Tolerance and detoxification mechanisms in marine diatom Phaeodactylum tricornutum exposed to cadmium

Tolerance is one of the most important mechanisms for survival. Organisms living in polluted environments with heavy metals develop tolerance to these contaminants; this tolerance has been attributed to the ability to synthesize metal-binding substances or another different mechanism able to mediate metal detoxification. It has been demonstrated that high levels of metals induce the formation of these substances in plants, algae, and some fungi (phytochelatins), which are a protective mechanism against the toxicity of heavy metals. In this study we have addressed the capacity of the microalga Phaeodactylum tricornutum to produce metal-binding pep tides in response to stress induced by cadmium. Liquid chromatography using biogel P-60, thiol analysis in chromatographic fractions, and capillary electrophoresis provided evidence of phytochelatins production in this alga. Contamination of rivers and estuaries with heavy metals has been documented in numerous surveys. The toxic effects of high concentrations of these trace metals on aquatic organisms, including algae, have also been the subject of an extensive literature (Whitton and Say 1975; Vymazal 1987). Heavy metals are important pollutants in aquatic environments with adverse effects on the organisms. Tolerance is one of the most important mechanisms for survival. A variety of tolerance mechanisms against cytotoxic effects of heavy metals have been described (Maeda and Sakaguchi 1990). Thus, many organisms respond to this effect by synthesizing metal chelating-proteins or peptides. In plants, algae, and some fungi, these peptides were designated as: phytochelatins (PC), cadystins, Cd-binding peptides (Cd-BP), or y-glutamyl peptides (Steffens 1990). Because of their ability to bind heavy metal ions, these molecules are considered to play a role in cellular metal homeostasis and metal detoxification. Phytochelatins have the general structure (y-Glu-Cys)n-Gly, where n can range from 2 to 11 depending on the species and the conditions of their induction. In vascular plants, the biosynthesis of phytochelatins is catalyzed by a specific y-glutamylcysteinedipeptidyl transpeptidase, called phytochelatin synthase, which is activated in the presence of metal ions and uses glutathion (GSH) as a substrate. An additional component of these molecular complexes is acidlabile sulfur (Steffens et al. 1986). Incorporation of sulfide in PC-metal complexes can result in the formation of metal-S crystallites, which might be the storage form of metal (Ortiz et al. 1992). Evidence that microalgal Cd tolerance is achieved by a mechanism that includes binding of induced polypeptidesto this metal ion was first obtained with Euglena gracilis (Gingrich et al. 1986). Cadmium has been shown to increase sulfate reduction in plants (Nussbaum et al. 1988). Incorporation of acidlabile sulfur into Cd-peptide complexes increases the metalbinding affinity. The relevance of the presence of sulfide in PC-metal complexes to metal detoxification is substantiated by the observation that mutants of Schizosaccharomyces pombe that produce only PC-Cd complexes without sulfide are hypersensitive to Cd (Mutoh and Hayashi 1988). In the present study, the resistence of the marine diatom Phaeodactylum tricornutum Bohlin (Bacillariophyceae) to cadmium by means of phytochelatins and acid-labile sulfur was demonstrated. Materials and methods