Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Genetically modified organism wikipedia , lookup
Expression vector wikipedia , lookup
Genetic engineering wikipedia , lookup
Biochemistry wikipedia , lookup
Plant nutrition wikipedia , lookup
Metalloprotein wikipedia , lookup
Gene regulatory network wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Plant breeding wikipedia , lookup
Biosynthesis wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Life, 51: 183 – 188, 2001 c 2001 IUBMB Copyright ° 1521-6543/01 $12.00 + .00 IUBMB Critical Review Heavy Metal Detoxication 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 detoxi 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 detoxi 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 identi ed a number of genes important in the biosynthesis or function of PCs. PC-de cient mutants of Arabidopsis have con rmed both the role of glutathione as the substrate for PC biosynthesis and the role of PCs themselves in heavy metal detoxi cation in plants. PC synthase genes have been identi ed in Arabidopsis and other plant species as well as in a number of animal species, suggesting PCs play a wider role in metal detoxi 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 detoxi 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 identi 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 rst identi 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 identi 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 detoxi 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 identi cation of similar genes in animal species, described next, suggests that, at least in some animal species, both these mechanisms contribute to metal detoxi cation and/or metabolism. Signi cant recent advances in our understanding of 183 184 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 identi ed in S. pombe. Genes identi 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 rst identi ed in 1983 in the yeast, Schizosaccharomyces pombe (where they were called cadystins) (4 ) and have subsequently have been identi ed in a wide variety of plant species and in some other microorganisms (reviewed in ref. 5 ). Numerous physiological, biochemical, and genetic studies have con 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 identi 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 rst identi 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 nonspeci c manner. Despite numerous attempts in the ensuing years, a PC synthase gene had not been identi ed. Arabidopsis as a Model for Studies of Heavy Metal Detoxication 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 detoxi 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 signi 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 identi cation or analysis of genes involved in GSH and PC biosynthesis. Among the Cd-sensitive mutants of Arabidopsis we identi ed, some were partially or entirely de cient in PCs, thus demonstrating conclusively that PCs play a signi cant role in the detoxi cation of at least some heavy metals in plants. Complementation analysis demonstrated these mutants identi ed two different genes. The cad2-1 mutant is partially de cient in both PCs and GSH (9). GSH-de cient mutants of S. pombe are also PC-de cient and hypersensitive to Cd. The cad2-1 mutant has decreased ° -glutamylcysteine synthetase (GCS) activity, the rst of the two GSH biosynthetic enzymes (Fig. 1). The Arabidopsis GCS gene was identi ed through the isolation of cDNAs that could complement a GCS-de 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-de 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 identi 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-detoxi 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 identi ed among a host of other mutants (see later). Nonetheless, a sequence similar to CAD1 was identi 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 PCde cient, con 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 puri cation of epitope-tagged derivatives of SpPCS and AtPCS1 expressed in S. cerevisiae (15, 16), was used to demonstrate both were necessary and suf cient for GSH-dependent, metal ion-activated PC biosynthesis in vitro. The Arabidopsis genome contains a second gene, AtPCS2, with signi 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. 185 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 identi ed similar genes in the nematode, Caenorhabditis elegans, and the slime mould, Dictyostelium discoideum. In addition, using PCR, similar partial sequences have been identi 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 con rming these animal genes also encode proteins with PC synthase activity would suggest that PCs play a wider role in heavy metal detoxi cation than previously expected. Similar sequences are absent from the Drosophila melanogaster genome and have not been identi ed in any vertebrates. A super cial view of the limited selection of species in which such sequences have been identi 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. 186 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 arti 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 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 signi cant recent study has provided evidence for an alternative model that provides a solution to this dilemma (20). Using puri 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 detoxi 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, sul de ions form an essential component of PC-Cd complexes, and a number of Cd-sensitive mutants believed to be affected in aspects of sul de metabolism have been identi 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 sul 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 de cient in porphobilinogen synthase involved in biosynthesis of siroheme, which is a cofactor for sul te reductase (24 ). CONCLUDING REMARKS Molecular genetic approaches using model organisms have identi 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 signi 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 detoxi 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. REFERENCES 1. Sanita di Toppi, L., and Gabbrielli, R. (1999) Response to cadmium in higher plants. Environ. Exp. Bot. 41, 105 – 130. 2. Rauser, W. E. (1999) Structure and function of metal chelators produced by plants. The case for organic acids, amino acids, phytin and metallothioneins. Cell Biochem. Biophys. 3, 19 – 48. 3. Grill, E., Winnacker, E.-L., and Zenk, M. H. (1987) Phytochelatins, a class of heavy-metal-bindin g peptides from plants are functionally analogous to metallothioneins. Proc. Natl. Acad. Sci. USA 84, 439 – 443. 4. Kondo, N., Isobe, M., Imai, K., and Goto, T. (1983) Structure of cadystin, the unit peptide of cadmium-binding peptides induced in the ssion yeast Schizosaccharomyce s pombe. Tetrahed. Lett. 24, 925 – 928. 5. Cobbett, C. S. (2000) Phytochelatins and their roles in heavy metal detoxi cation. Plant Physiol. 123, 825 – 832. 6. Grill, E., Lof er, S., Winnacker, E.-L., and Zenk, M. H. (1989) Phytochelatins, the heavy-metal-bindin g peptides of plants, are synthesized from glutathione by a speci c ° -glutamylcystein e dipeptidyl transpeptidase (phytochelatin synthase). Proc. Natl. Acad. Sci. USA 86, 6838 – 6842. 7. Howden, R., and Cobbett, C. S. (1992) Cadmium-sensitive mutants of Arabidopsis thaliana. Plant Physiol. 100, 100 – 107. 8. Murphy, A., and Taiz, L. (1995) A new vertical mesh transfer technique for metaltolerance studies in Arabidopsis-ecotypi c variation and coppersensitive mutants. Plant Physiol. 108, 29 – 38. 9. Howden, R., Andersen, C. R., Goldsbrough , P. B., and Cobbett, C. S. (1995) A cadmium-sensitive, glutathione-de cient mutant of Arabidopsis thaliana. Plant Physiol. 107, 1067 – 1073. 10. May, M. J., and Leaver, C. J. (1994) Arabidopsi s thaliana ° -glutamylcysteine synthetase is structurally unrelated to mammalian, yeast and Escherichia coli homologs. Proc. Natl. Acad. Sci. USA 9, 10059 – 10063. 11. Cobbett, C. S., May, M. J., Howden, R., and Rolls, B. (1998) The glutathione-de cient, cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is de cient in ° -glutamylcysteine synthetase. Plant J. 16, 73 – 78. 12. Vernoux, T., Wilson, R. C., Seeley, K. A., Reichheld, J. P., Muroy, S., Brown, S., Maughan, S. C., Cobbett, C. S., van Montagu, M., Inze, D., May, M. J., and Sung, Z. R. (2000) The ROOT MERISTEMLESS1/CADMIUM SENSITIVE2 gene de nes a glutathione-dependen t pathway involved in initiation and maintenance of cell division during postembyroni c root development . Plant Cell 12, 97 – 109. 188 COBBETT 13. Howden, R., Goldsbrough, P. B., Andersen, C. R., and Cobbett, C. S. (1995) Cadmium-sensitive, cad1, mutants of Arabidopsis thaliana are phytochelatin de cient. Plant Physiol. 107, 1059 – 1066. 14. Ha, S.-B., Smith, A. P., Howden, R., Dietrich, W. M., Bugg, S., O’Connell, M. J., Goldsbrough , P. B., and Cobbett, C. S. (1999) Phytochelatin synthase genes from Arabidopsis and the yeast, Schizosaccharomyce s pombe. Plant Cell 11, 1153 – 1164. 15. Vatamaniuk, O. K., Mari, S., Lu, Y.-P., and Rea, P. A. (1999) AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proc. Natl. Acad. Sci. USA 96, 7110 – 7115. 16. Clemens, S., Kim, E. J., Neumann, D., and Schroeder, J. I. (1999) Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J. 18, 3325 – 3333. 17. Xiang, C., and Oliver, D. J. (1998) Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell 10, 1539 – 1550. 18. May, M. J., Vernoux, T., Leaver, C., van Montague, M., and Inze, D. (1998) Glutathione homeostasis in plants: implications for environmental sensing and plant development . J. Exp. Bot. 49, 649 – 667. 19. Chen, J., Zhou, J., and Goldsbrough, P. B. (1997) Characterization of phytochelatin synthase from tomato. Physiol. Plant. 101, 165 – 172. 20. Vatamaniuk, O. K., Mari, S., Lu, Y.-P., and Rea, P. A. (2000) Mechanism of heavy metal ion activation of phytochelatin(PC) synthase: blocked thiols are suf cient for PC synthase-catalyze d transpeptidation of glutathione and related thiol peptides. J. Biol. Chem. 275, 31451 – 31459. 21. Li, Z.-S., Lu, Y.-P., Zhen, R.-G., Szczypka, M., Thiele, D. J., and Rea, P. A. (1997) A new pathway for vacuolar cadmium sequestration in Saccharomyce s cerevisiae: YCF1-catalyzed transport of bis(glutathionato) cadmium. Proc. Natl. Acad. Sci. USA 94, 42 – 47. 22. Ortiz, D. F., Ruscitti, T., McCue, K. F., and Ow, D. W. (1995) Transport of metal-binding peptides by HMT1, a ssion yeast ABC-type vacuolar membrane protein. J. Biol. Chem. 270, 4721 – 4728. 23. Speiser, D. M., Ortiz, D. F., Kreppel, L., and Ow, D. W. (1992) Purine biosyntheti c genes are required for cadmium tolerance in Schizosaccharomyces pombe. Mol. Cell Biol. 12, 5301 – 5310. 24. Hunter, T. C., and Mehra, R. K. (1998) A role for HEM2 in cadmium tolerance. J. Inorg. Biochem. 69, 293 – 303. 25. Raskin, I., and Ensley, B. D. eds. (2000) Phytoremediation of Toxic Metals: Using Plants to Clean Up the Environment. Wiley, New York.