Other Metal Chelating Agents Thiol Peptides

Another group of agents with metal-chelating properties has been described in some species of ECM fungi, but unlike siderophores and LMW organic acids, they are more related to detoxification mechanisms than to metal ion nutrition or mineral solubilization. This group includes MTs, PCs, and GSH, all the thiol peptides (Gadd 1993; Meharg 2003; Pocsi et al. 2004; Bellion et al. 2006). These peptides act intracellularly; once the metal ions have entered the cell, they regulate or prevent the activity of metal ions in the cytosol through complexation. Thus, unlike siderophores and LMW organic acids that can represent avoidance mechanisms in relation to metal detoxification, these peptides represent intracellular detoxification mechanisms.

MTs are peptides of low molecular weight (6-7 kDa), rich in cysteine (Cys), highly ubiquitous - given that they have been identified in animals, plants, fungi, and some bacteria - with the capacity to chelate metals through the thiol (-SH) groups. Some MTs can contain up to 60 amino acids, of which fewer than half correspond to Cys; they do not exhibit aromatic amino acids or histidine. MTs can form clusters with a large amount of divalent or monovalent metal ions through the domains rich in Cys (Robinson et al. 1993). The biosynthesis of MTs is regulated at transcriptional level by a variety of factors, among them some metals: Cd, Zn, Hg, Cu, Au, Ag, Co, Ni, and Bi (Kagi and Shaffer 1988); and of these Cu, Zn, Cd, Hg, Bi, Ag, Au, as well as Pb and Pt can be indeed chelated (Koagi 1991). However, the preference for one or the other metal, both in the regulation of biosynthesis and in chelation, depends exclusively on the organism (Robinson et al. 1993; Hall 2002). MTs were described in ECM fungi in 1986 and their production was induced by the presence of Cd2+ in a P. tinctorius culture medium (Morselt et al. 1986). Later, the production of MTs by P. involutus and L. laccata was shown, regulated by high Cd2+ concentrations (Howe et al. 1997). Using HPLC, several thiol-containing compounds were investigated in P. involutus (Courbot et al. 2004), with GSH, g-glutamylcysteine, and a compound of low molecular weight being detected that the authors suggested to correspond to MTs; all of these increased by exposing the fungus to Cd2+. The genes that codify for MTs were characterized in P. involutus (Pimt1 gene) (Bellion et al. 2007); the gene expression was regulated by Cu and Cd, but not by Zn, and the overexpression of Pimt1 in a strain transformed from

H. cylindrosporum gave it an increased tolerance to Cu. Recently, these genes were also characterized in H. cylindrosporum and their regulation by Cu and Cd was shown, but not by Zn, Pb, or Ni (Ramesh et al. 2009).

PCs are peptides rich in Cys, with a general (g-Glu-Cys)n-Gly (n = 2-11) structure, of low molecular weight, which in fungi does not seem to exceed 2 kDa. Unlike MTs, PCs are not codified in the genome and their presence has been shown mainly in plants, where they have been related to the detoxification of Cd (Cobbett 2000; Mejare and Bulow 2001; Hall 2002). They are synthesized from GSH, through the action of an enzyme (PC synthase), whose activity is regulated by metal ions such as Hg, Ag, Cu, Ni, Au, Pb, Zn, but mainly by Cd, which is the strongest inducer of PC synthase (Gadd 1993; Cobbett 2000; Mejare and Bulow 2001). Until recently, there were no reports of their presence in ECM fungi, and even the synthesis of these peptides has been shown in only a very few fungal species (Bellion et al. 2006). Nevertheless, Collin-Hansen et al. (2007) described for the first time the presence of PCs in the well known edible wild mushroom Boletus edulis. In this fungus, metal exposure induced the production of thiol compounds such as GSH, and an accumulation of Cd in the caps of this macro-mycete was observed due to the formation of complexes with the PCs. When the roots of Picea abies mycorrhized with L. laccata and the pure cultures of fungus were exposed to Cd, an increase was detected in the GSH, but not in the MTs (Galli et al. 1993). Recently, the role of tripeptide GSH (g-Glu-Cys-Gly) was established as a key agent in the responses to various stress situations in fungi (Pocsi et al. 2004); and in the case of metal stress, GSH can act as a precursor to the synthesis of PCs, as a scavenger of reactive oxygen species or as a metal-chelating agent (Ott et al. 2002; Courbot et al. 2004; Hegedus et al. 2007).

PCs, like MTs, have not only been linked to metal detoxification, but also to the regulation of the homeostasis of essential metal ions and other biological processes, acting as xenobiotic detoxifying agents, or antioxidants (Gadd 1993; Rauser 1995; Cobbett 2000; Bellion et al. 2006). Furthermore, these thiol peptides have also been related to the accumulation capacity of metal ions in the fruiting bodies of some basidiomycetes when they grow in contaminated environments, and this represents an enormous risk to human health due to the high consumption of edible wild mushrooms in many countries (Gadd 1993). Although there is a large number of studies in the literature on the accumulation of metals, metalloids, and radio-nuclides by several mushrooms (saprophyte and ECM fungi), most aim at determining the metal concentrations in the caps and stipes of the macrofungi and in their growth substrates. Nevertheless, the physiological and biochemical mechanisms involved in the accumulation are still not well understood, and the participation of metal-chelating agents like siderophores, MTs, PCs, and GSH have been suggested and in some cases proven, as with B. edulis (Collin-Hansen et al. 2007). On the other hand, a correlation between the GSH concentration and the Hg2+ and Cd2+ contents was described in the fruiting bodies of various ECM fungi (Kojo and Lodenius 1989). Recently, Amanita stroboliformes and A. solitaria were described as hyperaccumulators of Ag, accumulating 2,500 times more Ag in their fruiting bodies than in the growth soil (Borovicka et al. 2007). Despite Ag accumulation mechanisms not being investigated in this study, it may be similar to that described for saprophyte Agaricus bisporus, where the participation of MTs was suggested (Byrne and Tusek-Znidaric 1990). The accumulation of 137Cs has been observed in several species of saprophytes and ECM fungi, edible wild mushrooms (Gaso et al. 2000). Among these, the ECM fungus Clavariadelphus truncatus was described as an accumulator of 137Cs, Rb, and Pb; and this was related to metal-chelating compounds (siderophore-type) detected in its fruiting bodies (Gaso et al. 2007).

Given the metal-accumulation capacity of some macromycetes, their use as bioindicators of contaminated environments has been suggested by several authors. However, the variability in accumulation among fungal species is enormous and depends on the developmental stage of the fruiting bodies, the type of metal, and a series of environmental factors (Mejstrik and Lepsova 1993; Gadd 2007). Moreover, some ECM fungi may accumulate large concentrations of metals in their fruiting bodies with respect to growth soils, even when they are grown in non-contaminated environments. By contrast, other species exclude or do not accumulate metals in their fruiting bodies despite growing in close proximity to mine tailings. For this reason, the use of some saprophytic or ECM macromycetes as bioindicators of a metal-contaminated terrestrial ecosystem must be considered with caution.

Some of the metal-chelating agents or mechanisms described here for ECM fungi have also been described for arbuscular mycorrhizal fungi (AMF) (Khan et al. 2000; Gohre and Paszkowski 2006). However, given the symbiotic dependency required in AMF, the experiments are often of greater complexity. Regarding the metal-chelating mechanisms, there is a great difference between both groups of fungi, which is represented by glomalin, described to date exclusively in AMF. Glomalin is a glycoprotein produced by the hyphae, capable of chelating metals such as Cu, Pb, and Cd, and because of this some researchers have highlighted the significant role of the glomalin-producing AMF in the stabilization of the contaminated soils and in the protection of their host plants (Gonzalez-Chavez et al. 2004; Khan 2006).

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