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Life, 51: 183 – 188, 2001
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IUBMB
Critical Review
Heavy Metal DetoxiŽcation in Plants: Phytochelatin
Biosynthesis and Function
Christopher S. Cobbett
Department of Genetics, University of Melbourne, Australia 3010
WORTH A SECOND LOOK
Our search for reviews for IUBMB Life includes essays appearing in media of more limited circulation that merit exposure to an international readership. One source of excellent
reviews is the Australian Biochemist, the newsletter for the
Australian Society for Biochemistry and Molecular Biology,
Inc. A review on heavy metal detoxiŽ cation in plants was published there in December 2000 (vol. 31, pp. 16 – 19). The author,
Christopher Cobbett, Kindly agreed to provide the version republished here. We thank him and the Society, particularly
the Newsletter Editor, Philip Nageley, for their cooperation,
which will include granting permission to reprint, where material from the original is reproduced in its original form.
—The Editors
Summary
Phytochelatins (PCs) are a family of peptides important in the
detoxiŽ cation of heavy metals such as cadmium in plants and some
microorganisms. PCs are synthesised enzymatically from glutathione. Molecular genetic studies, particularly in the yeast Schizosaccharomyces pombe and in the plant Arabidopsis thaliana, have
identiŽ ed a number of genes important in the biosynthesis or function of PCs. PC-deŽ cient mutants of Arabidopsis have conŽ rmed
both the role of glutathione as the substrate for PC biosynthesis and
the role of PCs themselves in heavy metal detoxiŽ cation in plants.
PC synthase genes have been identiŽ ed in Arabidopsis and other
plant species as well as in a number of animal species, suggesting PCs play a wider role in metal detoxiŽ cation than previously
anticipated. PC synthesis is regulated at a number of levels, most
importantly through the activation of PC synthase by metal ions.
This article reviews recent advances in our understanding of the
biosynthesis and function of PCs in plants and other organisms.
IUBMB Life, 51: 183 – 188, 2001
Received 22 March 2001; accepted 22 March 2001.
Address correspondence to Chris Cobbett, Department of Genetics, University of Melbourne, Australia, 3010. Fax: 61-3-83445138 .
E-mail: [email protected]
Keywords
Arabidopsis; glutathione; heavy metal detoxiŽ cation;
phytochelatins.
INTRODUCTION
Plants have evolved an extensive suite of adaptive responses
to heavy metal toxicity. These include the immobilisation, exclusion, chelation, and compartmentalisation of metal ions and often involve metal-binding ligands (1 ). A number of such ligands
have been identiŽ ed in plants and include organic acids, amino
acids, peptides, and polypeptides (2 ). Polypeptides include the
metallothioneins (MTs), small, gene-encoded, cysteine-rich
polypeptides, and the phytochelatins (PCs), which, in contrast,
are enzymatically synthesised, cysteine-rich peptides.
MTs were Ž rst identiŽ ed as Cd-binding proteins in mammalian tissues and appear to be ubiquitous in animal species.
Thus, early reports of metal-binding proteins in plants generally
assumed them to be MTs. However, in the absence of detailed
characterisation or primary amino acid sequences, many of these
metal-binding complexes may have been comprised, at least in
part, of PCs, particularly where they were identiŽ ed in studies
of plant responses to Cd.
After the structures of PCs had been elucidated and it was
found that these peptides appear to be ubiquitous in the plant
kingdom, it was proposed that PCs were the functional equivalent of MTs (3 ). However, numerous examples of MT-like genes,
and in some cases MT proteins, have now been isolated from
a variety of plant species, and it is apparent that plants express
both of these metal-binding ligands. It is likely that the two play
relatively independent functions in metal detoxiŽ cation and/or
metabolism. PCs have not been reported in an animal species,
supporting the notion that in animals, MTs may well perform
some of the functions normally contributed by PCs in plants.
However, the isolation of the PC synthase gene from plants and
the consequent identiŽ cation of similar genes in animal species,
described next, suggests that, at least in some animal species,
both these mechanisms contribute to metal detoxiŽ cation and/or
metabolism. SigniŽ cant recent advances in our understanding of
183
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COBBETT
Figure 1. Phytochelatin biosynthetic pathway. Abbreviations are: GSH, glutathione; PC, phytochelatin; GCS, ° -glutamylcysteine
synthetase; GS, glutathione synthetase; PCS, phytochelatin synthase. HMT1 is a vacuolar membrane transporter of PC– Cd complexes identiŽ ed in S. pombe. Genes identiŽ ed by corresponding mutants in Arabidopsis are shown in italics.
aspects of PC biosynthesis and function derived predominantly
from molecular genetic approaches using model organisms are
the focus of this review.
Phytochelatins are a Family of Enzymatically Synthesised
Peptides
PCs are a family of peptides described by the formula
(° -GluCys)n -Gly, where n is generally in the range 2 to 5. PCs
were Ž rst identiŽ ed in 1983 in the yeast, Schizosaccharomyces
pombe (where they were called cadystins) (4 ) and have subsequently have been identiŽ ed in a wide variety of plant species and
in some other microorganisms (reviewed in ref. 5 ). Numerous
physiological, biochemical, and genetic studies have conŽ rmed
that the tripeptide glutathione (GSH; ° -GluCysGly) is the substrate for PC biosynthesis. The pathway of PC biosynthesis is
shown in Fig. 1. Although a number of structural variants of
PCs, for example, (° -GluCys)n -¯-Ala, (° -GluCys)n -Ser, and
(° -GluCys)n -Glu have been identiŽ ed in some plant species,
they are assumed to be functionally analogous and synthesised
via essentially similar biochemical pathways (see ref. 2).
PCs are rapidly induced in vivo by a wide range of heavy
metal ions, including both cations such as cadmium, copper,
and mercury and anions such as arsenate. Experiments with
cell cultures demonstrated this induction was not dependent on
translation, indicating PCs were synthesised by constitutively
expressed enzyme. This activity, PC synthase, was Ž rst identiŽ ed by Grill et al. in 1989 (6 ) and has been characterised in a
number of subsequent studies (reviewed in ref. 5 ). The enzyme
is a ° -GluCys dipeptididyl transpeptidase (EC 2.3.2.15) and its
reaction involves the transpeptidation of the ° -GluCys moiety
of GSH onto a second GSH molecule to form PC(n D 2) or onto
a PC molecule to produce a PC(n C 1) oligomer. The enzyme is
active only in the presence of heavy metal ions, but a wide range
of metal ions are effective, re ecting the in vivo observations.
This raises the interesting question of how an enzyme can be
activated in such a relatively nonspeciŽ c manner. Despite numerous attempts in the ensuing years, a PC synthase gene had
not been identiŽ ed.
Arabidopsis as a Model for Studies
of Heavy Metal DetoxiŽcation
Decades of physiological and biochemical studies of plant
responses to heavy metals, particularly Cd, had resulted in only
limited understanding of the mechanisms involved. There had
been, unlike with other organisms, no systematic genetic approach to this problem. Comparisons of naturally derived, heavy
metal-tolerant plants or laboratory-selected, metal-tolerant plant
cell lines with their sensitive counterparts had also failed to identify the underlying mechanisms. In the late 1980s, Arabidopsis
rose to prominence as the pre-eminent model organism for
molecular genetic studies in plants, and we chose Arabidopsis to
undertake an analysis of heavy metal detoxiŽ cation mechanisms.
Our approach was to identify metal-sensitive, particularly
Cd-sensitive, mutants using a somewhat tedious root growth assay described by Howden and Cobbett (7 ). Subsequently, a more
rapid and sophisticated variation of this assay was described by
Murphy and Taiz (8). In parallel with this work in Arabidopsis,
other groups have used S. pombe as a model organism; together,
these molecular genetic approaches have contributed to signiŽ cant advances in our understanding of the biosynthesis and function of PCs. In addition to the isolation and characterisation of
mutants, the expression of plant cDNAs in strains of E. coli and
S. cerevisiae has been particularly useful in the identiŽ cation or
analysis of genes involved in GSH and PC biosynthesis.
Among the Cd-sensitive mutants of Arabidopsis we identiŽ ed, some were partially or entirely deŽ cient in PCs, thus
demonstrating conclusively that PCs play a signiŽ cant role in
the detoxiŽ cation of at least some heavy metals in plants. Complementation analysis demonstrated these mutants identiŽ ed two
different genes. The cad2-1 mutant is partially deŽ cient in both
PCs and GSH (9). GSH-deŽ cient mutants of S. pombe are also
PC-deŽ cient and hypersensitive to Cd. The cad2-1 mutant has
decreased ° -glutamylcysteine synthetase (GCS) activity, the
Ž rst of the two GSH biosynthetic enzymes (Fig. 1). The
Arabidopsis GCS gene was identiŽ ed through the isolation of
cDNAs that could complement a GCS-deŽ cient mutant of E. coli
(10) and the cad2-1 mutant has a small in-frame deletion in that
gene (11). Interestingly, the rootmeristemless1 (rml1) mutant,
the primary phenotype of which is an absence of postembryonic root cell division and development, also has a mutation in
the GCS gene demonstrating a previously undiscovered role for
GSH in root cell division (12).
Mutants at the cad1 locus in Arabidopsis are Cd-sensitive and
PC-deŽ cient but, in contrast to cad2-1 and rml1, have wild-type
levels of GSH (13). Cad1 mutants lack PC synthase activity in
vitro (13). Thus, it was likely that CAD1 was the structural gene
for PC synthase. The Arabidopsis CAD1 gene was isolated by using a positional cloning strategy (14). Simultaneously, the same
PHYTOCHELATINS AND HEAVY METAL DETOXIFICATION
gene (referred to as AtPCS1 ) (15) and a similar gene in wheat
(TaPCS1 ) (16) were identiŽ ed by two other groups through the
ability of cDNAs to confer resistance to Cd when expressed in
the yeast S. cerevisiae. Both of these latter studies used a variety
of yeast mutants to demonstrate the mechanism of Cd-resistance
conferred by these cDNAs was distinct from other recognised
Cd-detoxiŽ cation mechanisms in yeast, was dependent on GSH,
and mediated PC biosynthesis in vivo.
Despite numerous previous screens for Cd-sensitive mutants
in S. pombe, surprisingly no PC synthase mutants had been identiŽ ed among a host of other mutants (see later). Nonetheless, a sequence similar to CAD1 was identiŽ ed in the genome of S. pombe
and targeted deletions of that gene were constructed in two of
the studies referred to previously (14, 16). The resulting mutants
were, like the Arabidopsis cad1 mutants, Cd-sensitive and PCdeŽ cient, conŽ rming the analogous function of the two genes in
the different organisms. Expression of the CAD1(AtPCS1) and
SpPCS genes in E. coli (14) or puriŽ cation of epitope-tagged
derivatives of SpPCS and AtPCS1 expressed in S. cerevisiae
(15, 16), was used to demonstrate both were necessary and sufŽ cient for GSH-dependent, metal ion-activated PC biosynthesis
in vitro.
The Arabidopsis genome contains a second gene, AtPCS2,
with signiŽ cant identity to CAD1/AtPCS1 (14). This was unexpected because PCs were not detected in a cad1 mutant after prolonged exposure to Cd, suggesting the presence of only a single
active PC synthase in the wild-type (13). AtPCS2 is transcribed
and expression experiments have demonstrated it encodes a
functional PC synthase enzyme (unpublished data). The physiological function of this gene remains to be determined. In most
tissues, AtPCS2 is expressed at a relatively low level compared
with AtPCS1. However, because AtPCS2 has been preserved as a
functional PC synthase through evolution, it must presumably be
the predominant PC synthase in some tissue/s or environmental
conditions, thereby conferring a selective advantage.
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Phytochelatins May Be Expressed in Some Animal Species
Previously, PCs had not been detected in animal species and
had been assumed to be a metal response mechanism unique to
plants and some microorganisms. However, database searches
also identiŽ ed similar genes in the nematode, Caenorhabditis
elegans, and the slime mould, Dictyostelium discoideum. In addition, using PCR, similar partial sequences have been identiŽ ed
from the aquatic midge, Chironomus, and earthworm species
(unpublished data). We have recently shown that a cDNA corresponding to the D. discoidium gene is also able to confer
Cd-resistance when expressed in yeast. Further evidence conŽ rming these animal genes also encode proteins with PC synthase activity would suggest that PCs play a wider role in heavy
metal detoxiŽ cation than previously expected. Similar sequences
are absent from the Drosophila melanogaster genome and have
not been identiŽ ed in any vertebrates. A superŽ cial view of the
limited selection of species in which such sequences have been
identiŽ ed might suggest that organisms with an aquatic or soil
habitat are more likely to express PCs.
Interestingly, although PC(n D 2) has been described in the
yeast S. cerevisiae, there is no homologue of the PC synthase
genes in the S. cerevisiae genome. An alternative pathway for
PC biosynthesis in S. pombe has been proposed and it may be
that this pathway also functions in S. cerevisiae. However, the
cad1-3 mutant of Arabidopsis and the PC synthase deletion mutant of S. pombe both lack detectable PCs suggesting that such an
alternative pathway is of little physiological relevance in these
organisms.
PC Synthase Amino Acid Sequence Comparisons
A comparison of the amino acid sequences of the Arabidopsis
and S. pombe enzymes with similar sequences from C. elegans
and D. discoideum shows that the N-terminal regions are very
similar (40 – 50% identical) while the C-terminal sequences show
no apparent conservation of amino acid sequence (Fig. 2). The
Figure 2. Schematic comparison of phytochelatin synthase polypeptides from different organisms. At, A. thaliana (CAD1/
AtPCS1; GenBank accession numbers, AF135155 and AF085230); Sp, S. pombe (SpPCS; Z68144); Ce, C. elegans (CePCS1;
Z66513); Dd, D. discoidium (unpublished data). The total number of amino acids in each is shown on the right. Approximate positions
of all Cys residues are indicated by vertical bars. The conserved N-terminal domains exhibit at least 40% identical amino acids in
pair-wise comparisons of the three sequences. An arrowhead indicates the position of the Arabidopsis cad1-5 nonsense mutation.
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COBBETT
most obvious common feature of the C-terminal regions is the
occurrence of multiple Cys residues, often as pairs. For example, the C-terminal regions of the Arabidopsis and S. pombe
proteins have 10 and 7 Cys residues, respectively, of which 4
and 6, respectively, are as pairs. However, there is no apparent
conservation of the positions of these Cys residues relative to
each other.
Alignment of PCS sequences derived from various plant
genes shows a high degree of conservation of sequence across
the entire proteins. This is true for comparisons between genes
from monocot and dicot plants (16). As with the comparison between more distantly related species described previously, the
N-terminal halves of the proteins are more highly conserved
than the C-terminal halves. Nonetheless, in contrast to the former comparison, the C-terminal domains show 60 – 70% identical amino acids in pairwise comparisons between plant PCS
proteins and the Cys residues are highly conserved.
Regulation of PC Biosynthesis
PC biosynthesis results in a demand for cysteine and GSH.
Thus, one level of regulation observed is the coordinated tran-
scriptional regulation in response to Cd exposure of genes involved in sulfur transport and assimilation and in GSH biosynthesis in Brassica juncea and in Arabidopsis. In Arabidopsis,
the signal molecule, jasmonate, mediated an effect similar to Cd
exposure (17 ). However, whether or not jasmonate is itself the
signal in response to Cd has not yet been demonstrated. There
is also evidence for the posttranscriptional regulation of GCS
expression in addition to the well-established regulation of GCS
activity through feedback inhibition by GSH (18).
Nonetheless, the primary determinant of PC biosynthesis is
expected to be the activity of PC synthase. Kinetic studies using
plant cell cultures exposed to Cd demonstrated PC biosynthesis
occurs within minutes and is independent of de novo protein
synthesis, consistent with the observation of enzyme activation
in vitro. The enzyme appears to be constitutively expressed and
has been detected in both intact plants and plant cell cultures
grown in soil or artiŽ cial medium in the presence of only trace
levels of essential heavy metals (6, 11, 19). The observation that
levels of AtPCS1/CAD1 mRNA are not in uenced by exposure
to a range of heavy metals is consistent with constitutive expression. In contrast, TaPCS1 expression in wheat roots is induced
Figure 3. A schematic model for phytochelatin synthase function. The box represents the conserved N-terminal region of the enzyme with a tail depicting the variable C-terminal containing multiple Cys residues. 1, In the cytoplasm Cd binds with glutathione
(° -GluCysGly) thereby forming the Ž rst of two substrates; 2, Cd-glutathione complexes may bind to the Cys residues in the variable region of the enzyme thereby increasing substrate availability; 3, Gly is cleaved from a molecule of glutathione, the second
substrate, forming a ° -GluCys acyl-enzyme derivative; 4, the ° -GluCys moiety is transferred from the substituted enzyme intermediate to the Cd-glutathione substrate forming, 5, a Cd¢PC [Cd(° -GluCys)n Gly] product; 6, the Cd¢PC product is transported to
the vacuole or; 7, participates as a substrate in a subsequent reaction forming a [Cd(° -GluCys)nC1 Gly] product. (Adapted from 20.)
PHYTOCHELATINS AND HEAVY METAL DETOXIFICATION
on exposure to Cd (16), suggesting that in some organisms regulation of PC synthase activity may involve multiple mechanisms.
Early models for the activation of PC synthase assumed a
direct interaction between metal ions and the enzyme but raised
the question of how the enzyme might be activated by such a
wide range of metals. A signiŽ cant recent study has provided
evidence for an alternative model that provides a solution to this
dilemma (20). Using puriŽ ed recombinant AtPCS1 these authors demonstrate that, in contrast to earlier models of activation,
metal binding to the enzyme per se is not responsible for catalytic activation. The kinetics of PC synthesis are consistent with
a mechanism in which heavy metal glutathione thiolate (e.g.,
Cd¢GS2 ) and free GSH act as ° -Glu-Cys acceptor and donor
(Fig. 3). The observation that S-alkylglutathiones can participate
in PC biosynthesis in the absence of heavy metals is consistent
with a model in which blocked glutathione molecules (metal
thiolates or alkyl substituted) are the substrates for PC biosynthesis. Thus, the role of metal ions in enzyme activation is as an
integral part of the substrate rather than interacting directly with
the enzyme itself. In this way, any metal ions that form thiolate
bonds with GSH have the capacity to activate PC biosynthesis.
There is likely to be a role in enzyme activation for the multiple Cys residues in the variable C-terminal domain. The cad1-5
mutation of Arabidopsis is a nonsense mutation that would result
in premature termination of translation downstream of the conserved domain (14). The truncated polypeptide is predicted to
lack 9 of the 10 Cys residues in the C-terminal domain (Fig. 2).
This mutant enzyme retains the greatest residual activity of all
the cad1 mutants analysed (as measured by in vivo PC levels and
sensitivity to Cd) and the mutant activity is expressed only in the
presence of Cd (13). Thus, it appears that the C-terminal domain
is not absolutely required for either catalysis or activation. Because the truncation of the cad1-5 mutant polypeptide produces
a mutant phenotype, the C-terminal domain clearly has some
role in activity. This domain probably acts to enhance activity
by binding metal ions or metal glutathione complexes bringing
them into closer proximity to the catalytic domain (Fig. 3).
PC–Cd Complexes are Sequestered to the Vacuole
In both plant and yeast species, heavy metals, Cd in particular,
are sequestered to the vacuole. In S. cerevisiae, YCF1 (21) and
in S. pombe, HMT1 (22) encode members of the ABC family of
membrane transporters that transport GSH – Cd and PC – Cd complexes, respectively, into the vacuole and play important roles
in Cd detoxiŽ cation. There is also increasing evidence that vacuolar localisation of heavy metal ions plays an important role
in naturally evolved, heavy metal-tolerant plants (reviewed in
ref. 2). In the vacuole, sulŽ de ions form an essential component
of PC-Cd complexes, and a number of Cd-sensitive mutants believed to be affected in aspects of sulŽ de metabolism have been
identiŽ ed in yeast species. These include a number of adenine
auxotrophs in S. pombe that, in addition to an inability to convert aspartate to intermediates in adenine biosynthesis, are also
unable to utilise cysteine sulŽ nate, a sulfur-containing analog
187
of aspartate, to form other sulfur-containing compounds (23).
Another is the hem2 mutant of C. glabrata that is deŽ cient in
porphobilinogen synthase involved in biosynthesis of siroheme,
which is a cofactor for sulŽ te reductase (24 ).
CONCLUDING REMARKS
Molecular genetic approaches using model organisms have
identiŽ ed some genes important in the PC biosynthetic pathway and others contributing to the subsequent function of PCs.
In particular, the isolation and characterisation of PC synthase
from plants and other organisms has been a signiŽ cant advance.
The concept of using plants in the bioremediation of polluted
environments (phytoremediation ) has gained considerable currency in recent years and is being developed as a viable process
using species that naturally hyperaccumulate particular metal
ions (25). Using model organisms to identify genes with important functions in metal detoxiŽ cation or transport will provide
directions in exploring the basis of metal hyperaccumulation
mechanisms and will contribute to the development and adaptation of phytoremediation as a useful process.
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