Lichen Substances Affect Metal Adsorption in Hypogymnia physodes

Abstract

Lichen substances are known to function as chelators of cations. We tested the hypothesis that lichen substances can control the uptake of toxic metals by adsorbing metal ions at cation exchange sites on cell walls. If true, this hypothesis would help to provide a mechanistic explanation for results of a recent study showing increased production of physodalic acid by thalli of the lichen Hypogymnia physodes transplanted to sites with heavy metal pollution. We treated cellulose filters known to mimic the cation exchange abilities of lichen thalli with four lichen substances produced by H. physodes (physodic acid, physodalic acid, protocetraric acid, and atranorin). Treated filters were exposed to solutions containing seven cations (Ca²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Mg²⁺, Mn²⁺, and Na⁺), and changes to the solution concentrations were measured. Physodalic acid was most effective at influencing metal adsorption, as it increased the adsorption of Fe³⁺, but reduced the adsorption of Cu²⁺, Mn²⁺, and Na⁺, and to a lesser extent, that of Ca²⁺ and Mg²⁺. Reduced Na⁺ adsorption matches with the known tolerance of this species to NaCl. The results may indicate a possible general role of lichen substances in metal homeostasis and pollution tolerance.

Introduction

Lichens are notable for the production of unique lichen substances, including more than 800 aliphatic, cycloaliphatic, aromatic, and terpenic compounds (Huneck and Yoshimura, 1996). Lichens are also sensitive to a variety of air pollutants and are widely used as biomonitors of air quality. Recently, Białońska and Dayan (2005) linked lichen secondary chemistry with the use of lichens as biomonitors in a study of the common lichen Hypogymnia physodes. This lichen has often been employed as a model species in ecophysiological and biomonitoring studies because of its abundance and its moderate sensitivity to SO₂ and heavy metals. In Europe, H. physodes has declined in many areas with significant pollutant load. Białońska and Dayan (2005) found that thalli of H. physodes transplanted to areas polluted with heavy metals and inorganic S compounds exhibited significantly lower concentrations of atranorin, physodic acid, and 3-hydroxyphysodic acid and significantly higher concentrations of physodalic acid. They postulated that physodalic acid might participate somehow in the stress response of H. physodes to toxic effects of heavy metals and acidic inorganic S compounds. This was the first time that the content of lichen substances had been linked to pollution tolerance. However, the authors did not provide any mechanistic hypothesis to explain any such relationship.

Our hypothesis is that the presence of lichen compounds significantly reduces toxic effects of heavy metals to living cells by controlling their extracellular adsorption and thereby influencing their intracellular uptake. Specifically, we tested the hypothesis that physodalic acid affects the adsorption of metal ions at cation exchange sites and thereby contributes to the heavy metal tolerance of H. physodes. To test our hypothesis, we used cellulose filters coated with physodalic acid and other lichen substances of H. physodes (atranorin, physodic acid, and protocetraric acid) as a standardized model system containing numerous hydroxyl groups as cation exchange sites (Hauck et al., 2007). Lichen cell walls and exopolysaccharide matrices harbor many hydroxylic and carboxylic binding sites for cations, from which delayed intracellular uptake is possible (Hauck et al., 2006). Metal ions chosen for the study included the transition metals Cu²⁺, Fe²⁺, Fe³⁺, and Mn²⁺ and the alkaline earth metals Ca²⁺ and Mg²⁺, which (1) occurred in measurable concentrations in the stemflow of trees with H. physodes in ecosystem studies conducted in Europe and North America (Hauck, 2003); (2) are essential macro- or micronutrients; and (3) are, in the case of the transition metals, toxic to H. physodes when concentrations significantly exceed the amount required for nutrition (Hauck, 2003). The alkali metal Na⁺ was studied in addition, as it is often the most common metal in precipitation.

If our hypothesis is correct, the presence of lichen compounds on cellulose filters will significantly alter the adsorption of metal cations from solution. We were especially interested in the effects of physodalic acid on cation uptake inasmuch as this compound was specifically mentioned by Białońska and Dayan (2005) to be involved in the stress response of H. physodes.

Methods and Materials

Hypogymnia physodes (L.) Nyl. is a common foliose lichen of the temperate and boreal zones of the northern hemisphere, which primarily occurs as an epiphyte (McCune, 2002). Seven lichen substances have been identified in H. physodes (McCune, 2002), which are either located in the cortex (atranorin, chloroatranorin) or in the medulla (physodic, 3-hydroxyphysodic, physodalic, protocetraric, 2′-O-methylphysodic acids). The two latter substances are found only in minor amounts (McCune, 2002). The cortical lichen substances of H. physodes are depsides, whereas the medullary ones belong to the depsidones (Huneck and Yoshimura, 1996).

Pure atranorin, physodic acid, physodalic acid, and protocetraric acid from the collection of S. Huneck were dissolved in acetone (2.5 mM) and applied to ash-free cellulose filter paper (Blue Ribbon Filters, Schleicher & Schuell, Dassel, Germany) by shaking filter strips (ca. 20 cm², 160 mg) in the lichen substance solution for 1 hr. Cellulose filters consist of randomly interlaced fibers that are littered with exchange sites that bind to metal ions, similar to the cell wall surfaces of lichens (Hauck et al., 2007). Untreated filter strips were used as controls. Two strips of impregnated filters per replicate were exposed for 1 hr to 25 ml of micromolar solutions of CuCl₂, FeCl₂, FeCl₃, MnCl₂, CaCl₂, MgCl₂, or Na₂SO₃; the concentrations applied (Table 1) referred to the optimum measuring range of the AAS (Vario 6, Analytik Jena, Jena, Germany; Cu, Fe, Mn, Ca, Mg) or inductively coupled plasma-atomic emission spectrometry (Spectraflame, Spectro Analytical Instruments, Fitchburg, MA, USA; Na) used for chemical analysis. After incubation, the filter paper was removed with forceps and metal concentrations in the solution were analyzed. The experiment was run in five replicates. Statistical analyses were conducted with SAS 6.04 software (SAS Institute, Cary, NC, USA). Duncan’s multiple range test was applied to test for significance of differences between means after testing for normal distribution with the Shapiro–Wilk test.

Table 1 Cation adsorption from micromolar salt solutions by filter paper soaked with 2.5 mM of different lichen substances

Results

For all but one of the cations studied, the presence of lichen compounds on filter strips resulted in significantly reduced removal of the cations from solution, indicating that lichen compounds prevent the binding of cations to exchange sites on the filters. Physodalic acid was especially inhibitory inasmuch as filters coated with this lichen compound adsorbed from solution significantly less Cu²⁺, Mn²⁺, Na⁺, Ca²⁺, and Mg²⁺ than control samples of filters not soaked with a lichen substance (Table 1). Adsorption of Mn²⁺ was also reduced by protocetraric acid, as was that of Ca²⁺ by atranorin. All studied lichen substances also inhibited the adsorption of Na⁺. Lichen compounds caused significantly enhanced adsorption of only one metal cation, and this was Fe³⁺. While the concentrations of Fe²⁺ were not affected by any of the lichen substances, Fe³⁺ was nearly completely removed from solution by filters coated with physodalic acid. Physodic and protocetraric acids significantly increased removal of Fe³⁺ from the incubation medium by 43–44% (Table 1).

Discussion

Our results provide a mechanistic explanation for the hypothesis of Białońska and Dayan (2005) that enhanced synthesis of physodalic acid in polluted areas is a stress response of H. physodes to heavy metal pollution. High ambient concentrations of both Cu²⁺ and Mn²⁺ have been shown to limit the abundance of H. physodes in the field (Hauck, 2003; Hauck and Paul, 2005). An inhibition of the adsorption of these ions in the apoplast would, therefore, increase the tolerance of H. physodes to Cu²⁺ and Mn²⁺.

The strong reduction of the Fe³⁺ concentration in the incubation medium by physodalic acid suggests that this lichen substance forms complexes with Fe³⁺. Evidence for the existence of such complexes with Fe³⁺ has been found for several lichen substances (Engstrom et al., 1980; Hauck et al., 2007) belonging, like physodic acid, to the depsidones (fumarprotocetraric and norstictic acids), the depsides (divaricatic acid), the pulvinic acid derivatives (pulvinic and rhizocarpic acids), or the anthraquinones (parietin). Complex formation with transition metals has also been established for other combinations of lichen substances and metal ions (e.g., Cu²⁺) and was mostly discussed in the light of immobilization of potentially toxic ions (Takani et al., 2002).

Occurrence of Fe³⁺-adsorbing lichen substances in lichens of sites with low Fe availability, but lack in most lichen species of Fe-rich substrate, suggests that Fe³⁺ bound in complexes with lichen substances is subsequently taken up into the cytoplasm (Hauck et al., 2007). The only Fe³⁺ adsorbing lichen substances known, so far, from lichen species of Fe-rich substrate, viz. norstictic and rhizocarpic acids, simultaneously reduce the adsorption of Fe²⁺ (Hauck et al., 2007). Increased uptake of Fe³⁺ in H. physodes by physodalic acid and other lichen substances would increase its tolerance to Mn²⁺, as Mn-related damage depends on the Mn/Fe ratio (Hauck and Paul, 2005).

If our hypothesis is correct, the increased production of physodalic acid by H. physodes in polluted areas observed by Białońska and Dayan (2005) is a response to pollution stress. However, the reduced concentration of other lichen substances (atranorin, physodic acid, and 3-hydroxyphysodic acid) also requires explanation. The decline of physodic acid in thalli of H. physodes transplanted to sites with heavy metal pollution could be due to conversion into the chemically related physodalic acid. Protocetraric acid, which is even more closely related to physodalic acid than physodic acid (Huneck and Yoshimura, 1996), was not included in the analyses of Białońska and Dayan (2005) and should be investigated for possible stress responses. Inhibition of Na⁺ adsorption by all investigated lichen substances (Table 1) agrees with the known tolerance of H. physodes to NaCl (Punz, 1979).

The putative ability of physodalic acid to increase heavy metal tolerance would explain why H. physodes was less subject to pollutant-caused declines in the past than other European Hypogymnia species, including Hypogymnia farinacea, Hypogymnia tubulosa, and Hypogymnia vittata, as H. physodes is the only European species with physodalic acid (McCune, 2002). The assumed protective effect of physodalic acid in H. physodes is probably limited because this substance is primarily produced by the fungal hyphae of the medulla and the algal layer (McCune, 2002), while the cortical atranorin did not affect the adsorption of transition metals (Table 1). The absence of a protective lichen substance from the cortex would explain the moderate heavy metal tolerance of H. physodes (Hauck, 2003; Hauck and Paul, 2005). Lecanora conizaeoides, a markedly Mn-tolerant lichen (Hauck and Paul, 2005), contains the depsidone fumarprotocetraric acid in the entire thallus. This substance also reduces Mn²⁺ adsorption and is even more effective at adsorbing Fe³⁺ than physodalic acid (Hauck et al., 2007). Though our experiment suggests a protective role of physodalic acid, it has to be kept in mind that adsorption rates on fungal and algal cell walls might differ from that of cellulose fibers. Moreover, other mechanisms including immobilization in polyphosphate bodies and phytochelatines are involved in the heavy metal tolerance of lichens (Hauck and Paul, 2005).