Genetic engineering in the improvement of plants for ...


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Genetic engineering in the improvement of plants for
phytoremediation of metal polluted soils
,D.van der Lelie
Department of Biochemistry and Biotechnology,University of Kuopio,PO Box 1627,FIN-70211,Finland
Department of Ecology and Ecotoxicology,Faculty of Biology,Free University of Amsterdam,De Boelelaan 1087,1081 HV Amsterdam,Netherlands
Limburgs Universitaire Centrum,Universitaire Campus,B-3590 Diepenbeek,Belgium
Vlaamse Instelling voor Technologisch Onderzoek (VITO),Boeretang 200,B-2400 Mol,Belgium
Received 29 August 1998;accepted 22 May 1999
``Capsule'':Plants can be used for phyto-extraction of heavy metals from polluted soil,with the possibility that genetic engineering may
increase ecacy.
Metal concentrations in soils are locally quite high,and are still increasing due to many human activities,leading to elevated risk
for health and the environment.Phytoremediation may o￿er a viable solution to this problem,and the approach is gaining
increasing interest.Improvement of plants by genetic engineering, modifying characteristics like metal uptake,transport and
accumulation as well as metal tolerance,opens up new possibilities for phytoremediation.So far,only a few cases have been
reported where one or more of these characteristics have been successfully altered;e.g.mercuric ion reduction causing improved
resistance and phytoextraction,and metallothionein causing enhanced cadmium tolerance.These,together with other approaches
and potentially promising genes for transformation of target plants are discussed.#2000 Elsevier Science Ltd.All rights reserved.
Metals are cycling at low rates within and between
bio-,geo-,atmos- and hydrospheric systems.Local
metal enrichments may result either fromnatural sources,
or from human activities,such as smelting,mining,pro-
cessing,agricultural and waste disposal technologies.Due
to technological advancements,metal releases by indus-
trial activities have strongly reduced;by consequence
metal concentrations in the air have signi®cantly
decreased.However,still accumulating metals in soils
are leading to higher risks due to leaching into ground
and surface water,uptake by plants and direct or indir-
ect intake by human population.When present at
increased levels of bioavailability,both essential (Cu,
Zn,Mn,Fe,Ni,Mo) and non-essential metals (e.g.Cd,
Pb,Hg,Cr) are toxic.
Microorganisms and plants possess a variety of mech-
anisms to prevent heavy metal poisoning.Examples are
active metal eux (particularly in eubacteria),synthesis
of metal-binding peptides like metallothioneins (MTs);
in blue-green algae,fungi and plants) and phytochelatins
(in plants and some fungi),vacuolar sequestration (in
fungi and plants),and several others,including extra-
cellular precipitation or chelation of free metal ions
(Macnair,1993;Silver,1996;Silver and Phung,1996).
Strains or ecotypes in strongly metal-enriched environ-
ments have usually evolved exceptionally high levels of
heavy metal tolerance (Baker and Brooks,1989).Adaptive
tolerance has been explored mainly in bacteria.It
usually relies on the presence of plasmid-encoded eux
systems (Tsai et al.,1992;Silver,1996;Silver and
Phung,1996),or on metal reductase activity (Cervantes
and Silver,1992;Misra,1992).The mechanisms of
tolerance in plants and fungi are largely unknown.In
the case of Zn and Cd,there is circumstantial evidence
of increased vacuolar transport (Ortiz et al.,1995;
Verkleij et al.,1998).
0269-7491/00/$ - see front matter#2000 Elsevier Science Ltd.All rights reserved.
PII:S0269- 7491( 99) 00141- 4
Environmental Pollution 107 (2000) 225±231
* Corresponding author.
E-mail address:sirpa.karenlampi@uku.® (S.Ka
Because of the adverse e￿ects of increased metal con-
centrations on most living organisms,techniques have
been developed to remediate contaminated soils.Current
remediation methods applicable to soils contaminated
with heavy metals are expensive,environmentally invasive,
and labor intensive.A remediation technique that is of
low cost,but protecting human health and the environ-
ment,would be a valuable addition to current remedia-
tion methods.Phytoremediation techniques,i.e.use of
green plants to remove,contain or render harmless
environmental contaminants,have gained an increasing
interest during the past few years.The term includes
several techniques,such as phytostabilization and phy-
toextraction.In phytostabilization,soil amendments
and plants are used to alter the chemical and physical
state of the heavy metal contaminants in the environ-
ment.A plant cover e￿ectively prevents contaminant
spread by minimizing wind erosion and surface run-o￿,
as well as by reducing percolation to the ground water.
Plants may also be used to remove contaminants from
soil by phytoextraction and then harvested for proces-
sing (Cunningham et al.,1995;Salt et al.,1995,1998;
Chaney et al.,1997).There are some promising results
suggesting that these techniques might become viable
alternatives to mechanical and chemical approaches in
remediation of metal contaminated soils.
Improvement of plants by genetic engineering opens up
new possibilities for phytoremediation of metal-polluted
soils.However,this approach can be fully exploited
only when the mechanisms of metal tolerance,accumu-
lation and translocation are better understood.
2.Ideal plant for phytoremediation
Populations of metal-tolerant,hyperaccumulating
plants can be found in naturally occuring metal-rich
sites (Baker and Brooks,1989).However,these plants
are not ideal for phytoremediation since they are usually
small and have a low biomass production.In contrast,
plants with good growth usually show low metal accumu-
lation capability as well as lowtolerance to heavy metals.
A plant suitable for phytoremediation should possess
the following characteristics:(1) ability to accumulate
the metal(s) intended to be extracted,preferably in the
above ground parts (plants which do not translocate
metals to the above-ground parts could be useful for
phytostabilization and landscape recreation);(2) toler-
ance to the metal concentrations accumulated;(3) fast
growth and highly`e￿ective'(i.e.metal accumulating)
biomass;and (4) easily harvestable.Chaney et al.(1997)
calculated that metal tolerance and hyperaccumulation
would be more important to phytoremediation than
high biomass production.
According to Chaney et al.(1997),for an e￿ective
development of phytoremediation,each element must
be considered separately because of its unique soil and
plant chemistry.On the other hand,metals rarely occur
alone and adaptive tolerance may be needed for several
metals simultaneously,even though phytoextraction of
only one metal would be the goal.In some cases it might
be desirable also to extract more than one metal at the
same time.
3.Genetically engineered plants with altered metal
tolerance or uptake
There are few published articles describing altered
metal tolerance or uptake in plants modi®ed with for-
eign genes (Table 1).They are discussed here in further
3.1.Mercuric ion reduction and resistance,
Bacteria can reduce a number of heavy metals to less
toxic states.Mercury resistance in Gram-negative bac-
teria is encoded by an operon,which includes ®ve to six
genes,among them a mercuric ion reductase gene
(merA).MerA is a soluble NADPH-dependent,FAD-
containing disul®de oxidoreductase.This enzyme con-
verts toxic Hg
to the less toxic metallic mercury
).Escherichia coli cells expressing merA gene were
shown to possess in addition to reduction of Hg
weak reduction activity toward Au
and Ag
mers and Sugarman,1974).The merA gene also weakly
increased Hg
tolerance of Saccharomyces cerevisiae
(Rensing et al.,1992).These studies suggested that merA
gene might a￿ect metal tolerance when expressed in
plant.Initial attempts to express the bacterial merA
gene from Tn21 in plants to produce Hg
were unsuccessful in spite of the use of very ecient
plant expression systems.No full-length merA RNA or
merA-encoded protein were detected.The original bac-
terial merA sequence is rich in CpG dinucleotide having
a highly skewed codon usage,both of which are par-
ticularly unfavorable to ecient expression in plants,
because they are exposed to methylation and sub-
sequent gene silencing (Rugh et al.,1998).Rugh et al.
(1996),therefore,constructed a mutagenized merA
sequence (merApe9),modifying 9%of the coding region
and transformed it to Arabidopsis thaliana.The seeds
germinated and the seedlings grew on medium contain-
ing up to 100 mM Hg,although the transgenic plants
expressed only low levels of merA mRNA.Transgenic
seedlings evolved two to three times the amount of Hg
compared to control plants.Plants were also resistant to
toxic levels of Au
.The paper of Rugh et al.(1996)
gives a good example of a successful modi®cation of a
bacterial metal tolerance gene for expression in plants.
Recently,Rugh et al.(1998) reported on the development
226 S.Ka
renlampi et al./Environmental Pollution 107 (2000) 225±231
of transgenic yellow poplar (Liriodendron tulipifera) for
mercury phytoremediation using merA gene (merA18)
modi®ed even further to optimize the codon usage in the
plant.Transgenic plants evolved 10 times the amount of
compared to control plants.So far,this system has
not been tested in ®eld conditions.This is,however,the
®rst clear indication that genetic engineering may
improve a plant's capacity to phytoremediate metal-
polluted soils.
3.2.MTs and Cd tolerance
MTs and phytochelatins in plants contain a high per-
centage of cysteine sulfhydryl groups,which bind and
sequester heavy metal ions in very stable complexes.
Phytochelatins bind Cu and Cd with high anity and
are induced by various metals (Rauser,1990;Ow,1993).
Phytochelatins may play a role in plant Cd tolerance.
Howden and Cobbett (1992) have isolated Arabidopsis
mutants with increased sensitivity to Cd while Cu toler-
ance was almost unchanged (Howden et al.,1995a,b).
These cad1-mutants were de®cient in PC synthesis and
showed greatly reduced levels of PC synthase activity.
MTs bind to Cu,Cd and Zn;they probably regulate
intracellular Zn concentrations and detoxify normally
lethal concentrations of Cd and Cu (Murphy et al.,
1997).MTs have the highest anity to Cu,and MT
expression correlates with Cu but not Cd tolerance in
Arabidopsis (Murphy and Taiz,1995).Various MT
genes Ðmouse MTI,human MTIA (alpha domain),
human MTII,Chinese hamster MTII,yeast CUP1,pea
PsMTAÐhave been transferred to Nicotiana sp.,Bras-
sica sp.or A.thaliana (Lefebvre et al.,1987;Maiti et al.,
1988,1989,1991;Misra and Gedamu,1989;Evans et
al.,1992;Yeargan et al.,1992;Brandle et al.,1993;Pan
et al.,1993,1994a,b;Elmayan and Tepfer,1994;Hattori
et al.,1994;Hasegawa et al.,1997).As a result,varying
degrees of constitutively enhanced Cd tolerance have
been achieved,being maximally 20-fold compared with
the control.Metal uptake was not markedly altered;in
some cases there were no di￿erences,in others maxi-
mally 70% less or 60% more Cd was taken up by the
shoots or leaves.Only one study has been reported on
a transgenic plant generated with MT of plant origin.
When pea (Pisum sativum) MT-like gene PsMTA was
expressed in A.thaliana,more Cu (several-fold in some
plants) accumulated in the roots of transformed than
of control plants (Evans et al.,1992).We have recently
isolated an MT gene from metal-tolerant Silene vul-
garis and transferred it into several metal-sensitive
yeasts (Tervahauta et al.,unpublished).Increases in
both Cd and Cu tolerance were observed in the mod-
i®ed yeasts.These studies suggest that MT gene may be
useful in improving metal tolerance of plants.How-
ever,it does not seem to have signi®cant e￿ects on
uptake or translocation of metals.
3.3.Ferric reductases and increased iron uptake
Because soil contains mainly insoluble Fe(III) oxides
and hydroxides,plants have adaptive mechanisms to
make Fe more available for uptake.Proton extrusion is
achieved by activation of an ATPase-driven proton
pump.This promotes Fe(III) solubility and reduction to
Fe(II) by plasma membrane-bound reductases.Fe(III)
reductases are activated under Fe de®ciency (Samuelsen et
al.,1998).FRO2 gene encoding a ferric-chelate reductase
has been isolated fromFe-de®cient roots of A.thaliana.It
belongs to a superfamily of ¯avocytochromes,which
transport electrons across membranes.FRO2 consists of
intramembranous binding sites for heme and cyto-
plasmic binding sites for NAPDH and FAD cofactors
that donate and transfer electrons (Robinson et al.,
1999).FRO2 gene was capable of restoring ferric-chelate
Table 1
Altered metal tolerance/uptake in transgenic plants
Gene Origin Host E￿ect
Metallothionein:Human Nicotiana tabacum Enhanced Cd tolerance (max 20 )
hMTI (human),Mouse Brassica sp.No major changes in metal uptake
MTIA (human) Chinese hamster Arabidopsis thaliana
MT-I (mouse) Pisum sativum
MTII (Chinese hamster) Yeast
PsMTA (Pisum sativum)
CUP1 (yeast)
Mercuric ion reductase:Shigella Arabidopsis thaliana Hg/Au resistance;increased Hg
merApe9,merA18 Liriodendron tulipifera evolution (max 10)
Fe(III) reductase Arabidopsis thaliana Arabidopsis thaliana Fe(III) reductase activity restored in de®cient mutant
FRO2 Saccharomyces cerevisiae Nicotiana tabacum Elevated Fe(III) reduction,Fe uptake increased
Ferritin Glycine max Oryza sativa Enhanced Fe uptake in seeds (3)
renlampi et al./Environmental Pollution 107 (2000) 225±231 227
reductase activity in an Arabidopsis mutant de®cient in
this enzyme (frd1) (Robinson et al.,1999).The same gene
also restored a mutant (frd1-1) with de®cient copper-
chelate reduction.Two Fe(III) reductases,FRE1 and
FRE2,have been isolated from S.cerevisiae (Dancis et
al.,1990;Georgatsou and Alexandraki,1994).Samuelsen
et al.(1998) transferred these genes together and
separately into tobacco and studied Fe(III) reductase
activity and Fe accumulation under Fe-sucient and
Fe-de®cient conditions in transgenic plants.In Fe-
de®cient conditions,FRE2 and double transformants
(FRE1 + FRE2) were more tolerant having higher Fe
concentrations in young leaves than the control and
FRE1 plants.When a normal Fe concentration was
present in the growth environment,Fe(III) reduction
was increased 4-fold in roots of double transformants
compared to control,and also the Fe content of leaves
was increased in the double transformants and FRE2
plants compared to controls and FRE1 plants.These
genes may be useful for generating crops with improved
nutritional quality and increased growth in Fe-de®cient
soils.Under suitable promoter the system may be uti-
lized to produce plants for phytoremediation of soils
polluted with more toxic metals such as Cu.
3.4.Ferritin and increased Fe uptake
Ferritin is an iron storage protein found in animals,
plants and bacteria.It comprises 24 subunits,which
may surround in a micellar up to 4500 ferric atoms
(Theil,1987).It provides iron for the synthesis of iron
proteins such as ferredoxin and cytochromes.It also
prevents damage from free radicals produced by iron/
dioxygen interactions.Ferritin has been found to pro-
vide an iron source for treatment of anemia in rat
(Beard et al.,1996).It was thus proposed that increase
of the ferritin content of cereals by genetic modi®cation
may help to solve the problemof dietary iron de®ciency.
To increase the Fe content of rice,Goto et al.(1999)
transferred soybean ferritin gene into the plant.Using
the rice seed storage protein glutelin promoter (GluB-1),
they could target the expression of ferritin in developing
seeds.The Fe content in transformed seeds was three-
fold compared to that in control seeds.
4.Potential genes to be transferred to improve metal
tolerance and/or accumulation
Several technical factors restrain the use of genetic
engineering of plants for phytoremediation.One of
the major factors is that there are only a few plant
systems of metal resistance and/or sequestration that
are suciently characterized to be used for this pur-
pose.The various systems of metal resistance and accu-
mulation are better known in microorganisms (Silver,
1996;Silver and Phung,1996),and the ®rst examples
of their potential use in phytoremediation of metal-
contaminated soils are emerging.Taking a gene from a
bacterium and transferring it into a plant is complicated the fact that the resistance is normally encoded
by a large plasmid containing an operon with many
genes involved in the resistance mechanism.So far,only
a single gene from such an operon has been transferred
(Rugh et al.,1998).In the following,some candidate
genes are discussed along with strategies for generating
more metal-tolerant/accumulator plants using genetic
engineering (Fig.1).Generally,each metal requires
speci®c molecular mechanism(s) for an ecient hyper-
accumulation and tolerance to make plants suitable for
Plants accumulating metals up to several percent of
their biomass (hyperaccumulators) should be good
sources for genes suitable for phytoremediation.Hun-
dreds of such plants have been found,including Thlaspi
caerulescens and Brassica juncea.Some of these hyper-
accumulator plants have been used for phytoremedia-
tion without genetic engineering.However,studies on
B.juncea showed that metal bioavailability was a major
problem in Pb extraction.EDTA was thus added in soil
to increase Pb availability (Blaylock et al.,1997);this
may,however,pose a risk to the environment.If the
metal availability could be locally improved by increasing
reductase activity or the amount of chelating agents,e.g.
phytosiderophores (Briat and Lobre
harmful e￿ects on the environment,these hyper-
accumulators might be used safely for phytoremedia-
4.1.Study the organism at high or low metal exposure
There is considerable literature about the induction of
various proteins by metals.The modern proteome and
DNA array technologies may be applied for searching
candidate genes/proteins for phytoremediation;some of
the proteins induced under metal stress may play a role
in metal tolerance or accumulation.However,there are
not many examples of a proven correlation between
protein induction and metal tolerance.MT gene
expression has been found to correlate with copper
tolerance in A.thaliana (Murphy and Taiz,1995).
Recently,Xiang and Oliver (1998) showed increased
transcription of the genes for glutathione synthesis,g-
glutamylcysteine synthetase and glutathione synthetase
as well as glutahione reductase by Cd and Cu.Gluta-
thione plays a pivotal role in protecting plants from Cu
and Cd.
Organic acids form complexes with metals.Ernst
(1976) observed high malate concentrations in Zn and
Cu tolerant plants;also the content of citrate was
increased.It has been proposed that metal tolerance could
be based on the complex formation.Hyperaccumulators
228 S.Ka
renlampi et al./Environmental Pollution 107 (2000) 225±231
are heavily loaded with these acids and acid anions
might have some function in metal storage or plant
internal metal transport.Free histidine has been found
as a metal chelator in xylem exudates in plants that
accumulate Ni and the amount of free histidine increases
in Ni exposure (Kra
mer et al.,1996).By modifying his-
tidine metabolism it might be possible to increase the
Ni-accumulating capacity of plants.
During the past few years several metal transporters
have been isolated from Arabidopsis:Zn transporters
ZIP1,3,4 (Grotz et al.,1998),Fe transporter IRT1
(Eide et al.,1996),and Cu transporter COPT1 (Kamp-
fenkel et al.,1995).Several transporters like ZIP1,ZIP3
and IRT1 are expressed in response to metal de®ciency.
IRT1 may also play a role in the uptake of other metals,
because Cd,Zn,Co and Mn inhibited Fe uptake of
IRT1 (Eide et al.,1996).Changing the regulation of the
expression of these transporters may modify the uptake
of metals to the cells or organelles in a useful way.
4.2.Candidate genes from metal-resistant and -sensitive
plant mutants
Recently,several mutants with altered response to
heavy metals have been isolated from A.thaliana.Cad-
mium-hypersensitive mutants with defect in phyto-
chelatin synthetase,and possibly in g-glutamylcysteine
synthetase and glutathione synthetase have been isolated
by Howden et al.(1995a,b).Chen and Goldsbrough
(1994) found an increased activity of g-glutamylcysteine
synthetase in tomato cells selected for cadmium toler-
ance.Some of these genes may prove useful in modifying
suitable target plants for phytoremediation,although
there are doubts about the usefulness of genes involved
in phytochelatin synthesis (De Knecht et al.,1992).
Further screening for hypertolerant and/or hyper-
accumulating mutants could be rewarding.Over-
expression of proteins involved in intracellular metal
sequestration (MTs,phytochelatin synthase,vacuolar
transporters) may signi®cantly increase metal tolerance,
but may not be useful for metal accumulation.These
proteins presumably improve accumulation only by
delaying the metal-responsive transcriptional down-reg-
ulation of plasma membrane transporter expression.
Substantially enhanced accumulation may only be
achieved by overexpression of plasma membrane trans-
porters put under the control of nonmetal-responsive,
or positively instead of negatively metal-responsive
promoters.Since tolerance and accumulation are largely
independent properties ( Thlaspi),they should
both be engineered to get a suitable plant for phytor-
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