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Heavy metals (copper, zinc, manganese, etc.) represent a dilemma for living organisms: they are essential, usually at low concentration, for the structure and/or function of many cellular components, but become toxic above threshold concentrations. Both prokaryotes and eukaryotes have thus developed homeostatic mechanisms to exert control over the intracellular concentration of metal ions in order to ensure the minimum amount required for normal metabolism and to avoid intoxication. The increasing concern about environmental pollution due to heavy metals, whether of natural or anthropic origin, justifies the numerous studies on the mechanisms of metal tolerance, in particular those involving mycorrhizal fungi. Mycorrhizal symbiosis has been recognized as a crucial determinant for plant growth and productivity, and there is mounting evidence that mycorrhizal fungi can alleviate situations of stress in host plants, including exposure to toxic metals (Meharg, 2003).
Over the past decade, Michel Chalot's group has already made some important contributions to the field of metal tolerance in ectomycorrhizal fungi. The occurrence of complex extra- and intracellular mechanisms has clearly been demonstrated through a combination of microbiological, biochemical and molecular biological approaches mainly applied to the model system Paxillus involutus after exposure to cadmium ions. These mechanisms involve nonspecific binding to cell walls, possibly intracellular metal chelation systems, responses to oxidative stress, and modulation of gene expression (Bellion et al., 2006 and references therein; Fig. 1). In this issue, Bellion et al. (pp. 151–158) add a further and important piece of information to our understanding of heavy metal tolerance in this ectomycorrhizal fungus. The authors have described a metallothionein-coding gene (Pimt1) identified from a transcriptomic approach, and functionally characterized it in terms of transcript profiles, heterologous complementation and transformation assays. The results highlight the key role of the gene in Cu and Cd tolerance.
‘… some attempts to increase heavy metal tolerance by manipulating the ability to synthesize intracellular metal chelators did not achieve the expected results.’
The enigmatic world of metallothioneins
Among the intracellular metal chelators, phytochelatins (PCs) and metallothioneins (MTs) are of primary importance in buffering the concentration of free metal ions such as Cu, Zn and Cd. Both are Cys-rich polypeptides that chelate metal ions through the formation of tetrahedrally coordinated metal-thiolate clusters. While PCs are synthesized through the ribosome-independent polymerization of reduced glutathione-derived γ-glutamyl-Cys units, MTs are small (60–80 amino acids) gene-encoded polypeptides (Cobbett & Goldsbrough, 2002). Metallothioneins have been classified into different families on the basis of both protein and gene sequences (http://www.expasy.org/cgi-bin/lists?metallo.txt) and are commonly thought to be involved in essential metal homeostasis, and possibly toxic metal detoxification and scavenging of free radicals. However, within different biological systems, MT expression has been shown to be induced not only by heavy metals, but also by other agents (e.g. hormones, drugs) and growth conditions (Haq et al., 2003). Considering their peculiar chemical properties – potent metal binding and redox capabilities – it is not surprising that MTs have been associated with a variety of cellular processes. For this reason, the primary role of these enigmatic proteins remains elusive.
From an evolutionary perspective, MTs or MT-like polypeptides have been found in all branches of the tree of life, with a remarkable conservation of the functional structure across the phyla. It has been hypothesized that ancestral MT structures evolved under selective pressure in an environment where toxic metals and free radicals were particularly abundant (Coyle et al., 2002). Modern MTs probably have a polyphyletic origin, and during evolution might have specialized in terms of metal-binding ability and functions within each life form to adapt to different environmental niches and/or specific endogenous metabolic requirements. Two main groups of MTs have been proposed on the basis of metal-binding ability: Cu-thioneins and Zn-thioneins. However, some Zn-thioneins exhibit dual behaviour. For example, MTs Cup1 and Crs5 from Saccharomyces cerevisiae yeast have been extensively related to Cu handling, but more recently Crs5 was defined as a dual metal-binding MT significantly closer to Zn-thioneins than to Cu-thioneins (Pagani et al., 2007). This feature, together with the slightly different gene-expression regulation and data obtained from mutant strains exposed to metal toxic concentrations, suggests physiological roles for Crs5 alternative to the Cup1 Cu-thionein, and a possible involvement in the response to Zn overload (Pagani et al., 2007). It is worth noting that increasing experimental evidence on yeast MTs indicates that some connections exist between the metabolism, homeostasis and responses to Cu and Zn. This is in agreement with the results obtained by Eide et al. (2005), who used a genome-wide approach to investigate metal homeostasis in yeast. The accumulation profile of 13 chemical elements (referred to as the ‘ionome’) was investigated in yeast mutants defective in >4000 different genes. Interestingly, the levels of multiple elements were altered in most mutants, suggesting extensive networks linking the metabolism of different metals (Eide et al., 2005).
Gene expression in heterologous systems: a powerful tool
Fungal MTs, research on which has been more sporadic, are usually described as Cu-binding proteins. However, few of them have been functionally characterized in terms of metal-coordination chemical properties and/or by means of complementation assays in metal-hypersensitive yeast mutants. Putative MTs have often been described within collections of expressed sequence tags (ESTs) simply on the basis of sequence similarity. This is the case for MT-like sequences found in mycorrhizal fungi, in particular in the ectomycorrhizal fungus Pisolithus tinctorius (Voiblet et al., 2001); the ericoid fungus Oidiodendron maius (Vallino et al., 2005); and the arbuscular mycorrhizal fungi Gigaspora rosea (Stommel et al., 2001), Gigaspora margarita (Lanfranco et al., 2002) and Glomus intraradices (González-Guerrero et al., 2006).
The P. involutus Pimt1 gene codes for a relatively short (34 amino acids) MT and contains just one domain bearing the classical C–X–C motifs, compared with the canonical and longer MTs that are usually composed of two Cys-rich domains. This feature has also been found in other fungal MTs; it appears that the length is not critical for metal binding, as an even shorter MT protein has proved to chelate metal ions efficiently (Tucker et al., 2004).
Bellion et al. (this issue) used heterologous expression systems to demonstrate unambiguously that the Pimt1 gene product can sequester metal ions, thereby conferring in vivo protection against metals, in particular Cu and Cd. Functional complementation assays were performed using three distinct metal-hypersensitive yeast mutants. A similar approach was used to characterize two MT-encoding genes from endomycorrhizal fungi (Lanfranco et al., 2002; González-Guerrero et al., 2006). This shows how the Genome Deletion Project on the Saccharomyces model system has also offered crucial tools for functional genomics studies in the field of metal tolerance. The availability of numerous well characterized metal-hypersensitive strains allows the in vivo dissection of metal-protection mechanisms, and eventually leads to the identification of the specific molecular roles played by the DNA sequence of interest.
In addition, the Pimt1 gene has been functionally characterized in vivo using another heterologous system: Hebeloma cylindrosporum transformants overexpressing Pimt1 showed increased Cu tolerance, fortifying the results obtained in yeast. This approach is of value as it also provides an opportunity to investigate heavy metal tolerance in a more congruent contest concerning multicellularity and nutritional strategies. The ectomycorrhizal H. cylindrosporum has a relatively long transformation history and, at present, is one of the most ‘genetically tractable’ mycorrhizal fungi (Combier et al., 2004 and references therein). DNA transfer via Agrobacterium tumefaciens, which is becoming the preferential transformation system of fungi because of its simplicity and high efficiency, also seems very promising for the successful genetic manipulation of other symbiotic fungi such as Tuber borchii, Laccaria bicolor and Pisolithus microcarpus. It would also be interesting to see Pimt1 overexpression in the homologous system, for which only biolistic transformation has been reported so far (Bills et al., 1995).
Metallothioneins: a role in developmental biology of fungi interacting with plants
There are increasing lines of evidence for the role of MTs or MT-like proteins in mutualistic and pathogenic interactions between plants and fungi. Fungal MT genes are often transcriptionally regulated during the life stages and, in particular, during plant colonization. The first report dates back to 1995, when two genes similar to MTs were described as expressed uniquely during appressorium formation by Colletotrichum gloeosporioides conidia induced by the host surface wax (Hwang & Kolattukudy, 1995). Similarly, two putative MT genes from the biotrophic pathogen Uromyces fabae are strongly upregulated in parasitic mycelium colonizing leaf tissues (Jakupoviæet al., 2006). As far as mycorrhizal fungi are concerned, Pimt1 was found by Johansson et al. (2004) in macroarray experiments as upregulated in ectomycorrhizal tissues compared with the saprotroph growth condition. In contrast, MTs from arbuscular mycorrhizal fungi appear to be downregulated when the fungus colonizes the root tissues (Lanfranco et al., 2002). It is worth noting that plant MTs are also upregulated in ectomycorrhizal associations of Betula pendula with Eucalyptus globulus and P. involutus with P. tinctorius, respectively (Voiblet et al., 2001; Johansson et al., 2004).
To further support the role of MTs in plant–fungus interaction, Tucker et al. (2004) described an unusual MT-like protein (22 amino acids long with only six cysteines) in the fungal pathogen Magnaporthae grisea, which showed a very high affinity to Zn. The gene has no effect on metal tolerance and, more interestingly, was shown to confer pathogenicity, probably by playing a role in the biochemical differentiation of the appressorium cell wall. Clearly, there is much more to learn concerning the involvement of MTs in the developmental biology of plant-interacting fungi.
Metallothioneins and bioremediation
Mycorrhizal fungi are a direct link between plants and soil, and are often needed to ensure plant survival in heavily polluted areas. Interestingly, this seems to be a common feature of mycorrhizal symbioses, independently of whether they are formed by ecto- or endomycorrhizal fungi (Meharg, 2003). To use mycorrhizal fungi for bioremediation and soil protection purposes, we clearly need to improve our understanding of the molecular mechanisms that underlie the metal-detoxification processes in these fungi. This would allow better exploitation of the mycorrhizal symbiosis. The characterization of a P. involutus MT is a new and important step in this regard. Data from yeast mutants, together with the fact that Pimt1 gene expression is highly responsive to Cu, allows Pimt1 to be defined as an important component in Cu detoxification in P. involutus. Transcriptional induction and yeast complementation, but not overexpression in Hebeloma, also suggests a role in the alleviation of Cd stress. Cadmium might also be detoxified by other intracellular mechanisms, as it is known that a variety of metal-protection strategies can coexist in a single organism; for example, Schizosaccharomyces pombe and Candida glabrata contain both PCs and MTs. The question is: does P. involutus have the genetic potential to synthesize additional intracellular chelators such as PCs? Biochemical studies seem to exclude this possibility at present (Bellion et al., this issue, and references therein).
Metallothionein overexpression often results in greater tolerance to heavy metals. However, it is worth noting that some attempts to increase heavy metal tolerance by manipulating the ability to synthesize intracellular metal chelators did not achieve the expected results. A lesson comes from another model system, Arabidopsis thaliana, where the overexpression of a PC synthase gene led to transgenic plants that were hypersensitive to Cd stress (Lee et al., 2003). The surplus synthesis of MTs and PCs, which is energy-demanding concerning nitrogen and sulphur, may have a negative impact on growth and also compromise detoxification capability.
An intriguing question, prompted by the symbiotic nature of P. involutus, is whether fungal-derived MTs can confer increased metal tolerance on host plants. Although it is commonly recognized that the preferential intracellular detoxification strategy of plants is based on PCs, plants do in fact possess their own MTs. At least in some cases, plant MTs seem responsible for providing metal-protective effects (van Hoof et al., 2001). The study of both plant and fungal MTs in the context of symbiotic systems is desirable to understand this process more fully. This may also highlight possible differences in the protection strategies triggered by ecto- or endomycorrhizal fungi.
As many organisms, including fungi, possess multiple MT sequences, a question arises: how many MT genes exist in the genome of a mycorrhizal fungus? No genome-sequencing project is currently dedicated to P. involutus, but EST collections have already revealed the existence of a second MT-like sequence (Bellion et al., this issue). The genome sequences of L. bicolor and G. intraradices (http://darwin.nmsu.edu/~fungi), as representative of ecto- and arbuscular mycorrhizal fungi, will soon be available and will undoubtedly give rise to important insights, at least into the genetic potential of the metal homeostasis and detoxification processes. Where multiple MT genes exist, it would be important to evaluate whether divergent structures mirror divergent functions. Further studies are also needed on the involvement of fungal MTs in cellular processes other than metal detoxification, and on investigating MTs from mycorrhizal fungi in a more natural context during the interaction with the host plant. A deeper knowledge of the molecular mechanisms of metal handling in mycorrhizas will eventually provide biological agents with superior remediation capabilities through the screening of naturally occurring plant–fungus combinations and/or through genetic improvement strategies.