Phyto Che Latins

8/12/2019 Phyto Che Latins

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Update on Heavy Metal Stress

Phytochelatins and Their Roles inHeavy Metal Detoxification

Christopher S. Cobbett*

Department of Genetics, University of Melbourne, Parkville, Victoria, 3052, Australia

Plants respond to heavy metal toxicity in a varietyof different ways. Such responses include immobili-zation, exclusion, chelation and compartmentaliza-tion of the metal ions, and the expression of moregeneral stress response mechanisms such as ethyleneand stress proteins. These mechanisms have beenreviewed comprehensively by Sanita di Toppi andGabbrielli (1999) for plants exposed to Cd, the heavy

metal for which there have been arguably the great-est number and most wide-ranging studies overmany decades. Understanding the molecular and ge-netic basis for these mechanisms will be an importantaspect of developing plants as agents for the phytore-mediation of contaminated sites (Salt et al., 1998).One recurrent general mechanism for heavy metaldetoxification in plants and other organisms is thechelation 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 beenrecognized in plants. The roles of several ligands

have been reviewed by Rauser (1999). Extracellularchelation by organic acids, such as citrate and malate,is important in mechanisms of aluminum tolerance.For example, malate efflux from root apices is stim-ulated by exposure to aluminum and is correlatedwith aluminum tolerance in wheat (Delhaize andRyan, 1995). Some aluminum-resistant mutants ofArabidopsis also have increased organic acid effluxfrom roots (Larsen et al., 1998). Organic acids andsome amino acids, particularly His, also have roles inthe chelation of metal ions both within cells and inxylem 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 ex-pression of MTs in plants, particularly Arabidopsis,have been reviewed elsewhere (Fordham-Skelton etal., 1998; Rauser, 1999). In contrast, the phytochela-tins (PCs), the subject of this Update, are enzymati-cally synthesized Cys-rich peptides. The most recentreview of PC structure, biosynthesis, and function inthis journal was by Rauser (1995). Other more recentreviews are by Zenk (1996) and Rauser (1999). Recentadvances in our understanding of aspects of PC bio-synthesis and function are derived predominantly

from molecular genetic approaches using modelorganisms.

PLANTS MAKE TWO TYPES OF PEPTIDE METAL-

BINDING LIGANDS: MTs AND PCs

The historical context of the identification of MTsand PCs in plants is worth discussing. MTs were firstidentified as Cd-binding proteins in mammalian tis-sues. Similar proteins have been identified in a largenumber of animal species (Kagi, 1991). Early reportsof metal-binding proteins in plants were generallyassumed to be MTs. However, in the absence ofdetailed characterization or primary amino acid se-quences, many of these metal-binding complexesmay have been comprised, at least in part, of PCs,particularly where they were identified in studies ofplant responses to Cd.

After the structures of PCs had been elucidated andit was found that these peptides are distributed

widely in the plant kingdom, it was proposed thatPCs were the functional equivalent of MTs (Grill etal., 1987). Subsequently, numerous examples of MT-like genes, and in some cases MT proteins, have beenisolated from a variety of plant species and it is nowapparent that plants express both of these Cys-containing metal-binding ligands. Furthermore, it islikely that the two play relatively independent func-tions in metal detoxification and/or metabolism.However, the extent to which this is true is not yetclear and will not become apparent until a completeset of MT-deficient mutants have been identified inArabidopsis. PCs have not been reported in an ani-

mal species, supporting the notion that in animals,MTs may well perform some of the functions nor-mally contributed by PCs in plants. However, theisolation of the PC synthase gene from plants and theconsequent identification of similar genes in animalspecies, described below, suggests that, at least insome animal species, both of these mechanisms con-tribute to metal detoxification and/or metabolism.Although PCs have also been classified as class IIIMTs and the use of the term phyto to designatethese compounds appears to be increasingly inaccu-rate, I believe phytochelatins has become so en-trenched in the literature that a more broadly encom-

passing designation is likely to cause only confusion.* E-mail [email protected]; fax 61393445139.Plant Physiology, July 2000, Vol. 123, pp. 825832, www.plantphysiol.org 2000 American Societyof Plant Physiologists 825www.plant.orgon February 27, 2014 - Published bywww.plantphysiol.orgDownloaded from

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PCs HAVE THE GENERAL STRUCTURE(-Glu-Cys)

n-Gly

Early analyses demonstrated PCs consisted of onlythe 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 increasingrepetitions of the -Glu-Cys dipeptide followed by aterminal Gly; (-Glu-Cys)n-Gly, where n has beenreported as being as high as 11, but is generally in therange of 2 to 5. PCs have been identified in a widevariety of plant species and in some microorganisms.They are structurally related to glutathione (GSH;-Glu-Cys-Gly) and were presumed to be the prod-ucts of a biosynthetic pathway. In addition, a numberof 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).

PCs ARE SYNTHESIZED FROM GSH

Numerous physiological, biochemical, and geneticstudies have confirmed that GSH (or, in some cases,related compounds) is the substrate for PC biosyn-thesis (Rauser, 1995, 1999; Zenk, 1996). Such studieshave used a variety of plant species, sometimes asintact plants but often in the form of in vitro cellcultures. Early studies with cell cultures demon-strated that induction of PCs in the presence of Cdcoincided with a transient decrease in levels of GSH.Furthermore, the exposure of either cell cultures orintact plants to an inhibitor of GSH biosynthesis,

buthionine sulfoximine, conferred increased sensitiv-ity to Cd with a corresponding inhibition of PC bio-synthesis. This could be reversed by the addition ofGSH to the growth medium.

By far the most detailed characterization of thepathway of PC biosynthesis has come from studies inthe fission yeast (Schizosaccharomyces pombe), and inArabidopsis. Genetic studies have confirmed GSH-

deficient mutants of the fission yeast and Arabidop-sis are also PC deficient and hypersensitive to Cd. Inparticular, the cad2-1 mutant of Arabidopsis is par-tially deficient in GSH and in -glutamyl-Cys syn-thetase (GCS) activity, the first of the two GSH bio-synthetic enzymes. The cad2-1 mutation is a 6-bp

deletion within an exon of the GCS gene affectingresidues in the vicinity of the presumed active site ofthe enzyme (Cobbett et al., 1998). The PC biosyn-thetic pathway is illustrated in Figure 1.

PC SYNTHASE IS ACTIVATED BYHEAVY METAL IONS

Grill et al. (1989) first identified an enzyme activityfrom cultured cells ofSilene cucubalisthat synthesizedPCs from GSH by transferring a -Glu-Cys moietyfrom a donor to an acceptor molecule. The reactioninvolved the transpeptidation of the -Glu-Cys moi-ety of GSH onto initially a second GSH molecule toform PC2or, in later stages of the incubation, onto aPC 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 a95,000-Mrtetramer with aKmof 6.7 mmfor GSH. Invitro the activity of the partially purified enzymewas 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 inducePC biosynthesis in vivo in plant cell cultures. In invitro reactions, PC biosynthesis continued until theactivating metal ions were chelated either by the PCs

formed or by the addition of a metal chelator such asEDTA (Loeffler et al., 1989). This provides a mecha-nism to autoregulate the biosynthesis of PCs in whichthe product of the reaction chelates the activatingmetal, thereby terminating the reaction.

Similar PC synthase activities have been detectedin pea (Klapheck et al., 1995), tomato (Chen et al.,1997), and Arabidopsis (Howden et al., 1995). Crude

Figure 1. Genes and functions contributing toCd detoxification in plants and fungi. The figureis a composite of different functions described in

different organisms. Enzyme abbreviations areshown in bold. GS, GSH synthetase; PCS, Phy-tochelatin synthase. Gene loci are shown inbold italics. CAD1 and CAD2 are in Arabidop-sis;hmt1,hmt2,ade2,ade6,ade7, andade8arein fission yeast; and hem2 is in Candida gla-brata. The details of sulfide metabolism on theright of the figure are not well understood.

Cobbett

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enzyme preparations from the roots of pea, whichnormally contain both GSH and homo-GSH (-Glu-Cys--Ala), could use GSH efficiently, but homo-GSH or -Glu-Cys-Ser less efficiently, as substratesfor PC synthesis. In the presence of both GSH andhomo-GSH, synthesis of homo-PCs was enhanced

(Klapheck et al., 1995). These observations were in-terpreted to indicate the enzyme had a -Glu-Cysdonor site that was relatively specific for GSH but aless specific acceptor site able to use each of the threesubstrates. Little is known about the tissue specificityof PC synthase expression and/or PC biosynthesis.In the only study of tissue-specific PC synthase ex-pression to date, the activity was detected in the rootsand stems of tomato plants but not in leaves or fruits(Chen et al., 1997).

PC SYNTHASE GENES WERE ISOLATED

INDEPENDENTLY IN THREE LABORATORIES

Despite the identification and purification of PCsynthase a decade ago, the isolation of the corre-sponding gene and a consequent detailed under-standing of the mechanism of PC biosynthesis haveeluded us until recently. PC synthase genes have

been isolated simultaneously by three researchgroups using different approaches. Two groups usedexpression of plant genes in Brewers yeast (Saccha-romyces cerevisiae) to identify genes involved in Cdresistance. One group identified an ArabidopsiscDNA (AtPCS1) that suppressed the Cd-sensitive

phenotype of both Brewers yeast yap1and ycf1mu-tants (Vatamaniuk et al., 1999).YAP1encodes a tran-scription factor that is required for the expression ofYCF1, a transporter responsible for the vacuolar se-questration of GSH-Cd complexes (Li et al., 1997).This group was searching for functional plant ho-mologs of YAP1 by using a two-step screening pro-cedure to identify Arabidopsis cDNAs that couldsuppress ayap1,but not aycf1,mutant. In this screenthey fortuitously identified AtPCS1. By expressing

AtPCS1in various mutant strains, they demonstratedthat the mechanism of AtPCS1-mediated Cd toler-ance functioned in bothyap1andycf1mutants, and inthe MT-deficient mutant, cup1. Thus the mechanismappeared to be distinct from other recognized Cd-detoxification mechanisms. Expression of AtPCS1mediated an increase in Cd accumulation indicatingit was probably involved in chelation or sequestra-tion. It also functioned in a vacuole-deficient mutant(pep5) demonstrating the AtPCS1 gene product wasnot a vacuolar membrane transport function similarto YCF1, for example. Consistent with the cDNAencoding PC synthase, the mechanism was not ex-pressed in a GSH-deficient mutant (gsh2) and medi-ated PC biosynthesis in vivo in Brewers yeast.

A second group identified a wheat cDNA (TaPCS1)that conferred increased Cd resistance when ex-

pressed in a wild-type (for Cd tolerance) strain of

Brewers yeast (Clemens et al., 1999). Similar to At-PCS1, the Cd resistance mediated by TaPCS1 wasassociated with an increase in Cd accumulation,functioned in a vacuole-deficient mutant (vps18) andwas GSH dependent, being diminished in the pres-ence of an inhibitor of GSH biosynthesis.TaPCS1alsomediated PC biosynthesis in vivo in Brewers yeast.

AtPCS1 has also been isolated through the cloningof theCAD1gene of Arabidopsis. Mutants at thecad1locus in Arabidopsis, like the cad2-1mutant, are Cdsensitive and deficient in the formation of Cd-

binding complexes and in PC biosynthesis. In partic-ular, PCs are undetectable in the cad1-3mutant afterprolonged exposure to Cd. In contrast to the cad2-1mutant, the cad1 mutants have wild-type levels ofGSH, suggesting a defect in PC synthase. Consistentwith this, PC synthase activity in crude extracts fromcad1mutants was less than 1% of the level in both thewild-type and the cad2-1 mutant (Howden et al.,1995). Thus it was likely that CAD1is the structuralgene for PC synthase (Fig. 1). The CAD1 gene has

been isolated using a positional cloning strategy (Haet al., 1999). The position of the gene was mappedusing molecular markers and a candidate gene iden-tified from the Arabidopsis genome initiativegenomic sequence. That this candidate was CAD1was confirmed by the identification of a differentmutation in that gene in each of the cad1mutants and

by complementation of the Cd-sensitive phenotypeofcad1-3by a genomic clone spanning this gene.

A similar sequence (SpPCS) was identified in thegenome of fission yeast and targeted deletions of that

gene were constructed in two of these studies (Cle-mens et al., 1999; Ha et al., 1999). The resulting mu-tants were, like the Arabidopsis cad1 mutants, Cdsensitive and PC deficient, confirming the analogousfunction of the two genes in the different organisms.Expression of theCAD1(AtPCS1) andSpPCSgenes inEscherichia coli (Ha et al., 1999) or purification ofepitope-tagged derivatives of SpPCS (Clemens et al.,1999) and AtPCS1 (Vatamaniuk et al., 1999) ex-pressed in Brewers yeast was used to demonstratethat both were necessary and sufficient for GSH-dependent, metal ion-activated PC biosynthesis invitro.

The Arabidopsis CAD1(AtPCS1) cDNA encodes apredicted 55-kD polypeptide of 485 amino acids. Acomparison of the Arabidopsis and fission yeastamino acid sequences showed that the N-terminalregions of the two are very similar (45% identical),whereas the C-terminal sequences show little appar-ent conservation of amino acid sequence (Fig. 2). Themost apparent common feature of the C-terminalregions is the occurrence of multiple Cys residues,often as pairs. The C-terminal regions of the Arabi-dopsis and fission yeast proteins have 10 and sevenCys residues, respectively, of which four and six,respectively, are as pairs. However, there is no ap-

parent conservation of the positions of these Cys

Phytochelatin Biosynthesis and Function

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residues relative to each other. The two plant PC

synthase sequences, TaPCS and AtPCS, can bealigned across their entire length (55% identity) (Cle-mens et al., 1999). The former contains 14 Cys resi-dues, including two pairs, in the C-terminal domain.

PC BIOSYNTHESIS MAY BE REGULATED INSEVERAL WAYS

There are a number of mechanisms by which the PCbiosynthetic pathway may be regulated. The first ofthese 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 shownthat PC biosynthesis and Cd tolerance can also beincreased (Yong et al., 1999; Zhu et al., 1999). Thissupports the idea that regulation of GSH biosynthesisis a plausible endogenous mechanism by which PCexpression might be modulated. In support of this isthe observation that a Cd-tolerant tomato cell line hasincreased GCS activity, although a causal link betweenphenotype and increased enzyme activity was notestablished (Chen and Goldsbrough, 1994).

Wild-type Indian mustard plants respond to expo-sure to Cd with increased levels of a GCS transcript(Schafer et al., 1998). Similarly, exposure of Arabi-dopsis plants to Cd and Cu causes an increase intranscript levels of the two genes in the GSH biosyn-thetic pathway and of GSH reductase (Xiang andOliver, 1998). The signal molecule, jasmonate, medi-ated a similar effect in the absence of heavy metalexposure although it has not been demonstrated thatthe effect of heavy metal stress on gene expression ismediated via jasmonate. There is also circumstantialevidence supporting post-transcriptional regulationof GCS expression in addition to the well-recognizedregulation of GCS activity through GSH feedbackinhibition (May et al., 1998; Noctor and Foyer, 1998).

Regulation of PC synthase activity is expected to bethe primary point at which PC synthesis is regulated.

Kinetic studies using plant cell cultures demon-

strated that PC biosynthesis occurs within minutes of

exposure to Cd and is independent of de novo pro-tein synthesis, consistent with the observation of en-zyme activation in vitro. The enzyme appears to beexpressed independently of heavy metal exposure. Ithas been detected in S. cucubalis cells grown in cul-ture medium (Grill et al., 1989), and in tomato (Chenet al., 1997) and Arabidopsis (Howden et al., 1995)plants grown in soil or agar medium, in the presenceof only trace levels of essential heavy metals. To-gether these observations indicate that PC synthase isprimarily regulated by activation of the enzyme inthe presence of heavy metals. In vivo studies haveshown that PC synthesis can be induced by a range of

metal ions in fission yeast and in both intact plantsand plant cell cultures (Rauser, 1995). This has beenmirrored by in vitro studies of PC synthase expressedinE. colior in Brewers yeast where the enzyme wasactivated to varying extents by Cd, Cu, Ag, Hg, Zn,and Pb ions (Clemens et al., 1999; Ha et al., 1999;Vatamaniuk et al., 1999).

A PC SYNTHASE MUTANT GIVES INSIGHT INTOTHE MECHANISM OF ENZYME ACTIVATION

The mechanism by which PC synthase is activatedwill undoubtedly prove an interesting one, particu-larly because it is relatively non-specific with respectto the activating metal ion, although some metals aremore effective than others. One model for the functionof PC synthase enzymes is that the conservedN-terminal domains possess the catalytic activity. Ac-tivation probably arises from metal ions interactingwith residues in this domain, possibly Cys or Hisresidues. 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 character-ization of mutantcad1alleles. One in particular,cad1-5,has a nonsense mutation that would result in prema-ture termination of translation downstream of the con-served domain (Ha et al., 1999). The truncated

polypeptide is predicted to lack nine of the 10 Cys

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, fissionyeast (SpPCS; accession no. Z68144); Ce, Caenorhabditis elegans(CePCS1; accession no. Z66513). The total number ofamino acids in each is shown on the right. Approximate positions of all Cys residues are indicated by vertical bars; adjacentCys residues are connected by a horizontal bar; and Cys residues conserved across the three sequences are highlighted withan asterisk. The conserved N-terminal domains exhibit at least 40% identical amino acids in pair-wise comparisons of thefour sequences. An arrowhead indicates the position of the Arabidopsis cad1-5nonsense mutation.

Cobbett

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residues in the C-terminal domain (Fig. 2). That thismutant enzyme is the least affected (as measured by invivo PC levels and sensitivity to Cd) and the mutantactivity is expressed only in the presence of Cd (How-den et al., 1995) confirms that the C-terminal domain isnot absolutely required for either catalysis or activation.

Since the truncation of the cad1-5mutant polypep-tide produces a mutant phenotype, the C-terminaldomain clearly has some role in activity. It is likelythat this domain acts as a local sensor by bindingheavy metal ions (presumably via the multiple Cysresidues, but possibly also others) and bringing theminto contact with the activation site in the catalyticdomain. This model is consistent with biochemicalstudies using epitope-tagged AtPCS, which demon-strated it binds Cd ions at high affinity (Kd 0.54 0.20 m) and high capacity (stoichiometric ratio 7.09 0.94) (Vatamaniuk et al., 1999). A schematic

illustration of this model is shown in Figure 3.Previous studies indicated PC synthase is ex-pressed constitutively and levels of enzyme are gen-erally unaffected by exposure of cell cultures or intactplants to Cd. This suggests the induction of PC syn-thase gene expression is unlikely to play a significantrole in regulating PC biosynthesis. This is supported

by northern or reverse transcriptase-PCR analysis ofthe expression ofAtPCS1/CAD1which showed thatlevels of mRNA were not influenced by exposure ofplants to Cd, even under conditions of severe stress(S.-B. Ha and C.S. Cobbett, unpublished data), thussuggesting an absence of regulation at the level of

transcription (Ha et al., 1999; Vatamaniuk et al.,1999). Interestingly, however, reverse transcriptase-PCR analysis ofTaPCS1expression in roots indicatedincreased levels of mRNA on exposure to Cd (Clem-ens et al., 1999). This suggests that, in some species,PC synthase activity may be regulated at both thetranscriptional and post-translational levels.

PC SYNTHASE GENES IN ANIMAL SPECIES?

Database searches also identified a similar gene inthe nematode, C. elegans. The predicted amino acidsequence of the N-terminal region shows 40% to 50%identical amino acids with the corresponding regions

of the plant and yeast gene products. Furthermore, theC-terminal domain, although showing no obvious se-quence 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 hasalso been identified in slime mold (Dictyostelium dis-coideum). In addition, using PCR similar sequences tothe conserved N-terminal regions of the three geneshave been identified from the aquatic midge,Chirono-mus oppositus, and earthworm species (W. Dietrich andC.S. Cobbett, unpublished data). As yet we have noevidence that these animal genes also encode a proteinwith PC synthase activity. However, in view of thehigh level of identity of the nematode gene productwith the conserved N-terminal domains of the yeastand plant enzymes, as well as the presence of a vari-able domain containing multiple Cys residues, itseems likely that it too encodes PC synthase. This,then, would suggest that PCs play a wider role inheavy metal detoxification than previously expected.A superficial view of the limited selection of species inwhich such sequences have been identified might sug-gest that organisms with an aquatic or soil habitat aremore likely to express PCs.

PC-Cd COMPLEXES ARE SEQUESTEREDIN VACUOLES

PC-Cd complexes are sequestered to the vacuole. Infission yeast this process has been most clearly dem-onstrated 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 chromatog-raphy. Thehmt1mutant is unable to form HMW com-plexes on exposure to Cd. The Hmt1gene encodes amember of the family of ATP-binding cassette (ABC)membrane transport proteins that is located in thevacuolar membrane (Ortiz et al., 1992). Both HMT1and ATP were required for the transport of PCs in theabsence of Cd and LMW PC-Cd complexes into vac-uolar membrane vesicles (Fig. 1). HMT1 did not trans-port Cd alone and the transport of PCs and PC-Cdcomplexes was not dependent on the proton gradientestablished across the vacuolar membrane by the vac-uolar proton-ATPase (Ortiz et al., 1995). Interestingly,in C. elegans, various mutations affecting ABC trans-porter proteins also confer heavy metal sensitivity(Broeks et al., 1996). This would not be unexpected ifPCs are expressed inC. elegansand are sequestered ina similarmanner at the cellular level.

Sequestration of PCs to the vacuole has also been

observed in plants. In mesophyll protoplasts derived

Figure 3. A model for the function of PC synthase. The C-terminaldomain acts as a local sensor of heavy metal ions, such as Cd. TheCys residues bind Cd ions, bringing them into closer proximity andtransferring them to the activation site in the N-terminal, cat alyticdomain. The activated N-terminal domain catalyzes the transfer ofthe -Glu-Cys moiety of a molecule of GSH onto a second molecule

of GSH or an existing PCn molecule to form a PCn1 product.


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from tobacco plants exposed to Cd almost all of boththe Cd and PCs accumulated was confined to thevacuole (Vogeli-Lange and Wagner, 1990) and anATP-dependent, proton gradient-independent activ-ity, similar to that of HMT1, capable of transporting

both PCs and PC-Cd complexes into tonoplast vesi-

cles derived from oat roots has been identified (Saltand Rauser, 1995).

Distinct from the PC transporter activity, an alter-native mechanism, identified in both fission yeastvacuolar membrane and oat tonoplast vesicles, is aCd/H antiporter activity dependent on the protongradient (Salt and Wagner, 1993; Ortiz et al., 1995)(Fig. 1). In addition, in Brewers yeast, YCF1, men-tioned above, is also a member of the ABC family oftransporters and transports both GSH conjugates and(GSH)2Cd complexes to the vacuole (Li et al., 1997).

SULFIDE IONS PLAY A ROLE IN PC FUNCTION

In some plants and in the yeasts, fission yeast, andC. glabrata, HMW PC-Cd complexes contain both Cdand acid-labile sulfide. In general the ratio of S2:Cdis higher in the HMW complex compared with theLMW complex. Those complexes with a compara-tively high ratio of S2:Cd consist of aggregates of20- diameter particles which themselves consist of aCdS crystallite core coated with PCs (Dameron et al.,1989; Reese et al., 1992). The incorporation of sulfideinto the HMW complexes increases both the amountof Cd per molecule and the stability of the complex.

Genetic evidence for the importance of sulfide in thefunction of PCs has been obtained from the analysis ofCd-sensitive mutants of fission yeast that are deficientin PC-Cd complexes. In one case the gene mutated inthe Cd-sensitive derivative, when cloned, was identi-fied as a gene involved in adenine biosynthesis. Sub-sequent genetic analysis demonstrated that differentsingle or double mutants deficient in steps in theadenine biosynthetic pathway lacked HMW com-plexes (Speiser et al., 1992). Biochemical characteriza-tion of the enzymes encoded by these genes indicatedthat this pathway, in addition to catalyzing the con-version of Asp to intermediates in adenine biosynthe-sis, could also utilize Cys sulfinate, a sulfur-containinganalog of Asp, to form other sulfur-containing com-pounds. These are believed to be intermediates orcarriers in the pathway of sulfide incorporation intoHMW complexes (Juang et al., 1993) (Fig. 1). Togetherthese observations confirm the importance of sulfidein the mechanism of PC detoxification of Cd. Whetheror not sulfide is involved in the detoxification of othermetal ions by PCs is unknown.

More recently, Cd-sensitive mutants isolated in fis-sion yeast and Candida glabratahave identified addi-tional functions which are probably also important insulfide metabolism. In fission yeast the hmt2mutanthyperaccumulates sulfide in both the presence and

absence of Cd (Vande Weghe and Ow, 1999). The

HMT2 gene encodes a mitochondrial sulfide/qui-none oxidoreductase, which was suggested to func-tion in the detoxification of endogenous sulfide. Therole of HMT2 in Cd tolerance is uncertain, but onepossibility is to detoxify excess sulfide generatedduring the formation of HMW PC-Cd complexes af-

ter Cd exposure (Fig. 1). In C. glabratathe hem2mu-tant is deficient in porphobilinogen synthase in-volved in siroheme biosynthesis (Hunter and Mehra,1998). Siroheme is a cofactor for sulfite reductaserequired for sulfide biosynthesis (Fig. 1). This defi-ciency may contribute to the Cd-sensitive phenotype.However, additional studies are required to establishthe precise influence of this pathway on PC function.

DO PCs DETOXIFY METALS OTHER THAN Cd?

Although both induction of PCs in vivo and acti-

vation of PC synthase in vitro are conferred by arange of metal ions, there is little evidence support-ing a role for PCs in the detoxification of such a widerange of metal ions. For metals other than Cd thereare few studies demonstrating the formation of PC-metal complexes either in vitro or in vivo. PCs canform complexes with Pb, Ag, and Hg in vitro (forexample, see Mehra et al., 1996; Rauser, 1999).Maitani et al. (1996) used inductively coupledplasma-atomic emission spectroscopy in combinationwith HPLC separation of native PC-metal complexesin the roots ofRubia tinctorum. PCs were induced tovarying 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 com-plexes identified in vivo were with Cd, Ag, and Cuions. PC complexes formed in response to Pb andarsenate but these complexes contained copper ionsand not the metal ion used for induction of synthesis.

The clearest evidence for the role of PCs in heavymetal detoxification comes from characterization ofthe PC synthase-deficient mutants of Arabidopsisand fission yeast. A comparison of the relative sen-sitivity of the Arabidopsis and fission yeast mutantsto different heavy metals revealed a similar but notidentical pattern (Ha et al., 1999). In both organismsPCs appeared to play an important role in Cd andarsenate detoxification and no apparent role in thedetoxification of Zn, Ni, and selenite ions. Minordifferences between the two organisms were ob-served with respect to Cu, Hg, and Ag.

From the preceding discussion it is apparent that themechanism of metal detoxification is more complexthan simply the chelation of the metal ion by PCs. Themetal ion must activate PC synthase, be chelated bythe PCs synthesized, and then presumably be trans-ported to the vacuole and possibly form a more com-plex aggregation in the vacuole with, for example,sulfide or organic acids. For Cd, the inability of theorganism to carry out of any of these steps decreases

the capacity of the organism for detoxification and

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thereby confers a sensitive phenotype. Whether thesame sequence of events occurs and is required forother metal ions is not clear. PCs appear to have arelatively insignificant role in the detoxification ofmetal ions such as Cu, Zn, Ni, and SeO3, in Arabidop-sis. Thus, for example, although PCs are induced by

Cu in vivo, PC synthase is activated effectively by Cuin vitro, and PCs can chelate Cu in vitro, it is unknownwhether PCs effectively chelate Cu in vivo or whetherPC-Cu complexes, if formed, can be sequestered in thevacuole. Zn and Ni appear to be relatively ineffectiveactivators of PC synthase in vitro. It may be that forthese there are alternative, more effective detoxifica-tion mechanisms, such as MTs or His, respectively.

WHAT ARE THE ROLES OF PCs?

PCs can be detected in plant tissues and cell cul-tures exposed only to trace levels of essential metals

and the level of PCs observed in cell cultures corre-lates with the depletion of metal ions from the me-dium. These observations have been interpreted toindicate a role for PCs in the homeostasis of essentialmetal ion metabolism (Rauser, 1995, 1999; Zenk,1996). In addition, in vitro experiments have shownthat PC-Cu and PC-Zn complexes could reactivatethe apo forms of the copper-dependent enzyme dia-mino oxidase and the Zn-dependent enzyme car-

bonic anhydrase, respectively (Thumann et al., 1991).Although these experiments demonstrate that PC-metal complexes are capable of donating metal ionsto metal-requiring enzymes, in each case the Cu or

Zn complexes were no more effective than the freemetal sulfate salt. In addition, roles for PCs in Fe orsulfur metabolism have also been proposed (Zenk,1996; Sanita di Toppi and Gabbrielli, 1999). However,there is currently no direct evidence that PCs havefunctions other than in metal detoxification.

Although PCs clearly can have a role in Cd detox-ification, for example, is this role of any physiologicalor ecological relevance? Whereas most experimentalstudies use Cd concentrations above 1 m(Sanita diToppi and Gabbrielli, 1999), it has been estimatedthat solutions of non-polluted soils contain Cd con-centrations ranging up to 0.3 m (Wagner, 1993).Wagner (1993) has also argued that, at low levels ofCd exposure, as represented by most soils, Cd would

be largely complexed with vacuolar citrate and onlyat high levels of Cd exposure (not generally found innatural environments) might PCs play a role.Counter to this argument is the observation that aPC-deficient mutant of Arabidopsis is highly sensi-tive to concentrations of Cd as low as 0.6 m(How-den et al., 1995). Even at concentrations of Cd wherethe mutant is not obviously sensitive, the wild typemay nonetheless have a selective advantage. Thissuggests that PCs may have a role in heavy metaldetoxification in an unpolluted environment. Theab-sence of PC-deficient mutants of other species makes

this question difficult to address.

FUTURE DIRECTIONS

The most significant recent advances in our under-standing of PC biosynthesis and function have comefrom molecular genetic studies using a variety ofmodel systems. These will continue to provide a

wealth of mutants for biochemical, molecular, andphysiological analysis. Interesting questions relatingto the roles of PC synthase and PCs themselves indifferent organisms, possibly including animal spe-cies, remain to be answered. The isolation of PCsynthase genes from a number of species will allow aconsiderably greater understanding of the mecha-nism of metal activation of PC biosynthesis and thecatalytic mechanism itself. There is considerable po-tential for the application of that understanding tooptimizing the process of phytoremediation.

Received November 9, 1999; accepted February 22, 2000.

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