Download Phytoremediation of Metals Using Transgenic Plants

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).
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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
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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-
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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
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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)
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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
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(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
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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). In fact, preliminary data suggest that
histidine overproducing plants have enhanced Ni
tolerance (Krämer and Chardonnens, 2001); (5) a
research area that may render a wealth of new
information in the coming years is molecular biology of the rhizosphere. Manipulation of the quality
and quantity of root-released compounds offer a
promising alternative strategy to affect metal uptake or exclusion. Together, these new developments likely will give rise to much new information about metal metabolism in plants in the near
future and may lead to the fruitful applications in
environmental cleanup, nutrition, and crop productivity.
ACKNOWLEDGMENTS
The authors’ work is supported by National
Science Foundation Grant MCB9982432 (E.P.S.),
Environmental Protection Agency Grant G8A11586
(E.P.S.), and National Science Foundation Grant
MCB0091163 (M.P.).
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