shown to result in loss of either Fe and Mn transport capacity, or Zn transport capacity (Rogers et al., 2000). With the overexpression of such engineered transporters, it may be possible to tailor transgenic plants to accumulate specific metals. C. Metal Metabolism Rather than accelerating existing processes in plants, an alternative approach is to introduce an entirely new pathway from another organism. This approach was taken by Richard Meagher and coworkers, who introduced two bacterial genes in plants that together convert methylmercury to volatile elemental mercury. MerB encodes organomercurial lyase, which converts methylmercury to ionic mercury or Hg(II); MerA encodes mercuric reductase, which reduces ionic mercury to elemental mercury or Hg(0) (Summers, 1986). Transgenic MerA A. thaliana plants showed significantly higher tolerance to Hg(II) and volatilized elemental mercury (Rugh et al., 1996). Transgenic MerB A. thaliana plants were significantly more tolerant to methylmercury and other organomercurials (Bizily et al., 1999). The MerB plants were shown to convert methylmercury to ionic mercury, a form that is ~100-fold less toxic to plants. MerA-MerB double-transgenics, obtained by crossing MerA and MerB transgenics, were compared with their MerA, MerB, and wild-type counterparts with respect to tolerance to organic mercury (Bizily et al., 2000). While MerB plants were 10-fold more tolerant to organic mercury than wild-type plants, MerA-MerB plants were 50-fold more tolerant. When supplied with organic mercury, MerA-MerB double transgenics volatilized elemental mercury, whereas single transgenics and wild-type plants did not; thus, MerA-MerB plants were able to convert organic mercury all the way to elemental mercury, which was released in volatile form. The same MerA/MerB gene constructs were used to create mercury-volatilizing plants from other species. Transgenic MerA and MerB tobacco and yellow poplar also showed enhanced mercury tolerance (Rugh et al., 2000). In an initial experiment to analyze the potential of these plants for phytoremediation, MerA tobacco plants removed 3- to 4-fold more mercury from hydroponic medium than untransformed controls (Meagher et al., 2000). To our knowledge, no reports have been published at this point involving the expression of metal hyperaccumulator genes in nonaccumulator species. However, an alternative approach has been used to transfer hyperaccumulation capacity to a nonaccumulator high biomass species. Brewer et al. (1999) used somatic hybridization (protoplast electrofusion) to create a hybrid between Thlaspi caerulescens and Brassica napus. Some of the hybrids showed high biomass combined with high metal tolerance and accumulation, making them attractive for metal phytoextraction. A different way of using genetic engineering to study metal metabolism is by creating hairy root cultures of plants using Agrobacterium rhizogenes. The resulting fast growing root culture can be grown in vitro indefinitely. Hairy root culture of Thlaspi caerulescens was shown to be more tolerant to Cd, and accumulated 1.5- to 1.7fold more Cd than hairy roots of nonaccumulator species (Nedelkoska and Doran, 2000). Agrobacterium rhizogenes infection may also be used to bring about root proliferation, and thus to increase the root surface area of a plant. The use of such plants may be attractive for rhizofiltration applications. D. General Oxidative Stress Resistance Mechanisms Overexpression of enzymes involved in general stress resistance mechanisms present an alter449 native approach to bring about metal tolerance. Several studies using this approach have led to promising results. Ezaki et al. (2000) reported that the overexpression of several genes involved in oxidative stress response (glutathione-S-transferase, peroxidase) resulted in enhanced Al tolerance. Oberschall et al. (2000) overexpressed an aldose/ aldehyde reductase responsible for detoxifying a lipid peroxide degradation product and found that the transgenics were more metal tolerant. The overexpression of glutathione reductase resulted in reduced Cd accumulation and enhanced Cd tolerance, as judged from chlorophyll content and chlorophyll fluorescence measurements (Pilon-Smits et al., 2000). Grichko et al. (2000) found that the overexpression of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase led to an enhanced accumulation of a variety of metals, as well as higher metal tolerance. ACC is the precursor for ethylene, the plant hormone involved in senescence. VI. PUTTING TRANSGENICS TO THE TEST — PHYTOREMEDIATION CASE STUDIES As listed above, several different strategies have been used successfully to create transgenics that show promising properties for phytoremediation, as judged from lab studies involving hydroponic systems or agar media. The next step is to test these transgenics on more realistic contaminated substrates, collected from the environment. So far, to our knowledge, no results from such studies have been published. Recently, a greenhouse study was performed to test transgenic phytochelatin-overproducing mustard plants (ECS/GS plants, see above) for their phytoextraction capacity, using metalcontaminated soil from Leadville, Colorado (Bennett et al., 2002). Both types of transgenics accumulated significantly higher levels of Cd and Zn in their shoots than untransformed plants. These results are encouraging because it suggests that metal accumulation results obtained from hydroponic studies are a valuable indication of transgenics’ metal accumulation potential from environmental soils containing metal mixtures. 450 VII. RISK ASSESSMENT CONSIDERATIONS Transgenic plants with altered metal tolerance, accumulation, or transformation properties are valuable for various reasons. They shed new light on basic biological mechanisms involved in these processes: which pathways are involved and which enzymes are rate limiting. Plants with altered metal accumulation properties may also be applicable, not only for phytoremediation but also to enhance crop productivity in areas with suboptimal soil metal levels, or as “fortified foods” for humans or livestock (Guerinot and Salt, 2001). When genetically engineered plants are used for any of these applications, a thorough risk assessment study should be performed in each case (Wolfenbarger and Phifer, 2000). Some of the possible risks involved are biological transformation of metals into forms that are more bioavailable, enhanced exposure of wildlife and humans to metals (in the case of enhanced metal accumulation in palatable plant parts, or volatilization), uncontrolled spread of the transgenic plants due to higher fitness (e.g. metal tolerance) or general weedy nature, and/or uncontrolled spread of the transgene by interbreeding with populations of wild relatives (for a comprehensive report on this topic, see Glass, 1997). These risks will have to be assessed on a case-by-case basis and weighed against the benefits, and against the risks of doing nothing or using alternative technologies. To our knowledge, no transgenics are used in the field for phytoremediation at this point. Therefore, the actual risks involved with the use of transgenic plants for phytoremediation have never been tested. However, theoretical calculations of risks associated with the use of mercuryvolatilizating plants have been done by Meagher and coworkers (Meagher et al., 2000; Rugh et al., 2000). According to their calculations, the mercury emitted by these plants would pose no significant threat to the environment and would be negligible compared with other sources of mercury, such as burning of fossil fuels and medical waste. Even if the level of volatile mercury at the phytoremediation site is 400-fold higher than background levels, that would still be 25 times below regulatory limits. In addition, the retention time of elemental mercury in the atmosphere, before precipitation, is 1 to 2 years during which the mercury is diluted to nontoxic levels. Norman Terry and coworkers have done a similar theoretical analysis of the risk of volatile Se emitted by plants (see Berken et al., this issue, and Lin et al., 2000), and came to the conclusion that the volatile Se will likely be beneficial rather than toxic, as it is likely to precipitate in Sedeficient areas. Metal accumulation in plant shoots brings along the risk of wildlife ingestion, and any increase in metal accumulation via biotechnology will lead to a proportional increase of this risk. On the other hand, if a site can be cleaned in a shorter time, the duration of exposure may be reduced when using transgenics. The risk of metal ingestion by wildlife may be minimized by fencing off the area, using deterrents such as periodic noise, and the use of less palatable plant species. The risk of transgenic plants or their genes “escaping” is not considered a significant problem by Meagher et al. (2000), because they generally offer little or no advantage over untransformed plants, either in a pristine or a contaminated environment. However, before using specific transgenics for phytoremediation in the field, this could be verified by a greenhouse or pilot field experiment, analyzing transgenic gene frequency over a number of generations, on polluted and uncontaminated soil. To further minimize the risk of outcrossing to wild relatives, transgenic plant species may be chosen that have no compatible wild relatives, male-sterile transgenics may be bred, and/or the plants may be harvested before flowering. Government regulation should not be a significant obstacle to the use of transgenic plants in phytoremediation in the U.S. (Glass, 1997). Permit applications should be filed to the U.S.D.A. approximately 4 months before the planned starting date, but in most jurisdictions approval for research field tests should be routine. VIII. PERSPECTIVES It has been shown in multiple studies that plant trace element metabolism can be genetically manipulated, leading to plants with altered metal tolerance, accumulation, and/or biotransformation capacity. When natural plant processes were accelerated by genetic engineering, the typical increase in metal accumulation per plant was 2- to 3-fold. This would potentially reduce the cost of phytoremediation to the same extent, if the same results hold true in the field. Furthermore, the introduction of a new pathway has led to plants that can detoxify Hg in ways that other plants cannot — this is potentially even more valuable. In the coming years some of these newly available transgenics will likely be put to the test in a more realistic phytoremediation setting. As more metal-related genes are discovered, facilitated by the genome sequencing projects, many new possibilities will open up for the creation of new transgenics with favorable properties for phytoremediation. In addition to constitutive overexpression of one gene, several genes may be overexpressed simultaneously, and the overexpression may be fine-tuned in specific tissues, under specific conditions, or in specific cellular compartments. Some promising strategies may be (1) the many newly discovered metal transporters, including the ones from hyperaccumulator plants (ZNT1, TgMTP1), may be overexpressed in high biomass plant species, targeted to different tissues and intracellular locations; (2) nicotianamine overproduction may be an interesting avenue to manipulate metal translocation and tolerance, as well as iron uptake in cereals, NA being the precursor of phytosiderophores. Overproduction of NA is feasible via overexpression of enzymes from the NA biosynthesis pathway, the genes for which have been cloned (Herbik et al., 1999; Higuchi et al., 1999; Ling et al., 1999; Takahashi et al., 1999); (3) overexpression of phytochelatin synthase (PS), the enzyme mediating PC synthesis from GSH, may further enhance metal tolerance and accumulation. The overexpression of PS is possible, because genes encoding PS have been cloned (Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et al., 1999). The overexpression of the vacuolar transporter responsible for shuttling the PC-metal complex into the vacuole may also enhance metal tolerance and accumulation; this too is possible because the A. thaliana gene encoding this transporter has been cloned (Lu et al., 1997); (4) overproduction of 451 histidine can be done because the genes involved in His biosynthesis have been cloned (Persans et al., 1999). 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