copper toxicity to five parmelia lichens in vitro

Copper toxicity to five Parmelia lichens in vitro

Joao Paulo Cabral *

Faculty of Sciences, Department of Botany, University of Oporto, Rua do Campo Alegre, 1191, 4150-181 Porto, Portugal

Center of Marine and Environmental Research (CIIMAR), University of Oporto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal

Received 21 May 2002; received in revised form 15 October 2002; accepted 15 October 2002

Abstract

Treatment of Parmelia caperata , P. perlata , P. subrudecta , P. sulcata and P. tiliacea with CuSO4 resulted in a time-

and copper-concentration-dependent decrease in the total and intracellular potassium concentrations of the thallus,

indicating that copper damaged the cytoplasmic membrane. Treatment with copper also resulted in a time-dependent

increase in the total copper concentration of the thallus. After 4 h of exposure to copper, the process of potassium efflux

was essentially completed but the absorption of copper was still increasing; moreover, the amount of copper bound to

the thallus exceeded twice the amount of potassium released from the thallus, suggesting that cupric ions reached

intracellular sites in the thallus, and K�/Cu2� exchange was not electroneutral. After 5 h of exposure to copper, the

extent of decrease in the total and intracellular potassium concentrations of the thallus was positively correlated with

copper absorption levels, but only at 0.05B/P B/0.10, suggesting that membrane damage was proportional to the

amount of bound copper, but other factors could have been operative, namely binding of copper to the cell wall.

Acetone extracts of untreated thalli contained low concentrations of amino acids, polyols, and sugars, but considerable

amounts of lichen substances: atranorin, caperatic, constictic, lecanoric, menegazziaic, protocetraric, salazinic, stictic,

and usnic acids. Titration of the extracts with copper and assay of the free Cu2� concentration revealed the presence of

copper-binding ligands, and several successive absorption cycles, most probably corresponding to the binding of Cu2�

to each of the lichen substances detected in the extracts. However, no significant correlation (P �/0.10) was found

between the Cu2�-complexing capacity of acetone extracts and copper-induced membrane damage. It was concluded

that in the studied Parmelia species, and in the experimental conditions used in this work, copper toxicity was not a

simple function of the Cu2�-binding properties of the lichen substances present in the thallus. Several hypotheses were

formulated to interpret the results.

# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Cupric ions; Lichen secondary metabolites; Membrane damage; Potassium efflux

1. Introduction

Most borderline (e.g. As, Cd, Co, Cr, Cu, Ni,

Sn, Zn) and soft (class B) (e.g. Ag, Au, Hg, Pb, Tl)

metal ions are toxic (at all concentrations, or

above a certain threshold concentration). Toxicity

* Present address: Departamento de Botanica, Rua do

Campo Alegre, 1191, 4150-181 Porto, Portugal. Tel.: �/351-

22-6002153; fax: �/351-22-6092227

E-mail address: [email protected] (J.P. Cabral).

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S0098-8472/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

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generally increases with increasing class B char-acter (Nieboer and Richardson, 1980). In particu-

lar copper, a borderline metal with a relatively soft

character, is highly toxic to plants, usually in the

micromolar range of exposure concentrations.

Copper inhibits root elongation and branching,

reduces transroot potential, increases lignin synth-

esis, blocks the photosynthetic electron transport,

and induces chlorophyll degradation and leakageof pool metabolites (Sandmann and Boger, 1980;

Arduini et al., 1995; Quartacci et al., 2001;

Patsikka et al., 2002). Although copper can inter-

fere with a number of physiological processes, the

primary site of copper toxicity is probably at the

cell membrane (Demidchik et al., 1997; Chen et al.,

2000). The lipid bilayer can be damaged by

displacement of endogenous-stabilizing cations(mainly Ca2� and Mg2�), by alterations in the

phospholipic composition, and/or by lipid perox-

idation (Avery et al., 1996; Demidchik et al., 1997;

Howlett and Avery, 1997; Murphy et al., 1999;

Quartacci et al., 2001). These result in a disorga-

nization of the packed phospholipidic structure,

with alterations in membrane fluidity and loss of

selective permeability (Quartacci et al., 2001).Copper can substitute essential cofactors and

bind to �/SH groups of proteins (including enzy-

matic systems, channels, and carriers) causing loss

of function (Nieboer and Richardson, 1980; Stau-

ber and Florence, 1987; Meharg, 1994; Demidchik

et al., 1997). Copper-induced membrane damage

usually results in the release of significant amounts

of K� and pool metabolites from the cells(Hassall, 1963; Joho et al., 1984; Ohsumi et al.,

1988).

Lichen mycobionts produce, mainly in the

symbiotic state, a special class of metabolites, the

lichen substances (Lawrey, 1986; Leuckert et al.,

1990; Ahmadjian, 1993; Elix, 1996). Lichen sub-

stances are phenolic secondary metabolites, have

very low or low solubility in water (Iskandar andSyers, 1971), but are highly soluble in organic

solvents (Fahselt, 1994; Elix, 1996). Lichen sub-

stances occur in the cortex or in the medulla of the

thallus, at the surface of the mycobiont hyphae,

and account for 0.1�/5% thallus dry weight (Fah-

selt, 1994). Several hundreds of lichen substances

have been identified in lichens, and most of the

lichen species accumulate these metabolites. Sev-eral roles have assigned to this special class of

molecules, including antimicrobial activity (Hu-

neck, 1968; Lawrey, 1986), antiherbivore roles

(Lawrey, 1986; Fahselt, 1994), antitumor activity

(Huneck, 1968), and light-screening ability (Law-

rey, 1986; Fahselt, 1994 Solhaug and Gausla,

1996).

Most lichen substances are acidic and whenisolated, in vitro, can bind metal ions, including

aluminum, calcium, copper, iron, and magnesium

(Syers, 1969; Iskandar and Syers, 1972; Purvis et

al., 1987). In lichens from cupriferous substrata,

the formation of complexes between lichen sub-

stances (norstictic and psoromic acids) and copper,

in the thallus, has been implicated in the resistance

of these lichens to copper toxicity (Purvis et al.,1985, 1987, 1990). These results suggested that

lichen substances might modulate copper toxicity.

Our initial working hypothesis was that copper

toxicity is a direct function of the Cu2�-binding

properties of the lichen substances present in the

thallus. To test this hypothesis, lichen substances

were extracted from the thallus of five Parmelia

species (growing in unpolluted sites), and thecopper-complexing capacity of these substances

determined by ion-selective electrode potentiome-

try. Copper toxicity was assessed by membrane

damage, and this was evaluated by determining, by

flame emission photometry and atomic absorption

spectrophotometry, the total and intracellular

potassium concentration of the thallus, after

copper treatment. In order to understand thekinetics of membrane damage, the binding of

copper to the thallus was also studied in identical

experimental conditions.

2. Materials and methods

2.1. Lichens and sample preparation

Five Parmelia species were used in this work,

Parmelia caperata (L.) Ach., P. perlata (Huds.)

Ach., P. subrudecta Nyl., P. sulcata Taylor, and P.

tiliacea (Hoffm.) Ach. These species were chosen

because they are all abundant in the country and

display a considerable diversity of lichen sub-

J.P. Cabral / Environmental and Experimental Botany 49 (2003) 237�/250238

stances present in the thallus. Lichens were col-lected in a rural unpolluted area of Oporto district

(northwest Portugal), and kept dry under normal

laboratory conditions for no more than a week.

The day before the experiment, the lichen was

placed on a moist paper filter. Adhering particles

and organisms were carefully removed from the

thallus with the aid of fine stainless steel tweezers

and a binocular microscope. The lichen waswashed twice in Mg2�-PIPES buffer, and placed

on a moist paper filter.

2.2. General layout of the experiments

Mg2�-PIPES buffer (10 mM free acid, 8.5 mM

Mg2�, 26 mM ionic strength; Cabral, 1992), pH

6.50, was used in all experiments since: (i) The use

of buffered solutions is indispensable in studies ofcopper toxicity (Baes and Mesmer, 1976; Buffle,

1990; Sadiq, 1992). (ii) At pH 6.50, most copper

exists as hydrated Cu2� ions (hydroxy and car-

bonate complexes are formed at higher pH), and

these is considered the main toxic species (see

Section 4). (iii) Lichen samples immersed in this

medium showed preserved vitality for several

hours (assessed by the potassium and mannitolconcentrations).

For membrane damage and copper binding to

lichen thallus experiments, samples (40 mg fresh

weight) were placed in small beakers containing 20

ml Mg2�-PIPES buffer. Volumes of a 100 mM

CuSO4 �/5H2O solution were then added, and the

beakers were incubated in a waterbath with

shaking (75 rpm min�1) at 25.0 8C. The use ofrather high copper concentrations (25�/500 mM)

was necessary in order to obtain significant

potassium loss from the cells within 6 h of copper

treatment. At the end of the incubation period, the

samples were removed from the solutions. Total

potassium, intracellular potassium, and total cop-

per in the samples were assayed by flame emission

photometry and atomic absorption spectrometry,respectively.

For copper binding to acetone extracts experi-

ments, the extracts (see below for method of

preparation) were diluted in Mg2�-PIPES buffer,

and transferred to a thermostatized beaker, at

25.0 8C. Small volumes of a concentrated CuSO4 �/

5H2O solution were added to the diluted extracts.After each addition, the free Cu2� concentration

was assayed by ion-selective electrode potentiome-

try. Acetone extracts were titrated immediately

after preparation.

2.3. Membrane damage

Membrane damage was assessed by measuringthe total and intracellular potassium concentra-

tions of samples exposed to copper. For measuring

total potassium concentration, lichen samples were

treated with 500 mM copper for 0, 10, 30, 60, 120,

240, 300, and 360 min. Controls were treated for 0

and 360 min. At the end of the incubation period,

samples were removed from the beakers, blotted

dry, and the total potassium concentration assayedby flame emission photometry, as described below.

For measuring intracellular potassium concentra-

tion, lichen samples were treated with 0, 25, 50,

100, 150, 200, 250, 300, 350, 400, 450, and 500 mM

copper for 300 min. At the end of the incubation

period, samples were removed from the beakers

and blotted dry. For the determination of intra-

cellular potassium, samples were immersed in 10ml distilled water for 30 min, to remove surface

and intercellular potassium, followed by two

treatments with 10 ml NiCl2 (20 mM) for 30 min

each, to remove cell wall bound potassium (Brown

and Beckett, 1984). At the end of the treatments,

samples were removed from the beakers, blotted

dry, and the potassium concentration was assayed

by flame emission photometry, as described below.For each copper concentration, the intracellular

potassium concentration was expressed as a per-

centage of the untreated samples. These percen-

tages were converted into probits, and plotted

against the log of copper dose. The resulting plots

showed a linear relationship. The copper concen-

tration needed to release 75% of intracellular

potassium (in relation to untreated samples),ED75, was calculated using the parameters of the

regression line.

2.4. Copper binding to the thallus

Lichen samples were treated with 500 mM

copper for 0, 10, 30, 60, 120, 240, 300, and 360

J.P. Cabral / Environmental and Experimental Botany 49 (2003) 237�/250 239

min. At the end of the incubation period, sampleswere removed from the beakers, blotted dry, and

the copper concentration of the samples was

assayed by atomic absorption spectrophotometry,

as described below.

2.5. Acetone extracts of the thallus

Some lichen substances are thermolabile, being

degraded at high temperatures (Culberson et al.,1977; Mirando and Fahselt, 1978). For this reason,

untreated lichen samples (ca. 845 mg fresh weight)

were dried at moderate temperatures (45 8C) for a

restricted period of time (18�/24 h; Culberson et

al., 1977). After drying, samples (ca. 300 mg dry

weight) were cut into pieces with the aid of

stainless steel scissors, and placed in a small

beaker. Samples were not ground since thisprocedure resulted in a significant contamination

of the extracts with intracellular constituents such

as potassium, amino acids, and chlorophylls (un-

published observations). Acetone (1.8 ml) was

then added, and the beaker incubated in a water-

bath with moderate shaking, at 25 8C for 1.5 h. At

the end of the incubation period, the acetone

extract was separated from the lichen fragments,and another volume of acetone was added. After

another period of incubation, the second acetone

extract was added to the first extract. The pooled

extract was stored in a closed tube, at room

temperature. Further treatments of the lichen

fragments with acetone did not result in significant

additional extraction of lichen substances (unpub-

lished observations).Acetone extracts were analyzed for amino acids,

polyols, sugars, and lichen substances. For the

assay of amino acids, polyols, and sugars, acetone

extracts were evaporated to dryness with gentle

heat, and distilled water was added to the residue.

Amino acids were assayed by the ninhydrin

method as described by Rosen (1957), using lysine

as the standard. Polyols were assayed using theperiodate method (Lewis and Smith, 1967; Dudley

and Lechowicz, 1987), with mannitol as the

standard. Sugars were assayed by the cysteine�/

sulphuric acid method (Chaplin, 1986) and by

the phenol�/sulphuric acid method (Dubois et al.,

1956), using glucose as the standard. Lichen

substances were assayed by UV spectrophotome-try and chromatography. For UV spectrophoto-

metry, acetone extracts were evaporated to dryness

with gentle heat, and the residue was dissolved in

methanol. The methanolic extracts were diluted in

buffer, prior to analysis. Spectra were recorded in

a double-beam model spectrophotometer (Jasco;

model V-530) in the 240�/350 nm range. Lichen

substances were identified by one-dimensionalthin-layer chromatography using the three-sol-

vent-system method standardized for lichen pro-

ducts (Culberson and Kristinsson, 1970;

Culberson, 1972; Santesson, 1973; White and

James, 1985; Orange et al., 2001). The acetone

extracts of the studied lichens and solutions of

pure lichen substances (atranorin, norstictic acid,

scrobiculin, stictic acid, and (�/)usnic acid) wereapplied to Merck silica gel 60 F-254 aluminum or

glass plates (20�/20 cm). Plates were developed in

the diagnostic solvents A, B and C. After the

chromatograms had air-dried, they were sprayed

with 10% H2SO4 and heated at 100 8C until

colours developed. Identification of lichen sub-

stances present in the extracts was carried out by

comparison with data reported in the literatureand the behaviour of pure compounds.

2.6. Copper binding to acetone extracts

The acetone extracts (180 ml) were diluted in

Mg2�-PIPES buffer (10 ml), and titrated with a

CuSO4 �/5H2O solution. Free Cu2� was assayed

with a copper-selective electrode (Metrohm Ltd.;

model 6.0502.140), together with a double junctionAg/AgCl/saturated KCl reference electrode (Me-

trohm; model 6.0726.100). Binding of Cu2� to P.

caperata , P. perlata , P. sulcata and P. tiliacea

acetone extracts was represented by successive

Langmuirian curves, each corresponding to an

absorption phase. The delimitation of the absorp-

tion phases was carried out by visual inspection of

the absorption isotherms (bound [Cu2�] vs. free(equilibrium) [Cu2�]), and confirmed by examina-

tion of the free [Cu2�]/bound [Cu2�] vs. free

[Cu2�] plots (Van der Berg and Kramer, 1979;

Hart, 1981; Neubecker and Allen, 1983).The

complexation capacity was calculated as the in-

verse of the slope values for each regression line,


free [Cu2�]/bound [Cu2�] vs. free [Cu2�], corre-sponding to the last absorption phase (Van der

Berg and Kramer, 1979; Hart, 1981; Neubecker

and Allen, 1983).

2.7. Assay of potassium by flame emission

photometry and copper by atomic absorption

spectrophotometry

The assay was carried out in digested subsam-

ples of dried lichen samples (at 60 8C). The

digestion was performed by adding 1 ml ofconcentrated HNO3 to the lichen subsamples,

and heating in a boiling waterbath, until dissolu-

tion. The determination of potassium was carried

out in a flame photometer (Jenway; model PFP7)

and copper in an atomic absorption spectrophot-

ometer (Philips; model PU 9200X). The accuracy

of this procedure was checked by comparison with

determinations carried out on reference lichenmaterial (Evernia prunastri ; reference material

IAEA-336; Stone et al., 1995). Values found for

the potassium and copper concentrations of re-

ference material were within the 95% confidence

interval of the mean certified values, indicating

that the procedure used in the determinations was

accurate.

2.8. Chemical and solutions

Solutions and standards were prepared with

deionised double distilled water or analytical grade

acetone or methanol. Mg2�-PIPES (1,4-piperazi-

nediethanesulfonate) buffer, pH 6.50, was pre-

pared by neutralizing the free acid (Sigma) with

MgO. Reference lichen material (E. prunastri ,

IAEA 336) was bought from Promochem Com-

pany. Atranorin was from Sigma and (�/)usnicacid from Fluka. Norstictic acid, scrobiculin, and

stictic acid were generous gifts from Dr. Thorsten

Lumbsch (Essen University, Germany).

2.9. Statistics

Correlation between variables was assessed by

determining the linear correlation coefficient,

using Microsoft Excel 2000 program.

3. Results

3.1. Membrane damage

Immersion of lichen samples in 500 mM copper

solutions resulted in a time-dependent decrease in

the total potassium concentration of the thalli, the

process being essentially completed after 4 h of

copper treatment (Fig. 1 and Table 1). Using the

sequential elution procedure of Brown and Beckett

(1984), results showed that after 5 h of exposure to

Fig. 1. Total potassium concentration of P. caperata (A), P.

perlata (B), P. subrudecta (C), P. sulcata (D), and P. tiliacea

(E) lichen samples, immersed in buffer, and exposed to 500 M

copper at 25.0 8C. Control (untreated) samples are displayed as

open circles. The mean number of determinations and the mean

coefficient of variation of the determinations was 6.6, 14%, 8.1,

32%, 3.9, 21%, 5.7, 18%, 3.7, 26%, for P. caperata , P. perlata ,

P. subrudecta , P. sulcata , and P. tiliacea , respectively.


500 mM copper, only a small fraction (4%, mean

value for the five species) of the potassium present

in the thallus was extracellular. Potassium leaked

from inside the cells after membrane damage was

therefore released into the suspending medium and

was not bound to cell walls and present in the

intercellular spaces. The observed decrease in total

potassium therefore reflected the extent of copper-

induced membrane damage.

Immersion of lichen samples in 25�/500 mM

copper solutions for 5 h resulted in a concentra-

tion-dependent decrease in the intracellular potas-

sium concentration of the thalli. Plots of

potassium concentration vs. copper concentration

were sigmoidal. Replotting y data in a probit scale

(or converting y values to probits), and converting

x values to log dose (Finney, 1952; Hewlett and

Plackett, 1979) yielded straight lines with correla-

tion coefficients higher than 0.768 (P B/0.001)

(Fig. 2). Using the probit transformation, the

sensitivity of an organism to an applied chemical

can be assessed by an appropriate effective dose

(ED; Table 1).

Results obtained by using total K� after 6 h

copper treatment and ED75 were correlated (r�/

0.973, P B/0.01). Using these two parameters, the

Table 1

Membrane damage and copper binding to the thallus

Species Total K�a (% control) ED75b (mg mg�1 dw) Copper boundc (mg mg�1 dw)

P. caperata 35 62.5 14.0

P. perlata 22 13.2 16.7

P. subrudecta 38 83.7 8.6

P. sulcata 16 11.6 19.7

P. tiliacea 20 16.0 14.1

a Total potassium concentration of the lichen sample exposed to 500 mM copper for 6 h (expressed as the percentage in relation to

untreated samples). Results are means from three to ten determinations. The mean coefficient of variation of the determinations was

29%.b Copper concentration needed to release 75% of intracellular potassium (expressed as the percentage in relation to untreated

samples), after 5 h copper treatment. Pooled results from three separate experiments. Values were calculated using the probit regression

equations.c Total copper concentration of the lichen sample exposed to 500 mM copper for 6 h. Results are means from 3 to 11 determinations.

The mean coefficient of variation of the determinations was 10%.

Fig. 2. Intracellular potassium concentration of P. caperata

(A), P. perlata (B), P. subrudecta (C), P. sulcata (D), and P.

tiliacea (E) lichen samples, immersed in buffer, and exposed to

25�/500 M copper at 25.0 8C for 5 h. K� is expressed as

percentage in relation to control samples, in a probit scale.

Pooled results from three separate experiments.


copper-induced membrane damage increased inthe following order: P. subrudecta , P. caperata , P.

perlata , P. tiliacea , and P. sulcata .

3.2. Copper binding to the thallus

Immersion of lichen samples in 500 mM copper

solutions resulted in a time-dependent increase in

the copper concentration of the thalli (Fig. 3). The

kinetics of accumulation was similar for all five

Parmelia species. After 6 h of exposure, the

absorption was essentially completed or near

completion. Plateau levels were however different

for the five species, and the highest (P. sulcata )

and the lowest (P. subrudecta ) differed more than

twofold (Table 2).

3.3. Chemical analysis of acetone extracts

Acetone extracts contained very low concentra-

tion of amino acids, polyols, and sugars. Acetone

extracts diluted in 10 ml buffer had the following

composition: amino acids ranged from 1.4 to 7.4

mg lysine equiv., polyols varied between 9.0 and

15.4 mg mannitol equiv., and sugars ranged from

5.5 to 37.0 mg glucose equiv. (by the cysteine�/

sulphuric acid method) or from 6.8 to 18.0 mg

glucose equiv. (by the phenol�/sulphuric acid

method).Typical UV spectra of the extracts in methanol

are shown in Fig. 4. Lichen substances identified in

the chromatograms (Table 2) are identical to those

reported for material collected in other areas of the

world, except for the lack of detectable amounts of

atranorin in P. subrudecta (Culberson, 1970;

Purvis et al., 1992).

3.4. Copper binding to acetone extracts

Acetone extracts of the five Parmelia species

contained ligands that bound copper. Plots of

bound Cu2� concentration vs. free (equilibrium)

Cu2� concentration (Fig. 5) were essentially

Langmuirian (type L isotherm of Giles et al.

(1960) system of classification). However, the

subtype of uptake isotherm varied among the

five tested Parmelia species. Binding of Cu2� to

P. caperata , P. perlata , P. subrudecta , P. sulcata

and P. tiliacea extracts appeared to be composed

of three, four, one, two, and two successive

adsorption phases, respectively. The copper-com-

plexing capacities of the extracts are displayed in

Table 2.

Fig. 3. Copper concentration of P. caperata (A), P. perlata (B),

P. subrudecta (C), P. sulcata (D), and P. tiliacea (E) lichen

samples, immersed in buffer, and exposed to 500 M copper at

25.0 8C. The mean number of determinations and the mean

coefficient of variation of the determinations was 5.9, 21%, 6.0,

12%, 3.7, 16%, 4.9, 13%, 3.6, 13%, for P. caperata , P. perlata ,

P. subrudecta , P. sulcata , and P. tiliacea , respectively.


4. Discussion

4.1. Membrane damage and copper binding to the

thallus

Copper damages the cytoplasmic membrane ofplant cells usually in the micromolar (1�/100) range

of concentrations. Murphy et al. (1999) found that

the addition of 30 mM Cu2� to Arabidopsis

seedlings growing in nutrient medium caused a

rapid K� efflux during the first 3 h of copper

treatment, followed by a slow rate of release

beginning at about 6 h of exposure to Cu2�.

Quartacci et al. (2001) studied the alterations

caused by exposure of wheat seedlings cells to

Table 2

Chemical characteristics of the acetone extracts

Species Copper-complexing capacitya Lichen substances

(mg copper 10 ml�1 extract) (mg copper extract 1 mg�1 dw)

P. caperata 146 10.1 Caperatic acid, protocetraric acid, usnic acid

P. perlata 197 16.3 Atranorin, constictic acid, menegazziaic acid, stictic acid

P. subrudecta 86 4.7 Lecanoric acid

P. sulcata 100 9.0 Atranorin, salazinic acid

P. tiliacea 152 11.6 Atranorin, lecanoric acid

a Acetone extracts were diluted in buffer and titrated with Cu2�. The complexation capacity was evaluated from the values of the

regression (free [Cu2�]/bound [Cu2�]) vs. free [Cu2�], corresponding to the last absorption cycle. Results are means of three to four

determinations. The mean coefficient of variation of the determinations was 15%.

Fig. 4. UV spectra of the extracts redissolved in methanol (and

diluted in buffer). P. caperata (A), P. perlata (B), P. subrudecta

(C), P. sulcata (D), and P. tiliacea (E). Absorbances are

represented in an arbitrary scale.

Fig. 5. Binding of Cu2� by P. caperata (A), P. perlata (B), P.

subrudecta (C), P. sulcata (D), and P. tiliacea (E) acetone

extracts (diluted in buffer). Results from typical experiments.

Absorption cycles are indicated by roman numbers.


Cu2� for 11 days. After copper treatment, rootswere harvested and incubated in distilled water for

24 h. Whereas seedlings grown with 0 or 0.3 mM

Cu2� and placed in distilled water released 30% of

the total K�, seedlings grown with 50 mM Cu2�

released 60% of cellular potassium, indicating that

this copper concentration damaged the cytoplas-

mic membrane of treated cells. Arduini et al.

(1995) studied the effects of Cu2� on pine seed-lings grown in nutrient solution. After 1, 3, 7, and

10 days of exposure to 0.012, 0.1, 1, or 5 mM

Cu2�, root tips were excised and placed in

aqueous solution of trypan blue. Penetration of

the dye was observed in most of the cells of

seedlings exposed to 1 mM Cu2� for 10 days,

indicating that the cytoplasmic membrane had

been damaged. In this work, it was necessary touse considerably higher (up to 500 mM) copper

concentrations, in order to cause a pronounced

release of potassium from the cells. This can be

due to the use of shorter exposure periods (few

hours instead of several days as often used with

plants) and/or an intrinsic relative resistance of the

studied lichen species to copper.

Immersion of lichen samples in 500 mM coppersolutions resulted in a pronounced release of K�

from the cells and a significant accumulation of

copper in the thalli. The kinetics of these two

processes were however different. Whereas the

decrease in thallus potassium was essentially

completed after 4 h copper treatment, the absorp-

tion of copper increased up to 6 h of incubation.

Moreover, the amount of copper bound to thethallus exceeded twice the amount of potassium

released from the thallus, suggesting that cupric

ions reached intracellular sites in the thallus, and

K�/Cu2� exchange was not electroneutral. In

normal physiological conditions, copper uptake

and trafficking in eukaryotic organisms appears to

follow a general pattern. Cu2� is reduced to Cu�

by a cytoplasmic membrane reductase (Hassettand Kosman, 1995; Hill et al., 1996). Cuprous ions

are then transported to the cytoplasm by specia-

lized transporter proteins (Lin et al., 1997; Fox

and Guerinot, 1998; Harris, 2000). Once inside the

cells, Cu� ions are bound to glutathione or to

specialized proteins (metallochaperones) (Fox and

Guerinot, 1998; Harris, 2000). These chaperones

guide copper to their appropriate biological part-ners (such as the cytochrome c oxidase and super-

oxide dismutase), and prevent intracellular copper

toxicity by decreasing the total cytoplasmic-free

copper to extremely low levels (Pufahl et al., 1997;

Portnoy et al., 1999; O’Halloran and Culotta,

2000). These processes occur in growing condi-

tions, at very low copper concentrations (usually

less than 1 mM Cu2�). In this work, very muchhigher (and stressing) copper concentrations were

used. Therefore, copper could have been accumu-

lated inside the cells as cuprous ions, as reported

for growing cells, and/or, if gross damage occurred

in the cytoplasmic membrane, copper could have

crossed the permeability barriers as unaltered

Cu2� ions.

The extent of released potassium (assessed byresidual K� and ED75) and the amount of copper

bound to the thallus were correlated but only at

0.05B/P B/0.10, suggesting that membrane da-

mage was proportional to the amount of bound

copper, but other factors were operative. Among

these, it is likely that a fraction of copper bound to

the thallus was extracellular, and therefore was not

directly involved in membrane damage.

4.2. Chemical composition of the acetone extracts

and copper-binding capacity

As expected from the method of preparation of

the extracts, acetone extracts contained very low

concentrations of pool and intracellular metabo-

lites such as amino acids, polyols, and sugars, but

significant concentration of lichen substances.Analysis of the extracts by thin-layer chromato-

graphy confirmed UV spectra (Hall, 1956; Rao et

al., 1967; Huneck, 1973). P. caperata extract

displayed maxima at 249�/254 and 283�/286 nm,

in accordance with the presence of protocetraric

acid (peaks at 239 and 314 nm) and usnic acid

(peaks at 232 and 282 nm). P. perlata extract

showed an inflexion at 242 nm and a smooth peakat 304�/310 nm, in accordance with the presence of

atranorin (peak at 252 nm; inflexion at 290 nm),

and b-orcinol depsidones*/constictic, menegaz-

ziaic, and stictic acids (peaks at 238 and 312

nm). P. subrudecta and P. tiliacea showed maxima

at 266�/267 and 296�/299 nm, close to the peaks of


orcinol depsides�/lecanoric acid, at 270 and 307

nm. P. sulcata extract displayed a maximum at

247�/250 nm and an inflexion at 294�/299 nm, in

accordance with the atranorin and salazinic acid

(maxima at 236.5 and 313 nm).

Titration of the extracts with copper and assay

of free copper by ion-selective electrode potentio-

metry revealed the presence of copper-binding

ligands. Although Cu2� coordinate more strongly

with nitrogen- and sulphur-containing ligands

than with oxygen-type ligands, cupric ions can

form stable chelates with ligands containing only

oxygen (Irving and Williams, 1953; Smith and

Martell, 1977). Data reported in the literature

show that Cu2� can form stable chelate com-

pounds with neighbouring hydroxyl, and/or car-

bonyl groups such as aldehyde, carboxylate, ester,

or ketone (Calvin and Wilson, 1945; Syers, 1969;

Iskandar and Syers, 1972; Casellato et al., 1977;

Smith and Martell, 1977). An analysis of the

molecular structure of the lichen substances pre-

sent in the extracts (Fig. 6) shows that these

molecules have neighbouring hydroxyl and/or

carbonyl groups, and therefore it is likely that

the copper-binding ligands detected in the extracts

were, partially or totally, the identified lichen

substances. This conclusion is further supported

by the presence of several lichen acids in the

extracts, and the existence of successive phases ofcopper binding.

4.3. Copper binding to acetone extracts and

membrane damage

Data reported in the literature indicate thatcopper-complexing compounds usually reduce

copper toxicity, since the main toxic species is

the free hydrated cupric ions (hydroxy complexes

are also toxic, but other inorganic copper com-

plexes are much less toxic), and most organic

copper complexes are non-toxic (however, some

lipophilic organocopper complexes can be very

toxic; see below) (Florence, 1983; Florence andStauber, 1986; Sadiq, 1992; De Filippis and

Pallaghy, 1994). However, in this work, no sig-

nificant correlation (r B/0.72, P �/0.10) was found

between the Cu2�-binding capacity of acetone

extracts and copper-induced membrane damage

(Table 2; Table 3), and therefore our initial

hypothesis was rejected. Whereas membrane da-

mage increased in the following order: P. subru-

decta , P. caperata , P. perlata , P. tiliacea , and P.

sulcata ; and Cu2�-complexing capacity decreased

in the following order: P. perlata , P. tiliacea , P.

caperata , P. sulcata , and P. subrudecta . It can,

therefore, be concluded that in the studied Parme-

Fig. 6. Molecular formula of selected lichen substances identified in the acetone extracts. One representative substance of each class is

shown.


lia species, and in the experimental conditions

used in this work, copper toxicity was not a simple

function of the Cu2�-binding properties of the

lichen substances present in the thallus, others

factors being operative.

The following hypotheses can be formulated to

interpret these results:

i) Lichen substances, isolated, in vitro could

bind considerable amounts of copper, but

when present in the living thallus, the binding

was not significant. In the experimental con-

ditions used-lichen samples immersed in water

for a restricted period of time, lichen sub-

stances present in the thallus could have

ineffective in binding considerable amounts

of copper. This interpretation is supported by

data reported by Brown (1976) showing that

the pretreatment of lichens with acetone prior

to metal exposure did not resulted in de-

creased metal binding (it should be stressed,

however, that in these experiments much

higher metal concentrations were used than

in the present work). According to Syers and

Iskandar (1973), lichen substances, although

having a low solubility in water, do form, in

natural conditions, soluble metal complexes,

which are directly involved in the biogeochem-

ical weathering of rocks and minerals. How-

ever, these are long-term processes and not

directly comparable with the time scale of the

experiments reported in this work, with in-

cubation periods only up to 6 h. Rapid

formation of soluble coloured complexes

with water suspensions of minerals and

rocks was described, but this occurred with

isolated lichen compounds or ground lichen

thalli.

ii) Lichen substances present in the thallus did

bind copper, but saturation was exceeded, and

most of the absorbed cupric ions remained

free, and reached target sites on the cytoplas-

mic membrane. According to this interpreta-

tion, at low copper concentrations, lichen

substances should have prevented copper

toxicity. This interpretation, however, is not

supported by the following results: (1) Probits

were linear functions of copper dose in theentire range of copper concentrations, suggest-

ing a similar mechanism throughout. (2)

Intracellular potassium after treatment with

a low copper dose (25 mM Cu2� correspond-

ing to 2 mg copper mg�1 dw) for 5 h was not

correlated with the copper-complexing capa-

city of the extracts (Fig. 2 and Table 2). The

existence of complexes between copper and

lichen substances in the thallus has indeed

been demonstrated, but only for species

adapted to high metal concentrations. Purvis

et al. (1985, 1987, 1990) studied several lichen

species occurring on copper-rich rocks, and

demonstrated the existence of copper�/norstic-

tic acid and copper�/psoromic acid complexes,

resulting in a green colouration of the thallus.

The Parmelia lichens used in this work were

not collected in copper-rich substrata. There-

fore, the concentration of lichen acids in the

thallus could have been insufficiently high to

bind most of the adsorbed copper in the

experiments.

iii) Some of copper�/lichen substance complexes

were toxic, and therefore increased, and not

decreased, copper availability. Data reported

in the literature do indicate that several

copper-complexing compounds can increasecopper toxicity by the formation of lipid

soluble complexes, which can rapidly pene-

trate the membranes. In addition, lipid-soluble

complexes carry into the membrane and

cytoplasm both copper and the ligand, which

may exert its own toxicity (Florence and

Stauber, 1986). Florence and Stauber (1986)

studied the toxicity of several copper com-plexes to the bacillariophyta Nitzschia closter-

ium and to the chlorophyta Chlorella

pyrenoidosa . It was found that, in general,

ligands which produced water-soluble com-

plexes with copper reduced its toxicity,

whereas ligands which strongly chelated cop-

per to yield lipid-soluble complexes, greatly

increased toxicity. Toxicity increased withincreasing lipophilicity of copper complexes.

The testing of these hypotheses will require the

study of the copper-binding characteristics of pure


lichen substances, and the assay of the toxicity ofcomplex copper�/lichen substances.

Acknowledgements

I am specially indebted to Dr. Olafsdottir

(University of Iceland, Iceland) for helpful discus-sions, and to Dr. Thorsten Lumbsch (University of

Essen, Germany) for giving samples of pure lichen

substances. The excellent technical assistance of

Mr. Sousa (Faculty of Sciences, Oporto Univer-

sity) on the atomic absorption spectrophotometer

was greatly appreciated.

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