Cadmium-Responsive Thiols in the Ectomycorrhizal Fungus Paxillus involutus

Abstract

Molecular and cellular mechanisms underlying the sustained metal tolerance of ectomycorrhizal fungi are largely unknown. Some of the main mechanisms involved in metal detoxification appear to involve the chelation of metal ions in the cytosol with thiol-containing compounds, such as glutathione, phytochelatins, or metallothioneins. We used an improved high-performance liquid chromatography method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (␥-glutamylcysteine, glutathione) to higher-molecular-mass compounds (phytochelatins). We found that glutathione and ␥-glutamylcysteine contents increased when the ectomycorrhizal fungus Paxillus involutus was exposed to cadmium. An additional compound with a 3-kDa molecular mass, most probably related to a metallothionein, increased drastically in mycelia exposed to cadmium. The relative lack of phytochelatins and the presence of a putative metallothionein suggest that ectomycorrhizal fungi may use a different means to tolerate heavy metals, such as Cd, than do their plant hosts.The response of ectomycorrhizal fungi to toxic metals is important, since these organisms are present at polluted sites, participate in crucial symbiotic relationships with trees that grow at these sites, and alleviate metal toxicity in the host plants (8,13,26). Paxillus involutus is an ectomycorrhizal fungus with a high tolerance to cadmium, one of the most toxic heavy metals, that can form symbioses with a broad range of host species. The uptake of metals by ectomycorrhizal fungi (2) and the effects of metals on biomass production (3) and nutrient uptake (1) in ectomycorrhizal fungi have been studied. The expression of enzymes involved in antioxidative response mechanisms is regulated by Cd in ectomycorrhizal fungi (12,22) and in mycorrhizal roots of Pinus sylvestris associated with P. involutus (26).Thiol compounds, including reduced glutathione (␥-glutamyl cysteinyl glycine), phytochelatins (PCs), and metallothioneins, are essential components of Cd detoxification pathways in various organisms (5-7, 11). Thiol composition of ectomycorrhizal fungi has not been studied in detail, although P. involutus and Laccaria laccata accumulate glutathione when exposed to Cd (7,22). Reduced glutathione is the most abundant nonprotein thiol component of eukaryotic cells, acts as a free radical scavenger, and reacts with various oxidants to produce oxidized glutathione (17). Phytochelatins are a family of small cysteinerich peptides capable of binding heavy metal ions via their SH group. The general structure of this set of peptides is [␥-GluCys] n -gly (n ϭ 2 to 11). Phytochelatins are enzymatically synthesized from glutathione and have been found in some fungi, algae, and all plant species examined so far (for reviews, see references 4, 5, and 23). Phytochelatin synthase (␥-glutamylcysteine dipeptidyl transpeptidase; EC 2.3.2.15) catalyzes the addition of the ␥-glutamylcysteine (␥-GluCys) moiety of glutathione onto another glutathione molecule (to produce PC...

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