Mechanisms of Metal Tolerance in ECMF

Metal tolerance may be defined as the genetically conditioned ability to grow and reproduce in environments with high concentrations of potentially toxic metals (Hartley et al. 1997). A metal-tolerant organism must be able to maintain metal homeostasis in the presence of high metal concentrations, controlling the concentrations of free metal ions in the cytosol. A multitude of mechanisms were proposed to contribute to metal tolerance in ECMF (Hartley et al. 1997; Leyval et al. 1997). Avoidance mechanisms reduce the exposure of ECMF cells to toxic metals and limit metal entry into the cell (Gadd 1993; Hartley et al. 1997). Avoidance mechanisms include the extracellular biochemical transformations of metals discussed above and the regulation of metal uptake: extracellular ligation of metal ions to di- and tricarboxylic acids and chelation with siderophores; extracellular metal immobilization by adsorption to cell walls, pigments and extracellular polysacchar-ides (fungal slimes); extracellular precipitation as oxalates; restriction of net metal uptake by reduced influx or increased efflux, through changes in the activity or specificity of metal transport channels (Bellion et al. 2006).

Cytoplasmatic and vacuolar sequestration of metals reduces the concentration of free ions in the cytosol. Mechanism of cellular sequestration and detoxification of metals comprise cytoplasmatic chelation by thiols (metallothioneins, glutathione and similar oligopeptides) and metal sequestration in the vacuole. Metal coordination within cells of ECMF was recently analysed by Fomina et al. (2007).

Increased production of cellular redox buffers such as glutathione and of the enzyme superoxide dismutase protects the cells from metal-induced oxidative damage, adding another line of defence against metal toxicity (Ott et al. 2002; Bellion et al. 2006).

The basic mechanisms of cytoplasmatic metal homeostasis used by fungi are shared with other eukaryotes. Studies in model organisms indicate that more cellular functions and molecules are involved in fungal metal tolerance. Kennedy et al. (2008) found many genes involved in Cd tolerance through a screen of knockout mutants of Schizosaccharomyces pombe. Their results suggested inter alia an involvement of coenzyme Q10 (ubiquinone) in Cd tolerance.

Most metal tolerance mechanisms imply metabolic costs: oxalate and exopoly-saccharide production requires organic carbon and increased efflux by metal transport channels is energy dependent. Intracellular metal chelation with metallothioneins and glutathione requires considerable amounts of cysteine. Under acute metal stress, other cysteine-requiring cellular activities such as hydrophobin synthesis are down-regulated in support of intracellular thiol levels (Bellion et al. 2006).

Metal adsorption to cell walls does not require additional synthetic effort but might be of limited relevance due to rapid saturation of potential sorption sites in highly metalliferous soils (Jentschke and Godbold 2000). Down-regulation of the expression of metal influx channels would save metabolic energy and might be the most economic adaptation to constantly increased substrate metal concentrations. The evolution of higher specificity of metal influx channels might improve the discrimination of non-essential and essential elements, but evidence for such a process in ECMF is not yet available.

Hartley et al. (1997) and Meharg (2003) pointed out that ECM and ERM fungi share a long evolutionary history of exposition to toxic metal concentrations, since ECM forests with understoreys of ERM plants cover huge areas with highly acidic soils, and soil acidity increases the concentration of free ions of Al, Fe and Mn. Mechanisms of Al detoxification and of Fe and Mn homeostasis were supposed to confer cotolerance to other metal elements. More particularly, sites with geogeni-cally elevated levels or toxic trace metals are potential hotspots for the evolution of tolerance of certain trace metals. Serpentine soils, the most widespread type of geogenically trace metal enriched substrate, are characterized by elevated levels of Mn, Ni and Cr (Urban et al. 2008; Moser et al. 2008). Other types of metalliferous outcrops are relatively rare and more restricted in surface.

The weathering of soil minerals by certain ECM fungi is considered important for the acquisition of essential cationic elements (van Scholl et al. 2008). By fungal attack on rock, potentially toxic metal ions can be liberated too, depending on the chemical composition of the rock material. Similar mechanisms are involved in ECM weathering and in the detoxification of metal ions by fungi, most importantly the exudation of LMWOAs. Citrate is the main ligand of Al3+ in podzolized forest soils (Landeweert et al. 2001) and oxalic acid is the main component of mycogenic precipitates of various metals (Gadd 2007). Extracellular ligation and precipitation, two essential mechanisms of avoidance of metal toxicity, may thus be an evolutionary by-product of the involvement of ECMF in mineral transformations driven by nutrient foraging.

Hartley et al. (1997) and Meharg (2003) hypothesized that cotolerance against several metals was likely to occur and would facilitate the evolution of metal tolerance. This argument may apply at least to very common mechanisms involving simple molecules, for example the up-regulation of oxalic acid exudation. On the other hand, it was frequently observed that increased metal tolerance in ECMF is metal specific, and that specificity in metal tolerance and local metal exposition can be correlated. Metal tolerance in Suillus spp. was found strain and metal specific and could be linked to the respective histories of metal exposure (Adriaensen et al. 2005; Krznaric et al. 2009). Recently, it could be demonstrated that metallothio-neins of the ECMF Paxillus involutus and Hebeloma cylidrosporum and the regulation of metallothionein gene expression are metal specific (Bellion et al. 2007; Ramesh et al. 2009). More information on specific mechanisms of metal homeostasis is available for model organisms. A Cd regulated Cd efflux system based on a PIB-type ATPase (PCA1) was recently reported from Saccharomyces cerevisiae (Adle et al. 2006). Lin et al. (2008) discovered that a single amino acid change in the vacuolar Zn transporter ZRC1 changed the substrate specificity of the transporter from Zn to Fe.

11.3.3 Protection of Host Trees

Schramm (1966) investigated plant colonization of barren coal mine spoils in Pennsylvania, a highly acidic substrate with toxic metal concentrations. He observed that both planted and spontaneously established trees (Pinus spp., Quer-cus borealis, Betula populifolia) were consistently associated with ECMF. Fruit bodies of Pisolithus tinctorius, Thelephora terrestris, T. caryophyllea, Astraeus hygrometricus and Inocybe sp. appeared near young trees established on barren substrate. The majority of spontaneously established pine seedlings were mycor-rhized, despite apparent limitation of ECM inoculum. The non-mycorrhized seedlings were reported as stunted and chlorotic, in contrast to the mycorrhized seedlings. Ever since it was observed many times that trees mycorrhized spontaneously or inoculated with appropriate ECMF species resist much better to extreme soil conditions, including metal toxicity. Trees were considered to tolerate metal contamination by means of phenotypic plasticity and ECM symbiosis. It was hypothesized that the presumably shorter life cycles of ECM fungi would open up more opportunity for genetic adaptation. Adaptive genetic change in ECM communities was considered essential for the understanding of the survival of ECM tree species challenged with metal toxicity (Wilkinson and Dickinson 1995). This hypothesis raised numerous questions, some of them being still under discussion.

11.3.4 Are ECMF More Resistant to Toxic Metals Than Their Host Trees?

Hartley et al. (1997) reviewed available data and concluded that a wide range of metal sensitivity can be found in both trees and ECMF, but upper tolerance limits appear to be far lower in the tree species. Growth-inhibiting Cd concentrations range from 0.3 to 30 p.M for non-mycorrhized trees and 0.1 to 90 p.M for ECM fungi in liquid media. In case of Pb exposition, the respective values were 0.5-230 p.M for trees and 125-960 mM for ECMF in liquid media. Hartley et al. (1997) attributed the higher adaptability of ECMF as expressed by higher upper limits of metal resistance to the higher diversity of ECMF compared to ECM trees, and not to the presumably shorter fungal generation cycles (Wilkinson and Dickinson 1995).

11.3.5 Can Metal-Resistant ECMF Confer Resistance to Their Host Trees?

From a co-evolutionary point of view, it seems obvious that a metal-resistant ECMF which supplies mineral nutrients to its host and which depends on organic carbon obtained from the host would benefit more if it alleviates metal stress in its host tree, with other words, selection is likely to favour host protection by ECMF, especially in pioneer populations, where only one tree individual might be available as carbon source. However, there is some experimental evidence that the most resistant ECMF species is not necessarily the most protective one (Jones and Hutchinson 1986). Godbold et al. (1998) concluded that only in a small number of experiments, amelioration of metal toxicity could be demonstrated, and that this was the case for specific metals and certain fungi only. The statement that amelioration of metal toxicity is highly species, strain and metal specific is still valid. However, it has to be considered that many earlier experiments with negative results had used ECMF from unpolluted sites, while amelioration of metal toxicity under experimental conditions had been recorded most often when fungi from metalliferous were used. Later studies provided unequivocal evidence that certain ECMF protect their host trees highly efficiently against specific metals (Adriaensen et al. 2004, 2005, 2006; Krznaric et al. 2009). Given recent evidence that ECMF communities in highly metalliferous soils can be surprisingly diverse, the question arises, if all those metal-tolerant ECMF have a similarly beneficial effect on their host trees. (Colpaert and van Assche 1993) inferred from experimental results in a semi-hydroponic system that species with abundant mycelia have the most beneficial effect. Field observations suggest that species with abundant mycelia such as Suillus spp. can be highly metal tolerant and beneficial for their host trees (Colpaert 2008). However, if ECM trees are competent of rewarding the most beneficial ECMF through selective organic carbon allocation, the diversity of metal tolerant ECMF might be per se beneficial.

ECM-associated microbes are likely to be important, too. Coinoculation with the ECM-associated bacterium Pseudomonas putida improved the growth promoting effect of Amanita rubescens on Pinus sylvestris exposed to Cd (Kozdroj et al. 2007).

11.3.6 ECMF and Host Tree Nutrient Status and Metal Uptake

Many metal polluted sites are poor in essential nutrients, and toxic metals can interfere with the uptake of essential nutrients. It is not easy to disentangle the beneficial effects of improved access to nutrients and/or reduced metal uptake. If revegetation of a devastated area is the first goal, this distinction might seem secondary. If metal cycles and the potential contamination of food webs or applications such as phytoextraction are considered, the quantity of metal uptake is of high practical relevance. Experimental results on the transfer of metal elements to trees via ECM fungi are manifold, but in many cases the toxic element filter hypothesis (Turnau et al. 1996) might be applicable. ECM fungi can reduce levels of available metals in soil by precipitation and by binding to organic compounds (Huang 2008), they can control symplastic metal transfers and cell wall components with high-metal affinity are likely to reduce apoplastic transport to the fine root. A clear amelioration of Cd toxicity in Picea abies seedlings by Paxillus involutus was found (Godbold et al. 1998), but Cd uptake was not decreased. Shoot metal concentrations are not necessarily reduced due to ECM colonization, effects on total shoot metal contents can be very variable. Ahonen-Jonnarth and Finlay (2001) observed a positive growth response of Ni and Cd exposed Pinus sylvestris seedling upon inoculation with Laccaria bicolor. Shoot metal concentrations were not affected, resulting in enhanced total metal uptake. In accumulating tree species, evidence for an ECM filtering effect may be lacking too (Krpata et al. 2009). Again, the metal exposition histories of both the ECMF and the host tree are essential to interpret experimental results.

11.3.7 Can ECM Symbiosis Confer Resistance to Sensitive Host Tree Genotypes?

The role of host sensitivity in the success of ECM associations was rarely investigated. Brown and Wilkins (1985) found increased Zn tolerance due to ECM inoculation in both tolerant and non-tolerant Betula. The translocation of Zn to the shoots of Betula was reduced, but Zn accumulated in the ECM. The differences of tolerance of the trees as expressed in growth rate and the respective limitations of leaf Zn concentrations were largely maintained. Adaptive tolerance of metal toxic-ity was suggested to occur in populations of Pinus ponderosa (Wright 2007) and P. balfouriana (Oline et al. 2000) growing in serpentine soils, but the potential role of ECMF was not assessed. Kayama et al. (2006) observed significantly reduced ECM colonization in non-tolerant Picea abies planted into serpentine soil, while ECM colonization was not decreased in serpentine adapted Picea glehnii. Two alternative hypotheses are proposed here to be tested in future studies: (a) metal uptake exceeds a critical threshold despite ECM colonization due to the low tolerance of non-adapted trees; (b) the naive, non-adapted host tree fails to select the most beneficial, toxic metal filtering ECMF via selective carbon allocation. In both cases, the metal sensitive tree will fail to grow normally despite nutrients offered by metal tolerant ECMF, the consequent reduction of photosynthesis and carbon supply will reduce colonization intensity and growth of fungal mycelia and destabilize the symbiosis.

11.4 Population Genetics of Adaptive Metal Tolerance in ECMF

Metal-contaminated soils are an attractive model system to investigate environment-driven population genetic processes, and important new insights into the microevolution of adaptive metal tolerance were reported for a few ECM model species. In subpopulations of the ECM basidiomycete Suillus luteus growing in Zn polluted soils, considerable genetic diversity was found and no reduction of genetic diversity compared to control populations could be detected using AFLP (Muller et al. 2004) and microsatellite (Muller et al. 2007) population markers. In contrast to a priori expectations, there was no evidence for clustering of subpopulations from polluted vs. unpolluted sites, despite significant differences in metal tolerance. It was concluded that metal pollution had a limited effect on the genetic structure of S. luteus populations, and that extensive gene flow and a high frequency of sexual reproduction allowed rapid evolution of tolerance while maintaining high levels of genetic diversity (Muller et al. 2007).

Adaption to Ni toxicity in naturally metalliferous soils was demonstrated in the ECM ascomycete Cenococcum geophilum (Goncalves et al. 2009). Mean in vitro 50% growth-inhibiting concentrations of Ni were about seven times higher in isolates from serpentine (23.4 mg/ml) than in control isolates (3.38 mg/ml). Furthermore, a marginally significant (P = 0.06) trend towards a negative correlation between Ni tolerance and growth rates in non-toxic conditions was found. This trade-off had been postulated earlier (Hartley et al. 1997) in order to explain why tolerance of metal toxicity fails to become a frequent trait in non-exposed populations. Moderate costs of metal tolerance are compatible with the observation of considerable variation in metal tolerance in non-exposed populations (Colpaert 2008). In contrast to the results of Goncalves et al. (2009), Colpaert et al. (2005) found no reduction of growth rates at low Zn levels linked to reduced Zn uptake in Zn-tolerant strains of Suillus spp. However, in vitro experiments can at best partially reproduce selective forces in ECM symbioses in natural environments.

Results concerning the population genetics of serpentine colonizing C. geophi-lum are rather contradictory. Panaccione et al. (2001) detected genetic divergence between C. geophilum from serpentine and from control sites, while Goncalves et al. (2007) found no differences linked to serpentine, but this result might be due to a limited sample size. Furthermore, Douhan et al. (2007) reconfirmed that C. geophilum s.l. is a species complex and recommended caution when conducting population genetic studies in C. geophilum due to the risk of comparing unrelated isolates.

The above-mentioned results suggest that in both anthropogenically and geo-genically metal-contaminated soils, tolerance of very high levels of toxic metals can be acquired by adaptive evolution, with or without high rates of genetic exchange with non-exposed populations. It is not clear if adaptive evolution of metal tolerance is widespread among ECMF. Strains of Paxillus involutus collected from Zn polluted sites were as Zn sensitive as control strains (Colpaert 2008). In certain ECMF species, constitutive levels of metal tolerance seemingly suffice to survive and compete in contaminated sites, while in others the frequently observed variation of metal tolerance in non-exposed populations may be the base for rapid selection of highly metal-tolerant genotypes.

Adaptive evolution of metal tolerance might be compatible with different population genetic patterns, respectively, reproductive systems and life history traits. Suillus luteus is a panmictic, sexually reproducing ECMF, C. geophilum is a complex of cryptic species possibly lacking a sexual state. Population genetics might rather be conditioned by life cycle traits than by metal stress. The probability of evolving distinct, specialized genotypes and genetically isolated populations might rather be determined by the structure of reproductive systems than by the nature and intensity of environmental selection pressure. At present, there is no evidence for serpentine driven speciation in ECMF (Urban et al. 2008) and the genetic structure of metal-tolerant Suillus luteus is similar like in pseudo-metallo-phytes (Colpaert 2008).

11.5 Distribution of Metal Elements in ECMF

Most studies on the elemental composition of ECMF were based on sporophores, motivated by the interest in nutritional value and ecotoxicological concerns, since wild edible fungi are an important component of human diets in many parts of the world. Earlier studies on metal contents in macrofungi were reviewed by Kalac and Svoboda (2000). They summarized that BCFs in wild edible mushrooms were found high for Cd (50-300), which is probably the most problematic element in mushrooms, and Hg (30-500) but low for Pb (10~2-10~1). Melgar et al. (2009) confirmed that all fungal species investigated accumulated Hg (BCF > 1). Highest values were found in ECM Boletus pinophilus and B. aereus and in saprotrophic Agaricus macrosporus and Lepista nuda (mean BCF between 300 and 450 in the hymeno-phore). Other ECM species had generally lower BCF values than saproptrophs.

Borovicka and Randa (2007) found Fe accumulation in Hygrophoropsis aur-antiaca and Zn accumulation in ECM Russula atropurpurea. Generally, lower Se acumulation was found in checked ECMF compared to saprotrophs, but some of the highest Se concentrations were recorded in ECMF (Boletus edulis, Boletus pino-philus, Amanita strobiliformis, Albatrellus pes-caprae).

Metal hyperaccumulation by fungi was rarely reported. Stijve et al. (1990) measured As hyperaccumulation in Sarcosphaera coronaria (100-7,000 ppm) and Borovicka et al. (2007) reported Ag hyperaccumulation in Amanita strobiliformis (mostly 200-700 mg/kg, highest value 1,253; mg/kg; BCF 800-2,500). Ag is microbicidal at low concentrations. Very high BCFs (about 1,000) were reported for the K analogue Rb in Suillus grevillei (Chudzynski and Falandysz 2008).

Bioavailability, nutritional value and toxicity of metals and metalloids in ECMF depend on speciation. Slejkovec et al. (1997) found some relation between As speciation and phylogenetic relationships in mushrooms. Mutanen (1986) reported low bioavailability of fungal Se. The metalloid Se is of interest due to frequent Se deficiency of human nutrition. Serafín Muñoz et al. (2006) suggested that a major part of Se in Pleurotus ostreatus is bound to the cell wall component chitin. Serafín Munoz et al. (2007) demonstrated a speciation-dependent protective role of Se against oxidative damage induced by Cd and Ag in liquid cultures of Pleurotus ostreatus.

Krpata et al. (2009) compared Zn and Cd concentrations in fruit bodies of ECM fungi and in leaves of their host trees, metal-accumulating Populus tremula, in locations highly contaminated by Pb/Zn smelting. BCFs were based on total metal concentrations in the mor-type organic layer (BCFtot) or on NH4NO3-extractable metal concentrations in mineral soil (BCFlab). When plotted on log-log scale, a linear model described well the decrease of BCFs with increasing soil metal concentrations. A better correlation was found for BCFlab than for BCFtot. The observation of decreasing BCFs with increasing substrate metal concentrations is not uncommon in fungi (Gast et al. 1988) and plants (Langer et al. 2009). Differences between fungal genera were found in Zn-BCFs but not in Cd-BCFs. Tricho-loma scalpturatum (762 ppm), Scleroderma verrucosum (598-777 ppm) and Amanita vaginata (403-571 ppm) had highest Zn concentrations, Laccaria laccata (12.3-93.3 ppm) and Amanita vaginata (10.1-48.5 ppm) had highest Cd values. Concentrations of Zn were in the range reported for fungal fruit bodies, Cd concentrations were highly elevated. Cd levels above 10 ppm are typically found in contaminated sites (Gast et al. 1988; Svoboda et al. 2006).

Accumulation and BCFs of Zn and Cd in the host trees were of the same order of magnitude as in the ECM fungi. Studies on metal element concentrations in sporophores demonstrated differences in metal affinities between various fungal species and strains and high metal specificity in fungal BCFs. Fungi growing in substrates with excessive metal concentrations usually have drastically reduced BCFs, probably a result of physiological control of metal uptake. The relative contributions of phenotypic plasticity and population genetic factors to the control of metal uptake rates as expressed by BCFs may vary according to the species concerned. Colpaert et al. (2005) found reduced Zn uptake in Zn-tolerant strains of Suillus spp. at low and high Zn concentrations and concluded that partial Zn exclusion contributed most to Zn tolerance. Zn tolerance as expressed by reduced Zn accumulation was specific, the concentrations of other micronutrients were not affected. Despite reduced Zn uptake in tolerant strains, the Zn concentrations in the mycelia of all treatments were very high, reaching up to 15.57 mg/g, a concentration representative of plant hyper-accumulators. Turnau et al. (2001) used micro-proton-induced X-ray emission (PIXE) true elemental maps to quantify metals in cryo-fixed S. luteus mycorrhizas collected from Zn wastes. They found similarly elevated Zn concentration in Suillus rhizomorphs, in average 12.83 mg/g. Colpaert and van Assche (1992) detected high concentrations of Zn in Suillus ECM grown in Zn-spiked substrate, while transfer to the host plant remained low. Representative Zn concentrations in Suillus sporophores are 30-150 mg/kg (Kalac and Svoboda 2000), a value about 100 times lower than in Suillus mycelia. Studies on metal concentrations in environmental samples of ECM mycelia are still scarce, despite growing awareness of their ecological significance (Finlay 2008). Wallander et al. (2003) investigated the elemental composition of ECM mycelia grown in contact with wood ash or apatite in forest soil. They measured high K accumulation by mycelium of Suillus granulatus and high concentrations of Ca, Ti, Mn and Pb in Paxillus involutus rhizomorphs. Piloderma croceum appears to accumulate and translocate Ca, an element that is scarce in podzols (Blum et al. 2002; Hagerberg et al. 2005).

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