Plant Physiol, July 2000, Vol. 123, pp. 825-832
UPDATE ON HEAVY METAL STRESS
Phytochelatins and Their Roles in Heavy Metal
Detoxification
Christopher S.
Cobbett*
Department of Genetics, University of Melbourne, Parkville,
Victoria, 3052, Australia
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INTRODUCTION |
Plants respond to heavy metal
toxicity in a variety of different ways. Such responses include
immobilization, exclusion, chelation and compartmentalization of the
metal ions, and the expression of more general stress response
mechanisms such as ethylene and stress proteins. These mechanisms have
been reviewed comprehensively by Sanita di Toppi and Gabbrielli (1999)
for plants exposed to Cd, the heavy metal for which there have been
arguably the greatest number and most wide-ranging studies over many
decades. Understanding the molecular and genetic basis for these
mechanisms will be an important aspect of developing plants as agents
for the phytoremediation of contaminated sites (Salt et al., 1998). One
recurrent general mechanism for heavy metal detoxification in plants
and other organisms is the chelation of the metal by a ligand and, in
some cases, the subsequent compartmentalization of the ligand-metal complex.
A number of metal-binding ligands have now been recognized in plants.
The roles of several ligands have been reviewed by Rauser (1999).
Extracellular chelation by organic acids, such as citrate and malate,
is important in mechanisms of aluminum tolerance. For example, malate
efflux from root apices is stimulated by exposure to aluminum and is
correlated with aluminum tolerance in wheat (Delhaize and Ryan, 1995).
Some aluminum-resistant mutants of Arabidopsis also have increased
organic acid efflux from roots (Larsen et al., 1998). Organic acids and
some amino acids, particularly His, also have roles in the chelation of
metal ions both within cells and in xylem sap (Kramer et al., 1996;
Rauser, 1999).
Peptide ligands include the metallothioneins (MTs), small gene-encoded,
Cys-rich polypeptides. Our current understanding of the functions and
expression of MTs in plants, particularly Arabidopsis, have been
reviewed elsewhere (Fordham-Skelton et al., 1998; Rauser, 1999). In
contrast, the phytochelatins (PCs), the subject of this Update, are enzymatically synthesized Cys-rich peptides. The
most recent review of PC structure, biosynthesis, and function in this journal was by Rauser (1995). Other more recent reviews are by Zenk
(1996) and Rauser (1999). Recent advances in our understanding of
aspects of PC biosynthesis and function are derived predominantly from
molecular genetic approaches using model organisms.
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PLANTS MAKE TWO TYPES OF PEPTIDE METAL-BINDING LIGANDS:
MTs AND PCs |
The historical context of the identification of MTs and PCs in
plants is worth discussing. MTs were first identified as Cd-binding proteins in mammalian tissues. Similar proteins have been identified in
a large number of animal species (Kägi, 1991). Early reports of
metal-binding proteins in plants were generally assumed to be MTs.
However, in the absence of detailed characterization 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
identified in studies of plant responses to Cd.
After the structures of PCs had been elucidated and it was found that
these peptides are distributed widely in the plant kingdom, it was
proposed that PCs were the functional equivalent of MTs (Grill et al.,
1987). Subsequently, numerous examples of MT-like genes, and in some
cases MT proteins, have been isolated from a variety of plant species
and it is now apparent that plants express both of these Cys-containing
metal-binding ligands. Furthermore, it is likely that the two play
relatively independent functions in metal detoxification and/or
metabolism. However, the extent to which this is true is not yet clear
and will not become apparent until a complete set of MT-deficient
mutants have been identified in Arabidopsis. 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 identification of similar genes in animal species,
described below, suggests that, at least in some animal species, both
of these mechanisms contribute to metal detoxification and/or
metabolism. Although PCs have also been classified as class III MTs and
the use of the term "phyto" to designate these compounds appears to be increasingly inaccurate, I believe "phytochelatins" has become so entrenched in the literature that a more broadly encompassing designation is likely to cause only confusion.
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PCs HAVE THE GENERAL STRUCTURE
( -Glu-Cys)n-Gly |
Early analyses demonstrated PCs consisted of only the three amino
acids: Glu, Cys and Gly with the Glu, and Cys residues linked through a
-carboxylamide bond. PCs form a family of structures with increasing
repetitions of the -Glu-Cys dipeptide followed by a terminal Gly;
(-Glu-Cys)n-Gly, where n has been
reported as being as high as 11, but is generally in the range of 2 to 5. PCs have been identified in a wide variety of plant species and in
some microorganisms. They are structurally related to glutathione (GSH;
-Glu-Cys-Gly) and were presumed to be the products of a biosynthetic
pathway. In addition, a number of structural variants, for example,
(-Glu-Cys)n-
-Ala,
(-Glu-Cys)n-Ser, and
(-Glu-Cys)n-Glu, have been identified in some
plant species (Rauser, 1995, 1999; Zenk, 1996).
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PCs ARE SYNTHESIZED FROM GSH |
Numerous physiological, biochemical, and genetic studies have
confirmed that GSH (or, in some cases, related compounds) is the
substrate for PC biosynthesis (Rauser, 1995, 1999; Zenk, 1996). Such
studies have used a variety of plant species, sometimes as intact
plants but often in the form of in vitro cell cultures. Early studies
with cell cultures demonstrated that induction of PCs in the presence
of Cd coincided with a transient decrease in levels of GSH.
Furthermore, the exposure of either cell cultures or intact plants to
an inhibitor of GSH biosynthesis, buthionine sulfoximine, conferred
increased sensitivity to Cd with a corresponding inhibition of PC
biosynthesis. This could be reversed by the addition of GSH to the
growth medium.
By far the most detailed characterization of the pathway of PC
biosynthesis has come from studies in the fission yeast
(Schizosaccharomyces pombe), and in Arabidopsis. Genetic
studies have confirmed GSH-deficient mutants of the fission yeast and
Arabidopsis are also PC deficient and hypersensitive to Cd. In
particular, the cad2-1 mutant of Arabidopsis is partially
deficient in GSH and in -glutamyl-Cys synthetase (GCS) activity, the
first of the two GSH biosynthetic enzymes. The cad2-1
mutation is a 6-bp deletion within an exon of the GCS gene affecting
residues in the vicinity of the presumed active site of the enzyme
(Cobbett et al., 1998). The PC biosynthetic pathway is illustrated in
Figure 1.
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Figure 1.
Genes and functions contributing to Cd
detoxification in plants and fungi. The figure is a composite of
different functions described in different organisms. Enzyme
abbreviations are shown in bold. GS, GSH synthetase; PCS,
Phytochelatin synthase. Gene loci are shown in bold italics.
CAD1 and CAD2 are in Arabidopsis;
hmt1, hmt2, ade2, ade6,
ade7, and ade8 are in fission yeast; and
hem2 is in Candida glabrata. The details of
sulfide metabolism on the right of the figure are not well
understood.
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PC SYNTHASE IS ACTIVATED BY HEAVY METAL IONS |
Grill et al. (1989) first identified an enzyme activity from
cultured cells of Silene cucubalis that synthesized PCs from GSH by transferring a -Glu-Cys moiety from a donor to an acceptor molecule. The reaction involved the transpeptidation of the -Glu-Cys moiety of GSH onto initially a second GSH molecule to form
PC2 or, in later stages of the incubation, onto a
PC molecule to produce an n + 1 oligomer. This -Glu-Cys
dipeptididyl transpeptidase (EC 2.3.2.15) has been named PC synthase.
The enzyme is a 95,000-Mr tetramer with a
Km of 6.7 mM for GSH. In vitro the
activity of the partially purified enzyme was active only in the
presence of metal ions. The best activator tested was Cd followed by
Ag, Bi, Pb, Zn, Cu, Hg, and Au cations. These metals also
induce PC biosynthesis in vivo in plant cell cultures. In in vitro
reactions, PC biosynthesis continued until the activating metal ions
were chelated either by the PCs formed or by the addition of a metal chelator such as EDTA (Loeffler et al., 1989). This provides a mechanism to autoregulate the biosynthesis of PCs in which the product
of the reaction chelates the activating metal, thereby terminating the reaction.
Similar PC synthase activities have been detected in pea (Klapheck et
al., 1995), tomato (Chen et al., 1997), and Arabidopsis (Howden et al.,
1995). Crude enzyme preparations from the roots of pea, which normally
contain both GSH and homo-GSH (-Glu-Cys--Ala), could use GSH
efficiently, but homo-GSH or -Glu-Cys-Ser less efficiently, as
substrates for PC synthesis. In the presence of both GSH and
homo-GSH, synthesis of homo-PCs was enhanced (Klapheck et al., 1995).
These observations were interpreted to indicate the enzyme had a
-Glu-Cys donor site that was relatively specific for GSH but a less
specific acceptor site able to use each of the three substrates. Little
is known about the tissue specificity of PC synthase expression and/or
PC biosynthesis. In the only study of tissue-specific PC synthase
expression to date, the activity was detected in the roots and stems of
tomato plants but not in leaves or fruits (Chen et al., 1997).
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PC SYNTHASE GENES WERE ISOLATED INDEPENDENTLY IN THREE
LABORATORIES |
Despite the identification and purification of PC synthase a
decade ago, the isolation of the corresponding gene and a consequent detailed understanding of the mechanism of PC biosynthesis have eluded
us until recently. PC synthase genes have been isolated simultaneously
by three research groups using different approaches. Two groups used
expression of plant genes in Brewer's yeast (Saccharomyces cerevisiae) to identify genes involved in Cd resistance.
One group identified an Arabidopsis cDNA (AtPCS1) that
suppressed the Cd-sensitive phenotype of both Brewer's yeast
yap1 and ycf1 mutants (Vatamaniuk et al., 1999).
YAP1 encodes a transcription factor that is required for the
expression of YCF1, a transporter responsible for the vacuolar
sequestration of GSH-Cd complexes (Li et al., 1997). This group was
searching for functional plant homologs of YAP1 by using a two-step
screening procedure to identify Arabidopsis cDNAs that could suppress a
yap1, but not a ycf1, mutant. In this screen they
fortuitously identified AtPCS1. By expressing
AtPCS1 in various mutant strains, they demonstrated that the
mechanism of AtPCS1-mediated Cd tolerance functioned in both
yap1 and ycf1 mutants, and in the MT-deficient
mutant, cup1. Thus the mechanism appeared to be distinct
from other recognized Cd-detoxification mechanisms. Expression of
AtPCS1 mediated an increase in Cd accumulation indicating it
was probably involved in chelation or sequestration. It also functioned
in a vacuole-deficient mutant (pep5) demonstrating the
AtPCS1 gene product was not a vacuolar membrane transport function similar to YCF1, for example. Consistent with the cDNA encoding PC synthase, the mechanism was not expressed in a
GSH-deficient mutant (gsh2) and mediated PC biosynthesis in
vivo in Brewer's yeast.
A second group identified a wheat cDNA (TaPCS1) that
conferred increased Cd resistance when expressed in a wild-type (for Cd
tolerance) strain of Brewer's yeast (Clemens et al., 1999). Similar to
AtPCS1, the Cd resistance mediated by TaPCS1 was
associated with an increase in Cd accumulation, functioned in a
vacuole-deficient mutant (vps18) and was GSH dependent,
being diminished in the presence of an inhibitor of GSH biosynthesis.
TaPCS1 also mediated PC biosynthesis in vivo in Brewer's yeast.
AtPCS1 has also been isolated through the cloning of the
CAD1 gene of Arabidopsis. Mutants at the cad1
locus in Arabidopsis, like the cad2-1 mutant, are Cd
sensitive and deficient in the formation of Cd-binding complexes and in
PC biosynthesis. In particular, PCs are undetectable in the
cad1-3 mutant after prolonged exposure to Cd. In contrast to
the cad2-1 mutant, the cad1 mutants have wild-type levels of GSH, suggesting a defect in PC synthase. Consistent with this, PC synthase activity in crude extracts from cad1
mutants was less than 1% of the level in both the wild-type and the
cad2-1 mutant (Howden et al., 1995). Thus it was likely that
CAD1 is the structural gene for PC synthase (Fig. 1). The
CAD1 gene has been isolated using a positional cloning
strategy (Ha et al., 1999). The position of the gene was mapped using
molecular markers and a candidate gene identified from the Arabidopsis
genome initiative genomic sequence. That this candidate was
CAD1 was confirmed by the identification of a different
mutation in that gene in each of the cad1 mutants and by
complementation of the Cd-sensitive phenotype of cad1-3 by a
genomic clone spanning this gene.
A similar sequence (SpPCS) was identified in the genome of
fission yeast and targeted deletions of that gene were constructed in
two of these studies (Clemens et al., 1999; Ha et al., 1999). The
resulting mutants were, like the Arabidopsis cad1 mutants, Cd sensitive and PC deficient, confirming the analogous function of the
two genes in the different organisms. Expression of the CAD1(AtPCS1) and SpPCS genes in
Escherichia coli (Ha et al., 1999) or purification of
epitope-tagged derivatives of SpPCS (Clemens et al., 1999) and AtPCS1
(Vatamaniuk et al., 1999) expressed in Brewer's yeast was used to
demonstrate that both were necessary and sufficient for GSH-dependent,
metal ion-activated PC biosynthesis in vitro.
The Arabidopsis CAD1(AtPCS1) cDNA encodes
a predicted 55-kD polypeptide of 485 amino acids. A comparison of the
Arabidopsis and fission yeast amino acid sequences showed that the
N-terminal regions of the two are very similar (45% identical),
whereas the C-terminal sequences show little apparent conservation of
amino acid sequence (Fig. 2). The most
apparent common feature of the C-terminal regions is the occurrence of
multiple Cys residues, often as pairs. The C-terminal regions of the
Arabidopsis and fission yeast proteins have 10 and seven Cys residues,
respectively, of which four and six, respectively, are as pairs.
However, there is no apparent conservation of the positions of these
Cys residues relative to each other. The two plant PC synthase
sequences, TaPCS and AtPCS, can be aligned across their entire length
(55% identity) (Clemens et al., 1999). The former contains 14 Cys
residues, including two pairs, in the C-terminal domain.
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Figure 2.
Schematic comparison of PC synthase polypeptides
from different organisms. At, Arabidopsis (CAD1/AtPCS1; GenBank
accession nos. AF135155 and AF085230); Ta, Triticum aestivum
(TaPCS1; accession no. AF093252); Sp, fission yeast (SpPCS; accession
no. Z68144); Ce, Caenorhabditis elegans (CePCS1; accession
no. Z66513). The total number of amino acids in each is
shown on the right. Approximate positions of all Cys residues are
indicated by vertical bars; adjacent Cys residues are connected by a
horizontal bar; and Cys residues conserved across the three sequences
are highlighted with an asterisk. The conserved N-terminal domains
exhibit at least 40% identical amino acids in pair-wise comparisons of
the four sequences. An arrowhead indicates the position of the
Arabidopsis cad1-5 nonsense mutation.
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PC BIOSYNTHESIS MAY BE REGULATED IN SEVERAL WAYS |
There are a number of mechanisms by which the PC
biosynthetic pathway may be regulated. The first of these is likely to
be regulation of GSH biosynthesis. Studies of transgenic Indian mustard (Brassica juncea) plants, in which the expression of the
enzymes of the GSH biosynthetic pathway was increased have shown that PC biosynthesis and Cd tolerance can also be increased (Yong et al.,
1999; Zhu et al., 1999). This supports the idea that regulation of GSH
biosynthesis is a plausible endogenous mechanism by which PC expression
might be modulated. In support of this is the observation that a
Cd-tolerant tomato cell line has increased GCS activity, although a
causal link between phenotype and increased enzyme activity was not
established (Chen and Goldsbrough, 1994).
Wild-type Indian mustard plants respond to exposure to Cd with
increased levels of a GCS transcript (Schafer et al., 1998). Similarly,
exposure of Arabidopsis plants to Cd and Cu causes an increase in
transcript levels of the two genes in the GSH biosynthetic pathway and
of GSH reductase (Xiang and Oliver, 1998). The signal molecule,
jasmonate, mediated a similar effect in the absence of heavy metal
exposure although it has not been demonstrated that the effect of heavy
metal stress on gene expression is mediated via jasmonate. There is
also circumstantial evidence supporting post-transcriptional regulation
of GCS expression in addition to the well-recognized regulation of GCS
activity through GSH feedback inhibition (May et al., 1998; Noctor and
Foyer, 1998).
Regulation of PC synthase activity is expected to be the primary point
at which PC synthesis is regulated. Kinetic studies using plant cell
cultures demonstrated that PC biosynthesis occurs within minutes of
exposure to Cd and is independent of de novo protein synthesis,
consistent with the observation of enzyme activation in vitro. The
enzyme appears to be expressed independently of heavy metal exposure.
It has been detected in S. cucubalis cells grown in culture
medium (Grill et al., 1989), and in tomato (Chen et al., 1997) and
Arabidopsis (Howden et al., 1995) plants grown in soil or agar medium,
in the presence of only trace levels of essential heavy metals.
Together these observations indicate that PC synthase is primarily
regulated by activation of the enzyme in the presence of heavy metals.
In vivo studies have shown that PC synthesis can be induced by a range
of metal ions in fission yeast and in both intact plants and plant cell
cultures (Rauser, 1995). This has been mirrored by in vitro studies of
PC synthase expressed in E. coli or in Brewer's yeast where
the enzyme was activated to varying extents by Cd, Cu, Ag, Hg, Zn, and
Pb ions (Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et al.,
1999).
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A PC SYNTHASE MUTANT GIVES INSIGHT INTO THE MECHANISM OF ENZYME
ACTIVATION |
The mechanism by which PC synthase is activated will
undoubtedly prove an interesting one, particularly because it is
relatively non-specific with respect to the activating metal ion,
although some metals are more effective than others. One model for the function of PC synthase enzymes is that the conserved N-terminal domains possess the catalytic activity. Activation probably arises from
metal ions interacting with residues in this domain, possibly Cys or
His residues. Five Cys (two of which are adjacent) (Fig. 2) and a
single His residue are conserved in this domain. This model is
supported by the molecular characterization of mutant cad1
alleles. One in particular, cad1-5, has a nonsense mutation that would result in premature termination of translation downstream of
the conserved domain (Ha et al., 1999). The truncated polypeptide is
predicted to lack nine of the 10 Cys residues in the C-terminal domain
(Fig. 2). That this mutant enzyme is the least affected (as measured by
in vivo PC levels and sensitivity to Cd) and the mutant activity is
expressed only in the presence of Cd (Howden et al., 1995)
confirms that the C-terminal domain is not absolutely required for
either catalysis or activation.
Since the truncation of the cad1-5 mutant polypeptide
produces a mutant phenotype, the C-terminal domain clearly has some role in activity. It is likely that this domain acts as a local sensor
by binding heavy metal ions (presumably via the multiple Cys residues,
but possibly also others) and bringing them into contact with the
activation site in the catalytic domain. This model is consistent with
biochemical studies using epitope-tagged AtPCS, which demonstrated it
binds Cd ions at high affinity (Kd = 0.54 ± 0.20 µM) and high capacity
(stoichiometric ratio = 7.09 ± 0.94) (Vatamaniuk et al.,
1999). A schematic illustration of this model is shown in Figure
3.
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Figure 3.
A model for the function of PC synthase. The
C-terminal domain acts as a local sensor of heavy metal ions, such as
Cd. The Cys residues bind Cd ions, bringing them into closer proximity
and transferring them to the activation site in the N-terminal,
catalytic domain. The activated N-terminal domain catalyzes the
transfer of the -Glu-Cys moiety of a molecule of GSH onto a second
molecule of GSH or an existing PCn molecule to
form a PCn+1 product.
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Previous studies indicated PC synthase is expressed constitutively and
levels of enzyme are generally unaffected by exposure of cell cultures
or intact plants to Cd. This suggests the induction of PC synthase gene
expression is unlikely to play a significant role in regulating PC
biosynthesis. This is supported by northern or reverse
transcriptase-PCR analysis of the expression of AtPCS1/CAD1 which showed that levels of mRNA were not influenced by exposure of
plants to Cd, even under conditions of severe stress (S.-B. Ha and C.S.
Cobbett, unpublished data), thus suggesting an absence of
regulation at the level of transcription (Ha et al., 1999; Vatamaniuk
et al., 1999). Interestingly, however, reverse transcriptase-PCR analysis of TaPCS1 expression in roots indicated increased
levels of mRNA on exposure to Cd (Clemens et al., 1999). This suggests that, in some species, PC synthase activity may be regulated at both
the transcriptional and post-translational levels.
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PC SYNTHASE GENES IN ANIMAL SPECIES? |
Database searches also identified a similar gene in the
nematode, C. elegans. The predicted amino acid sequence of
the N-terminal region shows 40% to 50% identical amino acids with the
corresponding regions of the plant and yeast gene products.
Furthermore, the C-terminal domain, although showing no obvious
sequence similarity to the plant or yeast gene products, contains 10 Cys residues including two pairs (Fig. 2). An expressed sequence tag
(GenBank accession no. AU061531) with similarity to PC synthase genes has also been identified in slime mold (Dictyostelium
discoideum). In addition, using PCR similar sequences to the
conserved N-terminal regions of the three genes have been identified
from the aquatic midge, Chironomus oppositus, and
earthworm species (W. Dietrich and C.S. Cobbett, unpublished
data). As yet we have no evidence that these animal genes also encode a
protein with PC synthase activity. However, in view of the high level
of identity of the nematode gene product with the conserved N-terminal
domains of the yeast and plant enzymes, as well as the presence of a
variable domain containing multiple Cys residues, it seems likely that it too encodes PC synthase. This, then, would suggest that PCs play a
wider role in heavy metal detoxification than previously expected. A
superficial view of the limited selection of species in which such
sequences have been identified might suggest that organisms with an
aquatic or soil habitat are more likely to express PCs.
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PC-Cd COMPLEXES ARE SEQUESTERED IN VACUOLES |
PC-Cd complexes are sequestered to the vacuole. In fission
yeast this process has been most clearly demonstrated through studies of the Cd-sensitive mutant, hmt1. In extracts of fission
yeast two PC-Cd complexes (referred to as high
Mr [HMW] and low Mr
[LMW]) can be clearly resolved using gel-filtration chromatography.
The hmt1 mutant is unable to form HMW complexes on exposure
to Cd. The Hmt1 gene encodes a member of the family of
ATP-binding cassette (ABC) membrane transport proteins that is located
in the vacuolar membrane (Ortiz et al., 1992). Both HMT1 and ATP were
required for the transport of PCs in the absence of Cd and LMW PC-Cd
complexes into vacuolar membrane vesicles (Fig. 1). HMT1 did not
transport Cd alone and the transport of PCs and PC-Cd complexes was not dependent on the proton gradient established across the vacuolar membrane by the vacuolar proton-ATPase (Ortiz et al., 1995).
Interestingly, in C. elegans, various mutations affecting
ABC transporter proteins also confer heavy metal sensitivity (Broeks et
al., 1996). This would not be unexpected if PCs are expressed in
C. elegans and are sequestered in a similar manner at the
cellular level.
Sequestration of PCs to the vacuole has also been observed in
plants. In mesophyll protoplasts derived from tobacco plants exposed to
Cd almost all of both the Cd and PCs accumulated was confined to the
vacuole (Vogeli-Lange and Wagner, 1990) and an ATP-dependent, proton
gradient-independent activity, similar to that of HMT1, capable of
transporting both PCs and PC-Cd complexes into tonoplast vesicles
derived from oat roots has been identified (Salt and Rauser, 1995).
Distinct from the PC transporter activity, an alternative mechanism,
identified in both fission yeast vacuolar membrane and oat tonoplast
vesicles, is a Cd/H+ antiporter activity
dependent on the proton gradient (Salt and Wagner, 1993; Ortiz et al.,
1995) (Fig. 1). In addition, in Brewer's yeast, YCF1, mentioned above,
is also a member of the ABC family of transporters and transports both
GSH conjugates and (GSH)2Cd complexes to the
vacuole (Li et al., 1997).
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SULFIDE IONS PLAY A ROLE IN PC FUNCTION |
In some plants and in the yeasts, fission yeast, and C. glabrata, HMW PC-Cd complexes contain both Cd and acid-labile
sulfide. In general the ratio of S2
:Cd is
higher in the HMW complex compared with the LMW complex. Those
complexes with a comparatively high ratio of
S2:Cd consist of aggregates of 20-Å diameter
particles which themselves consist of a CdS crystallite core coated
with PCs (Dameron et al., 1989; Reese et al., 1992). The incorporation
of sulfide into the HMW complexes increases both the amount of Cd per
molecule and the stability of the complex.
Genetic evidence for the importance of sulfide in the function of
PCs has been obtained from the analysis of Cd-sensitive mutants of
fission yeast that are deficient in PC-Cd complexes. In one case the
gene mutated in the Cd-sensitive derivative, when cloned, was
identified as a gene involved in adenine biosynthesis. Subsequent
genetic analysis demonstrated that different single or double mutants
deficient in steps in the adenine biosynthetic pathway lacked HMW
complexes (Speiser et al., 1992). Biochemical characterization of the
enzymes encoded by these genes indicated that this pathway, in addition
to catalyzing the conversion of Asp to intermediates in adenine
biosynthesis, could also utilize Cys sulfinate, a sulfur-containing
analog of Asp, to form other sulfur-containing compounds. These are
believed to be intermediates or carriers in the pathway of sulfide
incorporation into HMW complexes (Juang et al., 1993) (Fig. 1).
Together these observations confirm the importance of sulfide in the
mechanism of PC detoxification of Cd. Whether or not sulfide is
involved in the detoxification of other metal ions by PCs is unknown.
More recently, Cd-sensitive mutants isolated in fission yeast and
Candida glabrata have identified additional functions which are probably also important in sulfide metabolism. In fission yeast the
hmt2 mutant hyperaccumulates sulfide in both the presence and absence of Cd (Vande Weghe and Ow, 1999). The HMT2 gene
encodes a mitochondrial sulfide/quinone oxidoreductase, which was
suggested to function in the detoxification of endogenous sulfide. The
role of HMT2 in Cd tolerance is uncertain, but one possibility is to detoxify excess sulfide generated during the formation of HMW PC-Cd
complexes after Cd exposure (Fig. 1). In C. glabrata the hem2 mutant is deficient in porphobilinogen synthase
involved in siroheme biosynthesis (Hunter and Mehra, 1998). Siroheme is a cofactor for sulfite reductase required for sulfide biosynthesis (Fig. 1). This deficiency may contribute to the Cd-sensitive phenotype. However, additional studies are required to establish the precise influence of this pathway on PC function.
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DO PCs DETOXIFY METALS OTHER THAN Cd? |
Although both induction of PCs in vivo and activation of PC
synthase in vitro are conferred by a range of metal ions, there is
little evidence supporting a role for PCs in the detoxification of such
a wide range of metal ions. For metals other than Cd there are few
studies demonstrating the formation of PC-metal complexes either in
vitro or in vivo. PCs can form complexes with Pb, Ag, and Hg in vitro
(for example, see Mehra et al., 1996; Rauser, 1999). Maitani et al.
(1996) used inductively coupled plasma-atomic emission spectroscopy in
combination with HPLC separation of native PC-metal complexes in the
roots of Rubia tinctorum. PCs were induced to varying levels
by a wide range of metal ions tested. The most effective appeared to be
Ag, arsenate, Cd, Cu, Hg, and Pb ions. However, the only PC complexes
identified in vivo were with Cd, Ag, and Cu ions. PC complexes formed
in response to Pb and arsenate but these complexes contained copper
ions and not the metal ion used for induction of synthesis.
The clearest evidence for the role of PCs in heavy metal detoxification
comes from characterization of the PC synthase-deficient mutants of
Arabidopsis and fission yeast. A comparison of the relative sensitivity
of the Arabidopsis and fission yeast mutants to different heavy metals
revealed a similar but not identical pattern (Ha et al., 1999). In both
organisms PCs appeared to play an important role in Cd and arsenate
detoxification and no apparent role in the detoxification of Zn, Ni,
and selenite ions. Minor differences between the two organisms were
observed with respect to Cu, Hg, and Ag.
From the preceding discussion it is apparent that the mechanism
of metal detoxification is more complex than simply the chelation of
the metal ion by PCs. The metal ion must activate PC synthase, be
chelated by the PCs synthesized, and then presumably be transported to
the vacuole and possibly form a more complex aggregation in the vacuole
with, for example, sulfide or organic acids. For Cd, the inability of
the organism to carry out of any of these steps decreases the capacity
of the organism for detoxification and thereby confers a sensitive
phenotype. Whether the same sequence of events occurs and is required
for other metal ions is not clear. PCs appear to have a relatively
insignificant role in the detoxification of metal ions such as Cu, Zn,
Ni, and SeO3, in Arabidopsis. Thus, for example,
although PCs are induced by Cu in vivo, PC synthase is activated
effectively by Cu in vitro, and PCs can chelate Cu in vitro, it is
unknown whether PCs effectively chelate Cu in vivo or whether PC-Cu
complexes, if formed, can be sequestered in the vacuole. Zn and Ni
appear to be relatively ineffective activators of PC synthase in vitro.
It may be that for these there are alternative, more effective
detoxification mechanisms, such as MTs or His, respectively.
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WHAT ARE THE ROLES OF PCs? |
PCs can be detected in plant tissues and cell cultures exposed
only to trace levels of essential metals and the level of PCs observed
in cell cultures correlates with the depletion of metal ions from the
medium. These observations have been interpreted to indicate a role for
PCs in the homeostasis of essential metal ion metabolism (Rauser, 1995,
1999; Zenk, 1996). In addition, in vitro experiments have shown that
PC-Cu and PC-Zn complexes could reactivate the apo forms of the
copper-dependent enzyme diamino oxidase and the Zn-dependent enzyme
carbonic anhydrase, respectively (Thumann et al., 1991). Although these
experiments demonstrate that PC-metal complexes are capable of donating
metal ions to metal-requiring enzymes, in each case the Cu or Zn
complexes were no more effective than the free metal sulfate salt. In
addition, roles for PCs in Fe or sulfur metabolism have also been
proposed (Zenk, 1996; Sanita di Toppi and Gabbrielli, 1999). However,
there is currently no direct evidence that PCs have functions other than in metal detoxification.
Although PCs clearly can have a role in Cd detoxification, for example,
is this role of any physiological or ecological relevance? Whereas most
experimental studies use Cd concentrations above 1 µM
(Sanita di Toppi and Gabbrielli, 1999), it has been estimated that
solutions of non-polluted soils contain Cd concentrations ranging up to
0.3 µM (Wagner, 1993). Wagner (1993) has also argued that, at low levels of Cd exposure, as represented by most soils, Cd
would be largely complexed with vacuolar citrate and only at high
levels of Cd exposure (not generally found in natural environments) might PCs play a role. Counter to this argument is the observation that
a PC-deficient mutant of Arabidopsis is highly sensitive to
concentrations of Cd as low as 0.6 µM (Howden et al.,
1995). Even at concentrations of Cd where the mutant is not obviously sensitive, the wild type may nonetheless have a selective advantage. This suggests that PCs may have a role in heavy metal detoxification in
an unpolluted environment. The absence of PC-deficient mutants of other
species makes this question difficult to address.
| |
FUTURE DIRECTIONS |
The most significant recent advances in our understanding of PC
biosynthesis and function have come from molecular genetic studies
using a variety of model systems. These will continue to provide a
wealth of mutants for biochemical, molecular, and physiological
analysis. Interesting questions relating to the roles of PC synthase
and PCs themselves in different organisms, possibly including animal
species, remain to be answered. The isolation of PC synthase genes from
a number of species will allow a considerably greater understanding of
the mechanism of metal activation of PC biosynthesis and the catalytic
mechanism itself. There is considerable potential for the application
of that understanding to optimizing the process of phytoremediation.
| |
FOOTNOTES |
Received November 9, 1999; accepted February 22, 2000.
*
E-mail c.cobbett{at}genetics.unimelb.edu.au; fax
61-3-93445139.
| |
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TOP
INTRODUCTION
PLANTS MAKE TWO TYPES...
