As is a ubiquitous element present in the atmosphere as well as in the aquatic and terrestrial environments. Arsenite and arsenate are the major forms of As intoxication, and these anions are readily taken up by plants. Both anions efficiently induce the biosynthesis of phytochelatins (PCs) ([␥-glutamate-cysteine] n -glycine) in vivo and in vitro. The rapid induction of the metal-binding PCs has been observed in cell suspension cultures of Rauvolfia serpentina, in seedlings of Arabidopsis, and in enzyme preparations of Silene vulgaris upon challenge to arsenicals. The rate of PC formation in enzyme preparations was lower compared with Cd-induced biosynthesis, but was accompanied by a prolonged induction phase that resulted finally in higher peptide levels. An approximately 3:1 ratio of the sulfhydryl groups from PCs to As is compatible with reported As-glutathione complexes. The identity of the As-induced PCs and of reconstituted metal-peptide complexes has unequivocally been demonstrated by electrospray ionization mass spectroscopy. Gel filtration experiments and inhibitor studies also indicate a complexation and detoxification of As by the induced PCs.

The phytochelatin homologs homo-phytochelatins are heavy metal-binding peptides present in many legumes. To study the biosynthesis of these compounds, we have isolated and functionally expressed a cDNA GmhPCS1 encoding homo-phytochelatin synthase from Glycine max, a plant known to accumulate homo-phytochelatins rather than phytochelatins upon the exposure to heavy metals. The catalytic properties of GmhPCS1 were compared with the phytochelatin synthase AtPCS1 from Arabidopsis thaliana. When assayed only in the presence of glutathione, both enzymes catalyzed phytochelatin formation. GmhPCS1 accepted homoglutathione as the sole substrate for the synthesis of homo-phytochelatins whereas AtPCS1 did not. Homo-phytochelatin synthesis activity of both recombinant enzymes was significantly higher when glutathione was included in the reaction mixture. The incorporation of both glutathione and homoglutathione into homo-phytochelatin, n ‫؍‬ 2, was demonstrated using GmhPCS1 and AtPCS1. In addition to bis(glutathionato)⅐metal complexes, various other metal⅐thiolates were shown to contribute to the activation of phytochelatin synthase. These complexes were not accepted as substrates by the enzyme, thereby suggesting that a recently proposed model of activation cannot fully explain the catalytic mechanism of phytochelatin synthase ( Phytochelatins (PCs) 1 of the general formula (Glu-Cys) n -Gly (n ϭ 2-11) are the principal heavy metal-detoxifying compounds in the plant kingdom (1-4). These peptides are linear polymers of the ␥-glutamyl-cysteinyl (␥-Glu-Cys) portion of glutathione (GSH). Iso-phytochelatins are isoforms of phytochelatins (2) in which the terminal amino acid consists of serine (5), glutamic acid (6), glutamine (7), or in the case of the homophytochelatins, ␤-alanine (8, 9). Although the enzymology of phytochelatin formation by a constitutive phytochelatin synthase was clarified early on (10), the gene encoding this enzyme was discovered only recently (11-13). Remarkably, phytochelatin synthase activity has been even found to occur in a nematode Caenorhabditis elegans (14).Although the biosynthesis of phytochelatins seems to be clear on the enzymological and molecular level, there is little information available on the formation of iso-phytochelatins (2). Because iso-phytochelatins occur in only those plants that contain glutathione isomers, e.g. homoglutathione (hGSH) (8), hydroxymethyl-glutathione (5), or ␥-glutamyl-cysteinyl-glutamic acid (6), it could be supposed that these substances are involved in iso-phytochelatin biosynthesis. Furthermore, it was shown that several leguminaceous plants contain hGSH but not GSH (8,(15)(16)(17), and it could be, therefore, assumed that hGSH is the substrate for homo-phytochelatin biosynthesis. However, it has been reported that a crude enzyme preparation from Pisum catalyzes the formation of homo-phytochelatin from homoglutathione in the presence of GSH (9). In addition, using the same enzyme preparation from Pisum, and in the presence of GSH, the synthesis of (␥-Glu-Cys) ...

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A method based on pneumatically assisted electrospray ionisation tandem mass spectrometry (ESI MS-MS) was developed for the identification, sequencing and determination of phytochelatin (PC) peptides in plant tissue and plant cell cytosols. The ionization and fragmentation conditions were optimized using a series of (GluCys) 2 Gly (PC 2 ), (GluCys) 3 Gly (PC 3 ), and (GluCys) 4 Gly (PC 4 ) standards prepared from glutathione by enzymatically (g-glutamylcysteine dipeptyl transpeptidase) assisted biosynthesis in the presence of Cd 2+ . Phytochelatins were found to ionize readily to produce a characteristic mono-protonated ion. The collision-induced dissociation (CID) of this ion followed by mass spectrometry (MS-MS mode) allowed the determination of the amino acid sequence of each of the PCs. Calibration curves were linear up to a concentration of 2 mg ml 21 in the MS and MS-MS modes with the detection limits at the low ng ml 21 level. The method was applied to the determination of phytochelatin peptides biosynthesized by a number of plant cell cultures exposed to the Cd stress. The results agreed with those obtained by an independent procedure based on reversed-phase HPLC with post-column derivatization of the -SH groups with 5,5A-dithiobis-2-nitrobenzoic acid and spectrophotometric detection.

In a recent report, it was claimed that azuki beans (Vigna angularis) do not synthesize phytochelatins (PCs) upon exposure to cadmium, although glutathione (GSH), the substrate for PC synthesis, is present in this plant. This legume species thus would be the first exception in the plant kingdom that would fail to complex heavy metals by PCs. Here, we report that not GSH, but only homoglutathione can be detected in this plant and that homo-phytochelatins are formed when azuki beans are challenged with heavy metals such as cadmium. We also show that the 5,5Ј-dithiobis(2-nitrobenzoic acid)-oxidized GSH reductase recycling assay, used for GSH quantification in the recent study of heavy metal tolerance in azuki beans, reacts both with GSH and homoglutathione and therefore cannot be used when biological samples should be analyzed exclusively for GSH.