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 cere- visiae 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).