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

Highlights

• Cystoderma carcharias sporocarps express at least two metallothionein isoforms.

• CcMT1 appears dominating in the binding of hyperaccumulated Cd and Cu.

• CcMT2 appears specialized in Cu binding.

• Mutations of cysteinyl in CcMT2 diminish the Cu-protective effect in yeast model.

• Apparent Cd-MT complex seen in macrofungi in natural conditions for the first time.

Abstract

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.

Introduction

The natural capacity of macrofungi (mushrooms) to accumulate a wide range of heavy metals has been well known for decades. While many metals (e.g., Zn and Cu) are essential for cells in various metalloproteins, they can be toxic in excess. On the other hand, some metals (e.g., Ag, Cd, Pb) without a physiological function for an organism may exert toxic effects even in very low concentrations. Theintracellular mechanisms of metal detoxification in macrofungi generally involve efflux of the excess metal out of the cells or compartmentalization, especially in the case of Cd and Zn. In macrofungi, both metals are predominantly deposited in intracellular compartments, such as vacuoles (Sácký et al., 2014, Ruytinx et al., 2013) or, regarding Zn, in small vesicular compartments (referred to as “zincosomes”) in some cases (Sácký et al., 2014, Blaudez and Chalot, 2011). To handle intracellular metals (Cu and Ag in particular), fungi may further employ cytosolic metallothioneins (MTs) or MT-like peptides of sizes ranging from 26-amino acid (AA) in ascomycete Neurospora crassa to 283-AA (currently the largest known MT) in basidiomycete Tremella mesenterica (Leonhardt et al., 2019, Iturbe-Espinoza et al., 2016, Capdevila and Atrian, 2011), glutathione (Bellion et al., 2006), and rarely glutathione-derived phytochelatin peptides with (γGlu-Cys) repeats (Collin-Hansen et al., 2007). Moreover, non-MT Cd-binding peptides have been reported from two Cd-accumulating species: 13-kDa Cd-mycophosphatin in Agaricus macrosporus (Meisch and Schmitt, 1986) in which Cd binding has been attributed to a phosphate of phosphoserine and glutamyl (Glu) and aspartyl (Asp), and more recently nearly 25-kDa Lentinula edodes cadmium-binding protein (LECBP; Dong et al., 2019) in which the Cd-binding residues seem to be Asp, Glu, and histidinyl (His).

MTs are a ubiquitous, extremely heterologous group of cytosolic peptides rich in cysteinyl (Cys) residues that bind heavy metals through thiolate bonds; however, Zn-binding centers in some bacterial and plant MTs involve also His (Blindauer, 2011). Based on the distribution of Cys residues, MTs can be classified as members of one of the 15 families proposed by Binz and Kägi (1999), whilst a novel system suggests classification according to their preference to bind monovalent or bivalent metal ions (Palacios et al., 2011). The latter scheme recognizes strict Cu-thioneins and genuine Zn/Cd-thioneins as respective extremes, and intermediate MTs with no clear preference for Cu⁺ or Zn^2+/Cd²⁺ binding. Although MTs have been intensively investigated, the genes encoding macrofungal MTs have only been reported from a very few species, with the roles of their MTs in the handling of metals documented mostly in cultured mycelial isolates. Focusing on Basidiomycota, these involve Cu/Cd-inducible Paxillus involutus PiMT1 (Bellion et al., 2007), Cd/Zn-inducible HmMT1 and Ag-inducible HmMT3 in Hebeloma mesophaeum (Sácký et al., 2014), or Cu-responsive HcMT1 and rather Cd-responsive HcMT2 genes in Hebeloma cylindrosporum (Ramesh et al., 2009), the latter behaving in its expression profile like the Pisolithus albus PaMT1 (Reddy et al., 2016). The functions that MTs might provide in the biology of Cu (or chemically similar Ag) were investigated in Laccaria bicolor (Reddy et al., 2014), Suillus himalayensis (Kalsotra et al., 2018) and Amanita strobiliformis (Hložková et al., 2016, Osobová et al., 2011). Nevertheless, it appears that the multiple MT genes existing in macrofungi may play different roles as documented with Zn-binding AsMT3 in Amanita strobiliformis (Hložková et al., 2016) and its homologues later characterized from Suillus luteus as Cu-thioneins (Nguyen et al., 2017).

Our team has recently shown that the saprotrophic mushroom Cystoderma carcharias (Pers.) Fayod (Basidiomycota, Agaricales) from a smelter-polluted area accumulates remarkable concentrations of various metal(loid)s (Borovička et al., 2019). For its exceptional ability to accumulate Cd, with the highest detected concentration of 604 mg Cd kg⁻¹ dry weight (dwt) in sporocarps, C. carcharias can be considered a Cd hyperaccumulator. Preliminary metal speciation analyses in C. carcharias sporocarps suggested that this mushroom uses two fundamental mechanisms known to protect fungal cells against the toxicity of metal ions – compartmentalization in organelles (presumably in the vacuoles) and binding with MTs or MT-like peptides (Borovička et al., 2019). While the sporocarp Cu appeared bound predominantly in a complex with Cys-containing peptides, Zn and Pb were compartmentalized, and Cd was distributed between fractions corresponding to compartmentalized metal (60%) and Cys-containing peptide-associated Cd (40%).

It is worth noting that the roles of MTs in the detoxification of Cd in macrofungal sporocarps have so far only been inferred from data obtained with mycelia in laboratory conditions. In this study, we aimed to solve the question of whether or not a substantial proportion of Cd (and Cu) stored in the cells of C. carcharias sporocarps is bound with MTs in natural conditions. We thus isolated and functionally characterized two MT genes expressed in wild-grown C. carcharias sporocarps from the smelter-polluted area. The results presented here provide strong evidence that such storage of Cd (and Cu) in C. carcharias may involve binding with MTs, as has been previously hypothesized, e.g., with Cu in the ectomycorrhizal species Laccaria laccata and P. involutus (Howe et al., 1997) or seen with Ag and Cu in A. strobiliformis (Beneš et al., 2016, Osobová et al., 2011).