Phytochelatin Biosynthesis and Function in Heavy-Metal Detoxification Christopher S Cobbett

Phytochelatin biosynthesis and function in heavy-metal detoxification Christopher S Cobbett

Plants respond to heavy-metal toxicity via a number of important goal in developing plants for the phytoremedia- mechanisms. One such mechanism involves the chelation of tion of contaminated environments [3]. My review heavy metals by a family of peptide ligands, the phytochelatins. describes recent advances in our understanding of the Molecular genetic approaches have resulted in important genetic and molecular basis of the biosynthesis and func- advances in our understanding of phytochelatin biosynthesis. In tion of an important class of heavy-metal-binding ligands, particular, genes encoding the enzyme phytochelatin synthase the phytochelatins (PCs). have been isolated from plant and yeast species. Unexpectedly, genes with similar sequences to those encoding phytochelatin The PCs form a family of peptides that consist of repeti- synthase have been identified in some animal species. tions of the γ-Glu-Cys dipeptide followed by a terminal Gly — the basic structure being (γ-Glu-Cys)n–Gly [(PC) n], Addresses where n is generally in the range two to five. PCs have Department of Genetics, University of Melbourne, Parkville, been identified in a wide variety of plant species and in Victoria 3052, Australia; e-mail: [email protected] some microorganisms. They are structurally related to glu- γ Current Opinion in Plant Biology 2000, 3:211–216 tathione (GSH; -Glu-Cys-Gly) and were presumed to be the products of a biosynthetic pathway. Numerous physio- 1369-5266/00/$ — see front matter logical, biochemical and genetic studies have confirmed © 2000 Elsevier Science Ltd. All rights reserved. that GSH is the substrate for PC biosynthesis (Figure 1). Abbreviations PC structure and synthesis have been reviewed previously GCS γ-glutamylcysteine synthetase [2••,4]. In addition, a number of structural variants of PCs, GS glutathione synthetase γ β γ γ GSH glutathione such as ( -Glu-Cys)n– -Ala, ( -Glu-Cys)n–Ser and ( -Glu- PC phytochelatin Cys)n–Glu, have been identified in some plant species. PCR polymerase chain reaction RT reverse transcription PCs are rapidly induced in vivo by a wide range of heavy- metal ions. The enzyme PC synthase was first identified Introduction by Grill et al. [5] and has been characterised in a number of Heavy-metal toxicity can elicit a variety of adaptive subsequent studies [6,7]. The enzyme is a γ−Glu-Cys responses in plants. These responses have been compre- dipeptididyl transpeptidase (EC 2.3.2.15) and it catalyses hensively reviewed for plants exposed to cadmium [1••], the transpeptidation of the γ−Glu-Cys moiety of GSH the metal for which published studies are most extensive. either onto a second GSH molecule to form PC(n=2) or A ubiquitous mechanism for heavy-metal detoxification is onto a PC molecule to produce a PC(n+1) oligomer. PC the chelation of the metal ion by a ligand. A variety of synthase is activated by a variety of heavy-metal ions. metal-binding ligands have been described in plants, and their respective roles in heavy-metal detoxification have Genetic approaches to studying PC been reviewed [2••]. Such ligands include organic acids, biosynthesis and function amino acids, peptides and polypeptides. Understanding Significant recent advances in our understanding of the the genetic and molecular basis of such mechanisms is an molecular basis of PC biosynthesis and function have come

Figure 1

Phytochelatin biosynthetic pathway. Known Cd points of positive and negative regulation of enzyme activity or gene expression are ⊕ O Glu + Glu indicated by and , respectively.

– + A. thaliana (At), B. juncea (Bj) and T. aestivum + γGlu–Cys GSHPC PC–Cd Vacuole GCS GS PCS HMT1 (Ta) indicate where particular regulatory Cys influences have been observed in particular species. HMT1 is a vacuolar membrane transporter of PC–Cd complexes. JA, mRNA mRNA mRNA jasmonic acid; PCS, phytochelatin synthase. + JA + At At + Cd + Cd + Ta At/Bj At Gene Gene Gene Current Opinion in Plant Biology Table 1

Genes involved in phytochelatin biosynthesis and function.

Organism Gene/locus Activity/function References

PC biosynthesis Sp Gsh1 GCS/GSH biosynthesis At CAD2 GCS/GSH biosynthesis [8•] Sp Gsh2 GS/GSH biosynthesis At CAD1 PC synthase/PC biosynthesis [11,12••] Sp PCS1 PC synthase/PC biosynthesis [12••,14••]

PC function Sp Hmt1 PC–Cd vacuolar membrane ABC-type transporter [23] Sp Ade2,6,7,8 metabolism of cysteine sulfinate to products involved in sulphide biosynthesis; [26] also required for adenine biosynthesis. Sp Hmt2 Mitochondrial sulfide:quinone oxidoreductase/detoxification of sulphide [28] Ca Hem2 Porphobilinogen synthase/siroheme biosynthesis (cofactor for sulfite reductase) [27]

Other Cd-detoxification mechanisms Sc YCF1 GSH–Cd vacuolar membrane ABC-type transporter [22]

At, A. thaliana; Ca, C. albicans; Sc, S. cerevisiae; Sp, S. pombe. from molecular genetic studies using a number of model mutant screens. Biochemical confirmation of the activity of organisms. These approaches have centred on the identifi- the Arabidopsis and S. pombe gene products, purified as epi- cation of Cd-sensitive mutants of the plant Arabidopsis tope-tagged derivatives [13••,14••] or expressed in E. coli thaliana and the yeasts Schizosaccharomyces pombe and Can- [12••], demonstrated that each was sufficient for GSH- dida glabrata (Table 1). In addition, the expression of plant dependent, metal-ion-activated PC biosynthesis in vitro. cDNAs in strains of Escherichia coli and Saccharomyces cerevisiae has been particularly useful in the identification and An alignment of the amino acid sequences of the Ara- analysis of genes involved in functions related to heavy- bidopsis and S. pombe PC-synthase proteins shows a metal detoxification. Both of these approaches have been significant level of homology in the amino-terminal region, useful in identifying the genes that encode the enzymes with little apparent conservation of the carboxy-terminal involved in GSH biosynthesis ([8•]; and see [9,10]) and, region (Figure 2). In both sequences, the latter region con- more recently, the genes encoding PC synthase. tains numerous Cys residues, many as adjacent or neighbouring pairs, although the arrangement of these PC synthase genes in plants and yeast pairs is not conserved between the two sequences. The cadmium-sensitive cad1 mutants of Arabidopsis have wild-type levels of GSH but are PC-deficient and lack A second gene, AtPCS2, with significant homology to PC synthase activity in vitro. It was predicted that CAD1 CAD1/AtPCS1 has been found in the Arabidopsis genome is the structural gene for PC synthase [11] (Table 1). The [12••]. This discovery was unexpected because PCs were Arabidopsis CAD1 gene (also referred to as AtPCS1) has not detected in cad1 mutants after prolonged exposure to been isolated using a positional cloning strategy [12••]. Cd, suggesting the presence of a single active PC synthase cDNA clones of AtPCS1 [13••] and a similar gene in [11]. When AtPCS2 is expressed in yeast it confers wheat (TaPCS1) [14••] have also been identified by their Cd-resistance, indicating that its gene product is active ability to confer resistance to Cd when expressed in the (CS Cobbett, unpublished data). The function of this gene yeast S. cerevisiae. Both of the latter studies [13 ••,14••] remains to be determined. It seems likely that, in most tis- used various yeast mutants to demonstrate that the mech- sues, AtPCS2 is expressed at a relatively low level anism of Cd-resistance conferred by these cDNAs was compared with AtPCS1. Nevertheless, for it to have been distinct from other recognised Cd-detoxification mecha- preserved throughout evolution as a functional PC syn- nisms in yeast, was dependent on GSH, and mediated PC thase, AtPCS2 must presumably be the predominant PC biosynthesis in vivo. synthase in some tissue(s) or environmental conditions.

A gene, SpPCS1, that is similar to the plant PC-synthase Interestingly, although PC(n=2) has been described in the genes was identified in the genome of the fission yeast yeast S. cerevisiae, there is no homologue of the PC-synthase S. pombe (Figure 2). Targeted-deletion mutants of this gene genes in the S. cerevisiae genome. An alternative pathway for were also Cd-sensitive and PC-deficient, confirming the PC biosynthesis in S. pombe has been proposed [15], howev- analogous functions of the plant and yeast genes er, and it may be that this pathway functions in S. cerevisiae. [12••,14••] (Table 1). It is remarkable that such a Cd-sensi- Nevertheless, the cad1-3 mutant of Arabidopsis and the PC- tive mutant had not been isolated through various earlier synthase deletion mutant of S. pombe both lack detectable PCs, suggesting that such an alternative pathway is of little Figure 2 physiological relevance in these organisms.

At Regulation of PC biosynthesis Ta PC biosynthesis may be regulated by a number of mecha- Sp Amino- terminus terminus nisms. For example, in Brassica juncea, exposure to Cd Ce Carboxy- produces a requirement for both cysteine and GSH for PC Conserved Variable biosynthesis, that is met by coordinate transcriptional reg- Current Opinion in Plant Biology ulation of genes involved in sulphur transport and Schematic comparison of PC-synthase polypeptides from different assimilation [16,17] and GSH biosynthesis [18]. Similarly, organisms. The positions of Cys residues are indicated by vertical bars. exposure of Arabidopsis plants to Cd and Cu causes an The conserved amino-terminal domains exhibit at least 40% identical increase in the transcription of genes encoding GSH amino acids in pair-wise comparisons of the four sequences. At, A. thaliana (CAD1/AtPCS1; GenBank accession numbers, reductase and enzymes involved in the GSH biosynthetic AF135155 and AF085230); Ta, Triticum aestivum (TaPCS1; pathway: γ-glutamylcysteine synthetase (GCS) and glu- AF093252); Sp, S. pombe (SpPCS; Z68144); and Ce, C. elegans tathione synthetase (GS) [19••] (Figure 1). The signal (CePCS1; Z66513). molecule, jasmonate, mediates a similar effect in the absence of heavy-metal exposure, although it has not been demonstrated that jasmonate is directly involved in medi- and sensitivity to Cd), but this activity is expressed only ating the effects of heavy-metal stress on gene expression in the presence of Cd [11], demonstrating that the car- [19••]. There is also circumstantial evidence for the post- boxy-terminal domain is not absolutely required for transcriptional regulation of GCS expression [9], in either catalysis or activation. addition to the well-recognised regulation of GCS activity through GSH feedback inhibition [9,10] (Figure 1). It has been suggested that the carboxy-terminal domain acts as a local sensor by binding heavy-metal ions (pre- The importance of the regulation of GSH biosynthesis in sumably via the multiple Cys residues) and bringing them modulating PC expression is reinforced by the observation into contact with the activation site in the amino-terminal that PC biosynthesis and Cd tolerance are increased in trans- catalytic domain [12••]. This model is consistent with the genic B. juncea in which either GCS or GS was findings of biochemical studies using epitope-tagged over-expressed [20,21], although similar effects were not AtPCS1, which demonstrated that this enzyme binds Cd observed when GCS was over-expressed in poplar (see [10]). ions at high affinity (Kd = 0.54 ± 0.20 µM) and high capac- ity (stoichiometric ratio = 7.09 ± 0.94) [13••]. Although PC PC-synthase activity is expected to to be the major deter- synthase can bind Cd ions in the absence of GSH, an inter- minant of the rate of PC synthesis. Kinetic studies using esting and unresolved issue is whether Cd ions are solely plant cell cultures exposed to Cd demonstrated that PC involved in the activation of the enzyme or form an inte- biosynthesis occurs within minutes of exposure to the gral component of the substrate. In the former model, Cd heavy metal and is independent of de novo protein synthe- ions would interact with an activation site in the amino-ter- sis [2••]. PC synthase extracted from plant cells or tissues minal domain that is distinct from the GSH–PC is activated by various heavy-metal ions [5–7] (Figure 1). substrate-binding sites. In the latter model, GSH/PC–Cd Likewise, in in vitro studies of PC synthase expressed in complexes would be the substrate for the enzyme, and E. coli or in S. cerevisiae, the enzyme was activated to vary- individual Cd ions bound to the substrate would remain ing extents by Cd, Cu, Ag, Hg, Zn and Pb ions [12••–14••]. bound to the PC product. A schematic illustration of these alternative models is shown in Figure 3. The mechanism by which PC synthase is activated appears to be relatively non-specific with respect to the Previous studies indicated that PC synthase was activating metal ion, although some metals are more expressed constitutively and that the levels of this effective than others. One model for the function of PC enzyme in cell cultures or intact plants were generally synthase is that the conserved amino-terminal domain unaffected by exposure to Cd [5–7]. This is supported by confers the catalytic activity of this enzyme. Activation Northern or RT-PCR (reverse transcription polymerase probably arises from an interaction between residues in chain reaction) analysis of the expression of this domain and free metal ions or metal–GSH complex- CAD1/AtPCS1, which showed that levels of mRNA were es. This model is supported by evidence from the not influenced by exposure of plants to Cd, even under molecular characterisation of the cad1-5 mutant of Ara- conditions of severe heavy-metal induced stress bidopsis, which has a nonsense mutation that results in ([12••,13••]; CS Cobbett, unpublished data). Interesting- premature termination of translation after the conserved ly, however, RT-PCR analysis of TaPCS1 expression in amino-terminal domain [12••]. The truncated polypep- wheat roots indicated induction of mRNA on exposure tide is predicted to lack nine of the ten Cys residues in to Cd [14••] (Figure 1). This evidence suggests that, in the carboxy-terminal domain [12••]. This mutant retains some species, PC-synthase activity may be regulated at some activity (as measured by in vivo PC concentrations both the transcriptional and post-translational levels. Figure 3

Schematic models for the function of PC (a)Amino-terminal domain (b) Amino-terminal domain synthase. The carboxy-terminal domain acts as a PC PC local sensor of heavy-metal ions, such as Cd. PC Cd Cd The Cys residues bind Cd ions, bringing them GSH/PC into closer proximity and transferring them to the GSH + GSH/PC GSH + Cd amino-terminal, catalytic domain. The activated Cd amino-terminal domain catalyses the transfer of the γ-Glu-Cys moiety of a molecule of GSH onto a second molecule of GSH (or an existing PC GSH molecule) to form a PC product. In model (a), Cd–Cys Cd–Cys Cys Cys Cd alone interacts with the Cys residues and PC subsequently with an activation site in the amino- Cd–Cys Cd–Cys Cys Cys terminal domain that is distinct from the Carboxy-terminal GSH Carboxy-terminal GSH/PC-substrate-binding sites. The PC Cd–Cys domain Cd–Cys domain Cys Cys product is formed and binds Cd subsequently. In model (b), cytoplasmic Cd ions bound to GSH Current Opinion in Plant Biology or a previously formed PC interact first with the Cys residues in the carboxy-terminal domain and subsequently with the catalytic site in the amino- terminal domain where Cd remains an integral component of the substrate and, consequently, the product of the reaction. A variation of this model could involve GSH–Cd complexes binding at both the donor and acceptor sites.

PC synthase genes in animals? heavy-metal ions plays an important role in naturally Although PCs have not yet been identified in an animal evolved heavy-metal-tolerant plants [24,25]. In the vac- species there is a gene similar to AtPCS1 in the nematode, uole, sulphide ions are an essential component of PC–Cd Caenorhabditis elegans (Figure 2). The amino-terminal complexes, and a number of Cd-sensitive mutants region of the predicted gene product is equally similar to believed to be affected in aspects of sulphide metabolism the plant and yeast proteins. In contrast, the carboxy-termi- have been identified in yeast species [26] (Table 1). For nal domain has little obvious similarity to the plant or yeast example, in Candida glabrata, the Cd-sensitive hem2 gene-products, except that it contains multiple pairs of Cys mutant is deficient in porphobilinogen synthase, an residues. In addition, an homologous expressed sequence enzyme involved in the biosynthesis of siroheme, which is tag (GenBank accession number AU061531) has been iden- a cofactor for sulphite reductase [27]. A different mutant in tified in the slime mould Dictyostelium discoideum and, using S. pombe, hmt2, appears to lack a sulphide:quinone oxidore- PCR, sequences similar to the conserved amino-terminal ductase that is believed to be important for the regions of PC synthase genes have been identified from the detoxification of excess sulphide formed in response to Cd aquatic midge Chironomus and earthworm species (CS Cob- exposure and PC biosynthesis [28]. Further studies are bett, unpublished data). There is, as yet, no evidence that required to establish the pathways of sulphide metabolism, these animal genes encode PC-synthase activity. Nonethe- particularly in plants in which Cd-sensitive sulphide- less, it seems likely that they too encode PC synthase. The metabolism mutants have not been identified. existence of PC-synthase-like genes in animals suggests that PCs play a wider role in heavy-metal detoxification Roles for PCs in metal detoxification or than previously expected. A superficial view of the limited metabolism? selection of species in which such sequences have been The PC-deficient mutants of Arabidopsis and S. pombe are identified might suggest that organisms with an aquatic or most sensitive to Cd and the arsenate anion, and are less sen- soil habitat are more likely to express PCs. sitive to other heavy metals such as Cu and Hg [12••,14••]. Although this clearly demonstrates that PCs can have an Other aspects of cadmium detoxification important role in detoxification, it remains to be determined In both plant and yeast species, heavy metals (Cd in par- whether this is the function for which they evolved or an ticular) are sequestered to the vacuole. In S. cerevisiae, incidental role. Other proposed roles for PCs have included YEAST CADMIUM FACTOR 1 (YCF1) [22], and in their involvement in essential heavy-metal homeostasis, Fe S. pombe, HEAVY-METAL TOLERANCE 1 (Hmt1) [23], metabolism and sulphur metabolism [2••,4]. Although the encode members of the ATP-binding cassette (ABC) fam- evidence of a role for PCs in detoxification is strong, there is ily of membrane transporters that transport GSH–Cd and only circumstantial evidence in support of these alternatives. PC–Cd complexes, respectively, into the vacuole and play important roles in Cd detoxification (Table 1). There is Can PCs have a role in Cd detoxification at levels of Cd also increasing evidence that vacuolar localisation of exposure relevant to plants in a natural environment? It has been estimated that solutions of non-polluted soils contain 3. Salt DE, Smith RD, Raskin I: Phytoremediation. Annu Rev Plant Cd concentrations of less than 0.3 µM [29]. In contrast, most Physiol Plant Mol Biol 1998, 49:643-668. experimental studies use Cd concentrations more than 1 µM 4. Zenk MH: Heavy metal detoxification in higher plants – a review. Gene 1996, 179:21-30. [1••]. Wagner [29] has argued that at low levels of Cd nor- 5. Grill E, Loffler S, Winnacker E-L, Zenk MH: Phytochelatins, the heavy- mally present in most soils, Cd would largely be complexed metal-binding peptides of plants, are synthesized from glutathione by with vacuolar citrate, and only at high Cd concentrations (not a specific γ-glutamylcysteine dipeptidyl transpeptidase (phytochelatin generally found in natural environments) would PCs play a synthase). Proc Natl Acad Sci USA 1989, 86:6838-6842. role. Nonetheless, the sensitivity of the Arabidopsis cad1-3 6. Klapheck S, Schlunz S, Bergmann L: Synthesis of phytochelatins and homo-phytochelatins in Pisum sativum L.. Plant Physiol 1995, mutant to concentrations of Cd as low as 0.6 µM [11] at least 107:515-521. suggests that PCs may have a role in heavy-metal detoxifica- 7. Chen J, Zhou J, Goldsbrough PB: Characterization of phytochelatin tion in an unpolluted environment. Even at lower synthase from tomato. Physiol Plant 1997, 101:165-172. concentrations of Cd, at which sensitive plants may not be 8. Cobbett CS, May MJ, Howden R, Rolls B: The glutathione-deficient, easily distinguished from tolerant genotypes by obvious phe- • cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in γ-glutamylcysteine synthetase. Plant J 1998, 16:73-78. notypic differences, a plant expressing PCs may have a This paper describes the molecular characterisation of the first glutathione- selective advantage. It would be of interest to test the fitness deficient mutant identified in plants and provides an important genetic con- of the cad1-3 mutant compared to wild-type plants on a range firmation of the role of glutathione as the substrate for PC biosynthesis. of soils from natural environments. 9. May MJ, Vernoux T, Leaver C, Van Montague M, Inze D: Glutathione homeostasis in plants: implications for environmental sensing and plant development. J Exp Botany 1998, 49:649-667. Conclusions 10. Noctor G, Arisi A-CM, Jouanin L, Kunert K, Rennenberg H, Foyer CH: Glutathione: biosynthesis, metabolism and relationship to stress The identification of PC-synthase genes from plants and tolerance explored in transgenic plants. J Exp Botany 1998, other organisms is a significant breakthrough that will lead 49:623-647. to a better understanding of the regulation of a critical step 11. Howden R, Goldsbrough PB, Andersen CR, Cobbett CS: Cadmium- in PC biosynthesis. Nonetheless, we must keep in mind sensitive, cad1, mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol 1995, 107:1059-1066. the numerous other aspects of PC biosynthesis and func- 12. Ha S-B, Smith AP, Howden R, Dietrich WM, Bugg S, O’Connell MJ, tion, and the ways in which they, too, are regulated at a •• Goldsbrough PB, Cobbett CS: Phytochelatin synthase genes from cellular and physiological level in response to heavy-metal Arabidopsis and the yeast, Schizosaccharomyces pombe. Plant Cell 1999, 11:1153-1164. exposure. These include aspects of sulphur assimilation, One of a trio of papers describing the isolation of PC-synthase genes (see GSH and sulphide biosynthesis, PC compartmentalisation also [13••,14••]). Positional cloning of the CAD1 gene of Arabidopsis and the signal pathways through which metal toxicity leads and subsequent expression in E. coli confirmed that this gene, and an homologous gene in the yeast S. pombe, encodes PC synthase. to gene regulation. In the long term, we aim to understand 13. Vatamaniuk OK, Mari S, Lu Y-P, Rea PA: AtPCS1, a phytochelatin heavy-metal detoxification at the whole plant level and to •• synthase from Arabidopsis: isolation and in vitro reconstitution. exploit such knowledge. The concept of phytoremediation Proc Natl Acad Sci USA 1999, 96:7110-7115. One of a trio of papers describing the isolation of PC-synthase genes (see of contaminated soils has been increasingly supported by also [12••,14••]). An Arabidopsis PC synthase cDNA was identified through research in recent years. The understanding of heavy-metal its expression in yeast conferring increased Cd-tolerance. Importantly, using detoxification processes afforded by investigations in a purified, epitope-tagged derivative of AtPCS1, these authors demonstrated this enzyme is necessary and sufficient for PC biosynthesis and that the model systems such as Arabidopsis and S. pombe will, in the enzyme itself binds Cd ions. near future, allow us to explore the mechanisms by which 14. Clemens S, Kim EJ, Neumann D, Schroeder JI: Tolerance to toxic some species are capable of hyper-accumulation of metals •• metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J 1999, 18:3325-3333. such as Cd and how they may be best used for phytoreme- One of a trio of papers describing the isolation of PC-synthase genes (see diation. Ultimately, the genetic manipulation of such plants also [12••,13••]). A wheat cDNA encoding PC synthase was identified through its expression in yeast conferring increased Cd-resistance. may further enhance their usefulness for this purpose. Subsequent analysis of this gene, and homologous genes in Arabidopsis and S. pombe, confirmed its function. Acknowledgements 15. Hayashi Y, Nakagawa CW, Mutoh N, Isobe M, Goto T: Two pathways I wish to acknowledge the contributions of members of my laboratory and in the biosynthesis of cadystins (γ-EC)nG in the cell-free system valuable collaborations with colleagues. The work of my laboratory is of the fission yeast. Biochem Cell Biol 1991, 69:115-121. supported by the Australian Research Council. 16. Heiss S, Schafer HJ, Haag-Kerwer A, Rausch T: Cloning sulfur assimilation genes of Brassica juncea L.: cadmium differentially References and recommended reading affects the expression of a putative low-affinity sulfate transporter Papers of particular interest, published within the annual period of review, and isoforms of ATP sulfurylase and APS reductase. Plant Mol have been highlighted as: Biol 1999, 39:847-857. • of special interest 17. Lee SM, Leustek T: The effect of cadmium on sulfate assimilation •• of outstanding interest enzymes in Brassica juncea. Plant Sci 1999, 141:201-207. 1. Sanita di Toppi L, Gabbrielli R: Response to cadmium in higher 18. Schafer HJ, Haag-Kerwer A, Rausch T: cDNA cloning and expression •• plants. Environ Exp Bot 1999, 41:105-130. analysis of genes encoding GSH synthesis in roots of the heavy An excellent overview of the adaptive responses of plants to Cd toxicity. The metal accumulator Brassica juncea L.: evidence of Cd-induction of need for an integrated approach to understanding the complex physiological a putative mitochondrial gamma-glutamylcysteine synthetase mechanisms is emphasised. isoform. Plant Mol Biol 1998, 37:87-97. 2. Rauser WE: Structure and function of metal chelators produced 19. Xiang C, Oliver DJ: Glutathione metabolic genes coordinately •• by plants; the case for organic acids, amino acids, phytin and •• respond to heavy metals and jasmonic acid in Arabidopsis. Plant metallothioneins. Cell Biochem Biophys 1999, 31:19-48. Cell 1998, 10:1539-1550. A timely review examining the contributions of various heavy-metal-binding An exciting paper providing insights into the signal transduction and regula- ligands to detoxification processes in plants. tory pathways influencing GSH and, consequently, PC biosynthesis. The authors demonstrate that the transcription of the GSH biosynthetic genes is 24. Chardonnens AN, Tenbookum WM, Kuijper LDJ, Verkleij JAC, induced both in the presence of Cd and the signal molecule, jasmonate, sug- Ernst WHO: Distribution of cadmium in leaves of cadmium gesting a possible role for jasmonate in the signal transduction pathway for tolerant and sensitive ecotypes of Silene vulgaris. Physiol Plant Cd stress. 1998, 104:75-80. 20. Zhu YL, Pilon-Smits EAH, Jouanin L, Terry N: Overexpression of 25. Chardonnens AN, Koevoets PLM, van Zanten A, Schat H, Verkleij JAC: glutathione synthetase in Indian Mustard enhances cadmium Properties of enhanced tonoplast transport in naturally selected accumulation and tolerance. Plant Physiol 1999, 119:73-79. zinc-tolerant Silene vulgaris. Plant Physiol 1999, 120:779-785. 21. Yong LZ, Pilon-Smits EAH, Tarun AS, Weber SU, Jouanin L, Terry N: 26. Juang R-H, MacCue KF, Ow DW: Two purine biosynthetic enzymes Cadmium tolerance and accumulation in Indian Mustard is that are required for cadmium tolerance in Schizosaccharomyces enhanced by overexpressing γ-glutamylcysteine synthetase. Plant pombe utilize cysteine sulfinate in vitro. Arch Biochem Biophys Physiol 1999, 121:1169-1177. 1993, 304:392-401. 22. Li Z-S, Lu Y-P, Zhen R-G, Szczypka M, Thiele DJ, Rea PA: A new 27. Hunter TC, Mehra RK: A role for HEM2 in cadmium tolerance. pathway for vacuolar cadmium sequestration in Saccharomyces J Inorg Biochem 1998, 69:293-303. cerevisiae: YCF1-catalyzed transport of bis(glutathionato)- cadmium. Proc Natl Acad Sci USA 1997, 94:42-47. 28. Vande Weghe JG, Ow DW: A fission yeast gene for mitochondrial sulfide oxidation. J Biol Chem 1999, 274:13250-13257. 23. Ortiz DF, Ruscitti T, McCue KF, Ow DW: Transport of metal-binding peptides by HMT1, a fission yeast ABC-type vacuolar membrane 29. Wagner GJ: Accumulation of cadmium in crop plants and its protein. J Biol Chem 1995, 270:4721-4728. consequences to human health. Adv Agron 1993, 51:173-212.