Cadmium hyperaccumulating mushroom Cystoderma carcharias has two metallothionein isoforms usable for cadmium and copper storage.

Cystoderma carcharias is one of the few macrofungal species that can hyperaccumulate Cd. As we have previously documented in C. carcharias collected from a smelter-polluted area, it stores 40% of Cd and nearly 90% of Cu in sporocarps in complex(es) of identical size. In this paper we examined whether metallothionein (MT) peptides that bind Cd and Cu through cysteinyl-thiolate bonds were associated with the metals in these complexes. Screening of a sporocarp cDNA expression library in yeasts allowed the identification of two transcripts, CcMT1 and CcMT2, encoding functional 34-amino acid (AA) MTs sharing 56% identity and appearing to be encoded by duplicate genes. CcMT1 conferred reasonable tolerance to Cu and a substantially higher tolerance to Cd than CcMT2, while CcMT2 clearly protected the yeasts better against Cu toxicity. While size-exclusion chromatography revealed that CcMT1 was contained in all Cd/Cu complexes isolated from wild grown sporocarps, CcMT2 was detected in a much narrower subset of the fractions. The striking difference between the CcMTs is that CcMT1 lacks the third metal-biding cysteinyl (C) within an otherwise highly conserved-in-agaricomycetes-MTs C-AA4-C-AA-C-AA3-C-AA-C-AA4-C-AA-C motif. The elimination of the corresponding cysteinyl in CcMT2 only reduced the Cu-tolerant phenotype in yeasts to the levels observed with CcMT1. Altogether, these results indicate that CcMT2 is rather adjusted to perform Cu-related tasks and point to CcMT1 as the ligand for the storage of both Cd and Cu in C.carcharias, which is the first macrofungal species in which the potential of MT in Cd handling can be seen.

Arsenic accumulation and speciation in two cultivars of Pteris cretica L. and characterization of arsenate reductase PcACR2 and arsenite transporter PcACR3 genes in the hyperaccumulating cv. Albo-lineata.

Pollution and poisoning with carcinogenic arsenic (As) is of major concern globally. Interestingly, there are ferns that can naturally tolerate remarkably high As concentrations in soils while hyperaccumulating this metalloid in their fronds. Besides Pteris vittata in which As-related traits and molecular determinants have been studied in detail, the As hyperaccumulation status has been attributed also to Pteris cretica. We thus inspected two P. cretica cultivars, Parkerii and Albo-lineata, for As hyperaccumulation traits. The cultivars were grown in soils supplemented with 20, 100, and 250 mg kg-1 of inorganic arsenate (iAsV). Unlike Parkerii, Albo-lineata was confirmed to be As tolerant and hyperaccumulating, with up to 1.3 and 6.4 g As kg-1 dry weight in roots and fronds, respectively, from soils amended with 250 mg iAsV kg-1. As speciation analyses rejected that organoarsenical species and binding with phytochelatins and other proteinaceous ligands would play any significant role in the biology of As in either cultivar. While in Parkerii, the dominating As species, particularly in roots, occurred as iAsV, in Albo-lineata the majority of the root and frond As was apparently converted to iAsIII. Parkerii markedly accumulated iAsIII in its fronds when grown on As spiked soils. Considering the roles iAsV reductase ACR2 and iAsIII transporter ACR3 may have in the handling of iAs, we isolated Albo-lineata PcACR2 and PcACR3 genes closely related to P. vittata PvACR2 and PvACR3. The gene expression analysis in Albo-lineata fronds revealed that the transcription of PcACR2 and PcACR3 was clearly As responsive (up to 6.5- and 45-times increase in transcript levels compared to control soil conditions, respectively). The tolerance and uptake assays in yeasts showed that PcACRs can complement corresponding As-sensitive mutations, indicating that PcACR2 and PcACR3 encode functional proteins that can perform, respectively, iAsV reduction and membrane iAsIII transport tasks in As-hyperaccumulating Albo-lineata.

Functional analysis RaZIP1 transporter of the ZIP family from the ectomycorrhizal Zn-accumulating Russula atropurpurea.

A search of R. atropurpurea transcriptome for sequences encoding the transporters of the Zrt-, Irt-like Protein (ZIP) family, which are in eukaryotes integral to Zn supply into cytoplasm, allowed the identification of RaZIP1 cDNA with a predicted product belonging to ZIP I subfamily; it was subjected to functional studies in mutant Saccharomyces cerevisiae strains. The expression of RaZIP1, but not RaZIP1H208A or RaZIP1H232A mutants lacking conserved-among-ZIPs transmembrane histidyls, complemented Zn uptake deficiency in zrt1Δzrt2Δ yeasts. RaZIP1 substantially increased cellular Zn uptake in this strain and added to Zn sensitivity in zrc1Δcot1Δ mutant. The Fe uptake deficiency in ftr1Δ strain was not rescued and Mn uptake was insufficient for toxicity in Mn-sensitive pmr1Δ yeasts. By contrast, RaZIP1 increased Cd sensitivity in yap1Δ strain and conferred Cd transport activity in yeasts, albeit with substantially lower efficiency compared to Zn transport. In metal uptake assays, the accumulation of Zn in zrt1Δzrt2Δ strain remained unaffected by Cd, Fe, and Mn present in 20-fold molar excess over Zn. Immunofluorescence microscopy detected functional hemagglutinin-tagged HA::RaZIP1 on the yeast cell protoplast periphery. Altogether, these data indicate that RaZIP1 is a high-affinity plasma membrane transporter specialized in Zn uptake, and improve the understanding of the cellular and molecular biology of Zn in R. atropurpurea that is known for its ability to accumulate remarkably high concentrations of Zn.

Functional analysis of two genes coding for distinct cation diffusion facilitators of the ectomycorrhizal Zn-accumulating fungus Russula atropurpurea.

Russula atropurpurea can accumulate remarkably high concentrations of Zn in its sporocarps. We have previously demonstrated that 40 % of the intracellular Zn in this species is sequestered by MT-like RaZBP peptides. To see what other mechanisms for the handling of the accumulated Zn are available to R. atropurpurea, we searched its transcriptome for cDNAs coding for transporters of the cation diffusion facilitator (CDF) family. The transcriptome search enabled us to identify RaCDF1 and RaCDF2, which were further subjected to functional studies in metal sensitive Saccharomyces cerevisiae. The expression of RaCDF1 and its translational fusion with green fluorescent protein (GFP) protected the yeasts against Zn and Co, but not Cd or Mn, toxicity and led to increased Zn accumulation in the cells. The GFP fluorescence, observed in the RaCDF1::GFP-expressing yeasts on tonoplasts, indicated that the RaCDF1-mediated Zn and Co tolerance was a result of vacuolar sequestration of the metals. The expression of RaCDF2 supported Zn, but not Mn, tolerance in the yeasts and reduced the cellular uptake of Zn, which is congruent with the proposed idea of the Zn-efflux function of RaCDF2, supported by the localization of GFP-derived fluorescence on the plasma membrane of the yeasts expressing functional RaCDF2::GFP. Contrarily, RaCDF2 increased the sensitivity to Co and Cd in the yeasts and significantly promoted Cd uptake, which suggested that it can act as a bidirectional metal transporter. The notion that RaCDF1 and RaCDF2 are genuine CDF transporters in R. atropurputrea was further reinforced by the fact that the RaCDF-associated metal tolerance and uptake phenotypes were lost upon the replacement of histidyl (in RaCDF1) and aspartyl (in RaCDF2), which are highly conserved in the second transmembrane domain and known to be essential for the function of CDF proteins.

Intracellular sequestration of zinc, cadmium and silver in Hebeloma mesophaeum and characterization of its metallothionein genes.

Sequestration of intracellular heavy metals in eukaryotes involves compartmentalization and binding with cytosolic, cysteine-rich metallothionein (MT) peptides. We examined the roles of these processes in handling of zinc (Zn), cadmium (Cd) and silver (Ag) in sporocarps and a metal-exposed extraradical mycelium of Hebeloma mesophaeum, the Zn-accumulating ectomycorrhizal (EM) species frequently associated with metal disturbed sites. Size exclusion chromatography revealed that the majority of Zn and Cd in the sporocarps and mycelium was contained in a low molecular mass fraction attributable to compartmentalized metal. The staining of hyphal cells with the Zn-specific Zinquin and Cd-specific Leadmium fluorescent tracers labeled Zn and Cd in small, punctuated vesicles and vacuoles, respectively. By contrast, the sporocarp and mycelium Ag was associated with cysteine-rich, 5-kDa peptides. The peptides of the same size were also identified in minor Zn and Cd complexes from the metal-exposed mycelium. We have further isolated and characterized HmMT1, HmMT2 and HmMT3 genes coding for different 5-kDa MTs of H. mesophaeum collected at a lead smelter site. Heterologous complementation assays in metal-sensitive yeast mutants indicated that HmMTs encode functional, metal-specific peptides: only HmMT1 was able to complement sensitivity to Zn; HmMT1 conferred higher tolerance to Cd and Cu than HmMT2 or HmMT3; and both HmMT2 and HmMT3, but not HmMT1, conferred increased tolerance to Ag. The presence of HmMT1 and HmMT3, but not HmMT2, was also confirmed in a H. mesophaeum isolate from an unpolluted site. Gene expression analysis in the extraradical mycelium of this isolate revealed that the transcription of HmMT1 was preferentially induced in the presence of Zn and Cd, while Ag was a stronger inducer of HmMT3. Altogether, these results improve our understanding of the handling of intracellular Zn, Cd and Ag in Hebeloma and represent the first evidence suggesting involvement of MTs in sequestration of Zn in EM fungi.

Cadmium in the hyperaccumulating mushroom Thelephora penicillata: Intracellular speciation and isotopic composition.

Thelephora penicillata is an ectomycorrhizal mushroom that can accumulate extraordinarily high concentrations of Cd, As, Cu, and Zn in its fruit-bodies. To better understand its element accumulation ability, we compared the element concentrations in T. penicillata with 10 distinct ectomycorrhizal mushroom species growing at the same site (Karlina Pila, Czech Republic). On average, T. penicillata accumulated 330, 2130, 26, and 4 times more Cd, As, Cu, and Zn, respectively, than other mushrooms. Size-exclusion chromatography and an electrophoretic analysis of T. penicillata cell extracts indicate that intracellular Cd may be present mainly in >1 kDa, presumably compartmentalized, Cd species, and partially binding with 6-kDa cysteinyl-containing peptide(s) resembling metallothioneins. The cadmium isotopic composition of mushroom fruit-bodies, soil digests, and soil extracts was investigated by thermal ionization mass spectrometry (TIMS) with double spike correction. The isotopic composition (δ114/110Cd) of ectomycorrhizal mushrooms from Karlina Pila varied in a wide range of -0.37 to +0.14 ‰. However, remarkably low δ114/110Cd values were observed in the majority of the investigated mushrooms when compared to the relatively homogeneous Cd isotopic composition of bulk soil (δ114/110Cd = +0.09 ‰) and the comparatively heavy isotopic composition of soil extracts (mean δ114/110Cd values of +0.11 ± 0.01 ‰ and +0.22 ± 0.01 ‰, depending on the extraction method). The isotopic composition of Cd hyperaccumulated in T. penicillata essentially matched the mycoavailable soil Cd fraction. However, most isotopic data indicates isotopic fractionation at the soil/fruit-body interface, which could be of environmental significance.

A new mushroom hyperaccumulator: Cadmium and arsenic in the ectomycorrhizal basidiomycete Thelephora penicillata.

Fruit-bodies of six Thelephora species (Fungi, Basidiomycota, Thelephoraceae) were analyzed for their trace element concentrations. In Thelephora penicillata, extremely high concentrations of Cd and As were found, followed by highly elevated concentrations of Cu and Zn. The highest accumulation ability was found for Cd with a mean concentration of 1.17 ± 0.37 g kg-1 (dry mass) in fruit-bodies collected from 20 unpolluted sites; the mean As concentration was 0.878 ± 0.242 g kg-1. Furthermore, striking accumulation of Se (923 ± 28 mg kg-1) was found in one sample of T. vialis and elevated concentrations of S were detected in T. palmata (19.6 ± 5.9 g kg-1). The analyzed Thelephora species were sequenced and, based on the Maximum Likelihood phylogenetic analysis (ITS rDNA) of the genus, possible other Thelephora (hyper)accumulators were predicted on the basis of their phylogenetic relationship with the discovered (hyper)accumulators. The striking ability of T. penicillata to accumulate simultaneously Cd, As, Cu, and Zn has no parallel in the Fungal Kingdom and raises the question of a biological importance of metal(loid) hyperaccumulation in mushrooms.

Heterologous expression of Zn-binding peptide RaZBP1 from Russula bresadolae does not overcome Zn and Cd detoxification mechanisms in Hebeloma mesophaeum.

Homeostatic mechanisms preventing the toxicity of heavy metal ions in cells involve, among others, compartmentalization and binding with peptidaceous ligands, particularly the cysteinyl-rich metallothioneins (MTs). We have previously shown that in natural conditions Zn-overaccumulating ectomycorrhizal (EM) fungus Russula bresadolae stores nearly 40% of Zn bound with cysteinyl- and hystidyl-containing RaZBP peptides, which resemble MTs, while the detoxification of Zn and Cd in EM Hebeloma mesophaeum relies upon compartmentalization in small vesicles and vacuoles, respectively. Here, we examined the performance of RaZBP1 gene expressed in H. mesophaeum mycelium with respect to handling of Zn and Cd. Expression of RaZBP1 impaired growth of the mycelium on low-Zn medium by 60%, the growth was partly ameliorated upon the addition of Zn and remained considerable up to 2 mmol/L Zn, while the growth of the wild-type and control mycelia transformed with empty T-DNA was severely reduced in the presence of 0.5 mmol/L Zn; furthermore, RaZBP1 slightly added to Cd tolerance in the range of Cd concentrations of 0.625 to 8 µmol/L. Staining of Zn- or Cd-exposed hyphal cells with Zn- or Cd-specific fluorescent tracers did not indicate that the expression of RaZBP1 would redirect the flow of the metals away from their innate sinks. Size exclusion chromatography of extracted metal species revealed that the complexes corresponding to Zn/Cd-RaZBP1 are present only in minute levels. Considering that RaZBP1 inhibited growth at low Zn, and despite the benefit that it provided to H. mesophaeum in the presence of high Zn and moderate Cd, these data indicate that the binding of excess Zn and Cd with RaZBP1 is not a trait that would be outright transmitted to H. mesophaeum.

Different cadmium tolerance of two isolates of Hebeloma mesophaeum showing different basal expression levels of metallothionein (HmMT3) gene.

Hebeloma mesophaeum is an ectomycorrhizal fungus frequently associated with metal disturbed environments. In this work, we examined Ag, Cd, and Zn tolerance of H. mesophaeum isolates from heavy metal-polluted (isolate Prib) and clean (isolate Rez) sites. Both mycelia showed essentially the same level of Ag and Zn tolerance, but Prib was more Cd tolerant. In short-term exposures, Prib accumulated slightly less Cd than Rez. Size exclusion chromatography of cell-free extracts and fluorescence microscopy of hyphae with a Cd-specific fluorescent tracer revealed that substantial proportion of Cd was contained in the vacuoles in both isolates. Considering that the proportion of Cd associated with fractions attributable to Cd complexes with cytosolic, metallothionein (MT) peptides was higher in Prib, we examined the copy number and basal levels of HmMTs genes in Rez and Prib. While no difference between the isolates was observed in the gene copy numbers and basal levels of HmMT1 transcripts, the basal transcription of HmMT3 was 3-fold higher in Prib. These observations suggest that MTs provide in Prib better protection against Cd. Furthermore, the higher Cd tolerance in Prib can be to some extent also supported by the efflux or reduced uptake of Cd in the hyphae.

Zn overaccumulating Russula species clade together and use the same mechanism for the detoxification of excess Zn.

It has been firmly established that macrofungi can accumulate large amounts of heavy metals in their sporocarps. However, the mechanisms of the accumulation and storage are being uncovered only recently. We have previously documented that Russula bresadolae can accumulate over 1 g Zn kg-1 dry weight and that sequestration of a substantial proportion of overaccumulated Zn involves binding with peptides, RaZBPs, seen so far only in this species. In this work we examined Zn contents of 360 sporocarp collections from unpolluted environments covering 114 species of the genus Russula. Whilst the concentrations of Zn in most analysed species were in the range of 50-150 mg kg-1, the species of subgenera Brevipes and Compactae accumulate very low Zn (< 50 mg kg-1). We further identified five new Zn-overaccumulating species of subgenus Russula, which form with R. bresadolae a separate phylogenetic subclade in which the sporocarp Zn concentrations ranged from 326 to 845 mg kg-1. We demonstrate that R. pumila and R. ochroleuca express at least one ZBP gene and when expressed in metal-sensitive S. cerevisiae, all ZBPs protected the yeasts against Zn (and Cd) toxicity equally well. The respective ZBPs were confirmed in the native Zn-complexes of R. pumila and R. ochroleuca, which represented 80% of Zn extracted from their sporocarps. This study is the first extensive genus-wide report of metal accumulation in macrofungi, which further demonstrates that the Zn binding with cytosolic ZBP peptides is not a trait restricted only to R. bresadolae.

Disentangling the factors of contrasting silver and copper accumulation in sporocarps of the ectomycorrhizal fungus Amanita strobiliformis from two sites.

Amanita strobiliformis (European Pine Cone Lepidella) is an ectomycorrhizal fungus of the Amanitaceae family known to hyperaccumulate Ag in the sporocarps. Two populations (ecotypes) of A. strobiliformis collected from two urban forest plantations in Prague, Czech Republic, were investigated. The concentrations of Ag, Cu, Cd, and Zn were determined in the mushrooms. The metal mobility and fractionation in the soils was investigated by single extractions and sequential extraction. The soil distribution of A. strobiliformis mycelium was assessed by quantitative polymerase chain reaction (qPCR). The metal uptake from the soil into the mushroom sporocarps was traced by Pb isotopic fingerprinting. The findings suggested that A. strobiliformis (i) accumulates primarily Ag from the topsoil layer (circa 12cm deep) and (ii) accumulates Ag associated with the "reducible soil fraction". The concentrations of all metals, particularly Ag and Cu, were significantly higher in the A. strobiliformis sporocarps from one of the investigated sites (Klícov). The elevated concentrations of Ag in the sporocarps from Klícov can possibly be attributed to the higher Ag content in the topsoil layer found at this site. However, the simultaneously elevated concentrations of Cu in A. strobiliformis from Klícov cannot be explained by the differences in the geochemical background and should be attributed to biological factors.

Speciation analysis of elements accumulated in Cystoderma carcharias from clean and smelter-polluted sites.

Collections of Cystoderma carcharias sporocarps were sampled from clean and smelter-polluted sites and analyzed for Ag, As, Cd, Cu, Pb, Se, and Zn contents. Concentrations of all elements were significantly higher in samples from the smelter-polluted area. Except for As and Pb, all elements were effectively accumulated in the sporocarps at both clean and polluted sites. With the highest concentration of 604 mg Cd kg-1, C. carcharias can be considered as Cd hyperaccumulator. As revealed by HPLC-ICPQQQMS analysis, the As species in sporocarps from clean and polluted areas involved besides the major arsenobetaine a variety of known and unknown arsenicals; the occurrence of dimethylarsinoylacetate and trimethylarsoniopropionate is reported for the first time for gilled fungi (Agaricales). Size-exclusion chromatography of C. carcharias extracts supported by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC data indicated that detoxification of intracellular Cd and Cu may largely rely on metallothioneins (MT) or MT-like peptides, not phytochelatins.

Two P1B-1-ATPases of Amanita strobiliformis With Distinct Properties in Cu/Ag Transport.

As we have shown previously, the Cu and Ag concentrations in the sporocarps of Ag-hyperaccumulating Amanita strobiliformis are correlated, and both metals share the same uptake system and are sequestered by the same metallothioneins intracellularly. To further improve our knowledge of the Cu and Ag handling in A. strobiliformis cells, we searched its transcriptome for the P1B-1-ATPases, recognizing Cu+ and Ag+ for transport. We identified transcripts encoding 1097-amino acid (AA) AsCRD1 and 978-AA AsCCC2, which were further subjected to functional studies in metal sensitive Saccharomyces cerevisiae. The expression of AsCRD1 conferred highly increased Cu and Ag tolerance to metal sensitive yeasts in which the functional AsCRD1:GFP (green fluorescent protein) fusion localized exclusively to the tonoplast, indicating that the AsCRD1-mediated Cu and Ag tolerance was a result of vacuolar sequestration of the metals. Increased accumulation of AsCRD1 transcripts observed in A. strobiliformis mycelium upon the treatments with Cu and Ag (8.7- and 4.5-fold in the presence of 5 µM metal, respectively) supported the notion that AsCRD1 can be involved in protection of the A. strobiliformis cells against the toxicity of both metals. Neither Cu nor Ag affected the levels of AsCCC2 transcripts. Heterologous expression of AsCCC2 in mutant yeasts did not contribute to Cu tolerance, but complemented the mutant genotype of the S. cerevisiae ccc2Δ strain. Consistent with the role of the yeast Ccc2 in the trafficking of Cu from cytoplasm to nascent proteins via post-Golgi, the GFP fluorescence in AsCCC2-expressing ccc2Δ yeasts localized among Golgi-like punctate foci within the cells. The AsCRD1- and AsCCC2-associated phenotypes were lost in yeasts expressing mutant transporter variants in which a conserved phosphorylation/dephosphorylation site was altered. Altogether, the data support the roles of AsCRD1 and AsCCC2 as genuine P1B-1-ATPases, and indicate their important functions in the removal of toxic excess of Cu and Ag from the cytoplasm and charging the endomembrane system with Cu, respectively.

Metallothionein-like peptides involved in sequestration of Zn in the Zn-accumulating ectomycorrhizal fungus Russula atropurpurea.

Homeostatic mechanisms preventing the toxicity of free Zn ions in cells involve, among others, cytosolic Zn-binding ligands, particularly the cysteine-rich metallothioneins (MTs). Here we examined the Zn-binding peptides of Russula atropurpurea, an ectomycorrhizal fungus known for its ability to accumulate high amounts of Zn in its sporocarps. The Zn complexes and their peptide ligands were characterized using chromatography, electrophoresis after fluorescent labeling of cysteine residues, and tandem mass spectrometry. Functional complementation assays in Saccharomyces cerevisiae were used to obtain and characterize cDNA sequences. Zn-speciation analysis showed that nearly 80% of the Zn extracted from the sporocarps was associated with cysteine-containing peptides in a 5 kDa complex. Screening of an R. atropurpurea cDNA library for sequences encoding peptides capable of sequestering divalent heavy metals was conducted in the Cd-hypersensitive ycf1Δ yeast. This allowed identification of two cDNAs, RaZBP1 and RaZBP2, which protected the metal-sensitive yeast mutants against Cd and Zn, but not Co, Mn or Cu, toxicity. The corresponding RaZBP1 and RaZBP2 peptides consisting of 53 amino acid (AA) residues and sharing 77% identity showed only a limited sequence similarity to known MTs, particularly due to the absence of multiple Cys-AA-Cys motifs. Both RaZBPs were detected in a native Zn-complex of R. atropurpurea and the recombinant RaZBP1 was found associated with Zn and Cd in yeasts. Altogether, the results point to an important role of RaZBPs in the handling of a substantial portion of the Zn pool in R. atropurpurea.