Annals of Botany 82 : 735–746, 1998
Article No. bo980734
Ultrastructural Responses of the Lichen Bryoria fuscescens to Simulated Acid
Rain and Heavy Metal Deposition
S. T A R H A N E N
Department of Ecology and EnŠironmental Science, UniŠersity of Kuopio, P.O. Box 1627, FIN-70211 Kuopio,
Finland
Received : 14 April 1998
Returned for revision : 8 June 1998
Accepted : 17 July 1998
Effects of simulated acid rain and heavy metal deposition on the ultrastructure of the lichen Bryoria fuscescens
(Gyeln.) Brodo and Hawksw. were examined in a field study conducted in northern Finland. Lichens were exposed
to simulated rain containing two levels of a mixture of copper (Cu#+) and nickel (Ni#+) ions alone or in combination
with acid rain (H SO ) at pH 3 over 2 months in addition to ambient rainfall. The algal and fungal components
# %
responded differently to pH and there was an interaction with metal toxicity. The algal partner was the most sensitive
to acid rain and heavy metal combinations and had more degenerate cells than the fungal partner. Damage was
apparent in chloroplasts and mitochondria, where thylakoid and mitochondrial cristae were swollen. The fungal
partner was the more sensitive to high concentrations of metal ions in the absence of acidity, suggesting an
ameliorating interaction between the metals and acidity. For algae, critical metal concentrations in the thallus were
50 µg g−" for Cu and 7 µg g−" for Ni in the presence of acidity, and 20 µg g−" for Ni in the absence of acidity.
Detrimental effects of heavy metals on fungal ultrastructure were seen when thallus metal concentrations exceeded
400 µg g−" for Cu and 100 µg g−" d. wt for Ni. The results suggest that acid wet deposition containing metal ions may
reduce survival of lichens in northern environments.
# 1998 Annals of Botany Company
Key words : Copper, nickel, sulphuric acid, ultrastructure, Bryoria fuscescens (Gyeln.) Brodo and Hawksw., epiphytic
lichen, air pollution.
INTRODUCTION
Although studies concerned with the accumulation of heavy
metals in lichens have an extensive history (Garty, 1993),
few have concentrated on their physiological effects
(Puckett, 1976 ; Nieboer et al., 1979 ; Richardson et al.,
1979 ; Brown and Beckett, 1983, 1984) and virtually no
information is available on ultrastructural effects. Biological
availability and toxicity of many metals in the environment
are influenced by stability of the minerals, pH of the
substratum, and oxidation potential (Eh) (Purvis and Halls,
1996). Near smelters, the solubility and potential toxicity of
metal particles are often increased by SO emissions and
#
acidification. Numerous studies have shown that rain less
than pH 4 is toxic to the photobiont of lichens, reducing
photosynthetic rate and pigment concentration, and leading
to plasmolysis and death of the algal cells, especially if the
concentration of sulphate in the rain is high (Lechowicz,
1982 ; Roy-Arcand, Delisle and Brie' re, 1989). The toxic
effect of Cu-Ni smelting activities can be seen as impoverishment of lichen species at polluted sites (Gorshkov,
1993). Nevertheless, limited information is available from
the field concerning threshold concentrations of metals in
lichen thalli (Tyler, 1989).
Copper is an essential constituent of several enzymes
which catalyse redox reactions in a variety of metabolic
pathways. Nickel is required for the urease synthase pathway
of many plants and fungi (Hausinger, 1994), including
lichens (Rai, 1988 ; Vicente and Legaz, 1988), where the
0305-7364\98\120735j12 $30.00\0
enzyme plays an important role in nitrogen metabolism.
Like many trace elements, both Cu and Ni are toxic in
excess (Friedland, 1990). They inhibit growth and photosynthesis, and increase the permeability of the plasma
membrane, causing loss of K+ ions and a reduction in the
uptake of essential elements (Gadd, 1993 ; Lidon and
Henriques, 1993 ; L’Huillier et al., 1996). Ultrastructural
injuries arising from metal stress have been observed in
chloroplasts, mitochondria and nuclei of higher plants
(Angelov et al., 1993 ; L’Huillier et al., 1996). Moreover,
metal exposure can induce formation of osmiophilic vesicles,
the number of which increase in both microalgae (BallanDufrançais, Marcaillou and Amiard-Triquet, 1991 ; Nassiri
et al., 1997) and mycorrhizal fungi (Turnau, Kottke and
Dexheimer, 1994) in response to metal contamination.
In northern Finland and northwest Russia, the ion
leakage values of epiphytic lichens have been found to
increase near the Nikel and Monchegorsk smelters (Tarhanen et al., 1996) where Cu, Ni and SO concentrations are
#
elevated (Kryuchkov, 1993 ; Tuovinen et al., 1993). There
are no reports on interactive effects of acid rain and metals
on the ultrastructure of lichens. Because the lichen thallus
involves at least two genetically distinct symbionts, the
mycobiont (fungus) and the photobiont (alga), the response
of lichens to environmental stresses may be more complex
than the response of the individual partners. The aims of
this study were to assess the effects of acidity and Cu-Ni
ions, alone and in combination, on the ultrastructure of
algal and fungal cells, and to determine threshold concentrations for effects of pollutants on the symbionts.
# 1998 Annals of Botany Company
736
Tarhanen—Effect of Pollution on Bryoria Ultrastructure
MATERIALS AND METHODS
Experimental design
The study was conducted at the Kevo Subarctic Research
Station of the University of Turku in northern Finland
(69m 45h N, 27m 01h E) during the summers of 1992 and 1993.
The site was divided into two sub-areas on the basis of
vegetation. Sub-area 1 consisted of mixed mountain birch
(Betula pubescens subsp. tortuosa (Ledeb.) Nyman) and
Scots pine (Pinus sylŠestris L.) woodland and included 20
study plots (6i8 m#) each containing one pine and one
birch tree. Study plots were grouped in sets of four to form
five blocks with randomized design. The plots within each
block were randomly assigned to one of four treatments :
irrigated control (IC) at pH 5, acid rain at pH 3 (pH 3),
medium heavy metal treatment (CuNi1) and combined
medium heavy metal treatment and acid rain (CuNi1j
pH 3). In sub-area 2, the dominant tree species was mountain
birch (Betula pubescens subsp. tortuosa). This area had 30
study plots (3i5 m#) grouped into five blocks consisting of
six treatments, four as above and, in addition, a high heavy
metal treatment (CuNi2) and combined high heavy metal
treatment and acid rain (CuNi2jpH 3).
Exposure of lichens
Scots pine branches (Pinus sylŠestris L.) supporting
Bryoria fuscescens were collected from Kaamanen (69m 5h N,
27m 15h E) approx. 100 km south of the Kevo Station on 23
Jun. 1992 and 18 Jun. 1993). Lichens were transplanted into
each block and subjected to six different treatments : (1)
irrigated control (IC) ; (2) acid rain treatment at pH 3
(pH 3) ; (3) medium level of metal treatment with copper
and nickel treatment (CuNi1) ; (4) a combination of medium
level of metal and acid treatments (CuNi1jpH 3) in subarea 1 ; and (5) high level of metal treatments (CuNi2) ; and
(6) a combination of high level of metal and acid treatments
(CuNi2jpH 3) in sub-area 2. Treatment solutions, including control (IC), were adjusted with H SO to pH 5 to
# %
simulate the pH of natural rain water. Acid treatments were
adjusted to pH 3. In northernmost Europe, the acidity of
precipitation is mainly related to sulphate deposition.
Nitrogen deposition has little acidifying significance because
approx. 60 % of the total nitrogen (NO −jNH +) appears
%
$
as nitrate (Tuovinen et al., 1993). Copper and Ni were
added as CuSO ;5H O and NiSO ;6H O salts.
%
#
%
#
In addition to ambient rainfall, the study plots received
5 mm simulated rain twice a week between 11 Jun. and 28
Aug. 1992, and between 14 Jun. and 27 Aug. 1993. During
the 1992 irrigations 0n83 and 8n33 mg m−# Cu and 0n50 and
5n00 mg m−# Ni were applied in the medium (CuNi1) and
high metal treatments (CuNi2), respectively. In 1993,
12n5 mg m−# Cu and 7n5 mg m−# Ni comprised the high
(CuNi2) metal treatment, while the concentrations of metals
in the medium treatment, CuNi1, were the same as in 1992.
Ion concentrations in the high metal treatments were
enhanced to simulate better the pollution load near the
Monchegorsk and Nikel smelters (Kryuchkov, 1993), which
is approx. 600 times that at Kevo. Metal load in the medium
treatment was nearly 50 times the background level in the
study area (Laurila, Tuovinen and La$ ttila$ , 1991). Ambient
precipitation was approx. 300 and 170 mm during the
summer months (June–August) in 1992 and 1993 (FMI,
1992, 1993), respectively. Total loads of Cu, Ni and S
applied are given in Table 1. At the end of each experiment,
the lichens were air dried (Tarhanen et al., 1996) and sent to
the University of Kuopio, where they were immediately
prepared for electron microscopy. A field sample of B.
fuscescens was collected the beginning of the experiment in
1992, to compare the effects of control treatments.
Microscopy
For each treatment, five or six randomly chosen thalli
from each study plot were fixed in 2n5 % glutaraldehyde
(Electron Microscope Sciences, FT, Washington, PA, USA)
in 0n05  phosphate buffer (pH 7n0) and post fixed in 1 %
buffered OsO . Samples were dehydrated and embedded in
%
Ladd’s LX-112 resin as described in Tarhanen, Holopainen
and Oksanen (1997). Semi-thin sections of the thalli were
cut with an Ultracut E (Reichert-Jung AG, Wien, Austria)
onto microscope slides, stained with toluidine blue and
examined with a Zeiss light microscope (Germany). Ultrathin sections were cut from the same specimens, stained with
uranyl acetate and lead citrate and examined with a JEM
1200 EX electron microscope (Jeol, Tokyo, Japan) operating
at 80 kV.
The percentage of dead algal cells was calculated from the
total number of cells within the thallus on the light
microscopic sections. Highly plasmolysed, totally empty
and collapsed algal cells were designated as dead. Relative
volume densities occupied by different cell components
within a median section of an algal cell were examined by
using the point intercept method with the help of a grid
placed over the electron micrograph. The components of
the cells were divided into compartments of total cell,
protoplast and chloroplast volume. Volume densities for the
cell wall and periplasmic space were calculated with reference
to total cell volume. The relative volume of chloroplasts,
lipid bodies, mitochondria, nuclei, vacuoles and electronopaque deposits in vacuoles were estimated from protoplast
volume. In addition, percentages of thylakoids, stroma,
pyrenoid and starch grains were calculated with reference to
chloroplast volume (Tarhanen et al., 1997).
For qualitative analysis, the ultrastructural characteristics
of chloroplasts and mitochondria of algal cells were classified
by dividing them into three classes (0–2). Class 0 represented
healthy mature cells and the other classes expressed a degree
of deviation from healthy cells. The definition of a healthy
cell was based on the supposition that the chloroplast is
lobate, thylakoid membranes are intact and undulated, and
mitochondria are intact with cristae (Galun, Paran and BenShaul, 1970 ; Peveling, 1974). Ultrastructural characters of
fungal cell components, i.e. cell wall, cytoplasm, storage
bodies and vacuoles, were also studied and their overall
condition estimated.
Tarhanen—Effect of Pollution on Bryoria Ultrastructure
Data analyses
All statistical analyses were conducted with SPSS\PC
(version 5.0 or 7.5). To analyse the effect of block, year,
T     1. Irrigation period and composition of simulated rain
in the experimental plots in 1992 and 1993
mg m−#
Number of
applications
Treatment
1992
IC
pH 3
CuNi1
CuNi1jpH
CuNi2
CuNi2jpH
1993
IC
pH 3
CuNi1
CuNi1jpH
CuNi2
CuNi2jpH
3
3
3
3
Cu
18
18
18
18
19
19
19
19
219
219
22
22
22
22
21
21
18
18
263
263
Ni
S
pH
12
12
131
131
103
2466
119
2482
258
1879
5n5
3n1
5n3
3n1
4n8
3n0
11
11
159
159
177
2529
192
2544
340
2015
6n0
3n1
5n9
3n1
5n4
3n0
737
heavy metal and acidity and their interactions, on the lichen
ultrastructure, a general factorial analysis of variance was
used. For comparison between treatment groups, Duncan’s
multiple range test in one-way ANOVA was used. For
quantitative ultrastructural data, a total of 297 and 265
algal cells were examined in 1992 and 1993, respectively. To
avoid pseudoreplication (Hurlbert, 1984) means by plots
(n l 4–5) were used in the analysis of dead algal cells and
volume densities of different cell components. Ultrastructural characteristics of algal cells were analysed by
using means of thalli based on three cells in each. In total 162
and 145 thalli were examined in 1992 and 1993, respectively.
RESULTS
Irrigated control
IC, Irrigated control ; pH 3, acid rain treatment of pH 3 ; CuNi1,
medium Cu and Ni treatment ; CuNi1jpH 3, combined medium Cu
and Ni and acid rain treatment ; CuNi2, high Cu and Ni treatment ;
CuNi2jpH 3, combined high Cu and Ni and acid rain treatment.
The ultrastructure of algal cells in the water-control (IC)
treated and field-collected lichens was very similar. The
shape of the chloroplasts in the algal cells was predominantly
lobate with inner thylakoids being undulated (Table 2 ; Fig.
1 A). Some algal cells also showed less lobate chloroplasts
with straightened and regularly arranged thylakoids (Fig.
1 B) resembling young and maturing cells of Trebouxia spp.
described in previous studies (Galun et al., 1970 ; Peveling,
1974). These two chloroplast characters (i.e. shape and
thylakoid arrangement) were used, as a guideline, to separate
young and mature individuals. Starch grains were seen only
occasionally in the chloroplasts. Ribosomes, vacuoles,
T     2. Contingency table of classified characteristics of algal cell ultrastructure of the lichen Bryoria fuscescens in different
treatments in 1992 and 1993
Percentage (%) of thalli showing changes in algal cell structure
Chloroplast
shape
Treatment
1992
IC (control)
pH 3
CuNi1
CuNi1jpH
CuNi2
CuNi2jpH
Total
1993
IC (control)
pH 3
CuNi1
CuNi1jpH
CuNi2
CuNi2jpH
Total
No.
3
3
3
3†
Lobate Rounded
Swelling of
thylakoid
membranes
Thylakoid
arrangement
P
Wavy
Straight
P
Present Absent
Swelling of
mitochondria
P
Present Absent
P
18
18
17
17
21
17
108
89
67
47
71
38
41
11
33
53
29
62
59
ns
**
ns
**
**
89
78
35
88
38
35
11
22
65
12
62
65
ns
**
ns
**
**
22
44
0
59
24
29
78
56
100
41
76
71
ns
ns
*
ns
ns
0
22
35
41
24
59
100
78
65
59
76
41
ns
*
*
ns
***
16
19
19
18
21
5
99
81
100
68
61
81
60
19
0
32
39
19
40
ns
ns
ns
ns
—
81
79
74
28
57
20
19
21
26
72
43
80
ns
ns
**
ns
—
25
26
0
0
14
20
75
74
100
100
86
80
ns
ns
ns
ns
—
13
58
26
56
71
60
87
42
74
44
29
40
**
ns
**
***
—
† Expected value 5, treatment dropped from the contingency test for independence. Chi-square test for independence, ns, not significant ;
* P 0n05 ; ** P 0n01 ; *** P 0n001. No, Number of thalli ; IC, irrigated control ; pH 3, acid rain treatment, pH 3 ; CuNi1, medium Cu and Ni
treatment ; CuNi1jpH 3, combined medium heavy metal and acid rain treatment ; CuNi2, high Cu and Ni treatment ; CuNi2jpH 3, combined
high heavy metal and acid rain treatment.
738
Tarhanen—Effect of Pollution on Bryoria Ultrastructure
F. 1. Transmission electron micrographs of Bryoria fuscescens from the control treatments with no pollutants. A, Mature algal cell showing
lobate chloroplast (c) shape with undulated thylakoids. Algal cell wall (aw) ; central pyrenoid of the chloroplast with pyrenoglobuli (p) ; nucleus,
(n). i6200. B, Young algal cell from a field-collected lichen with no treatment. The chloroplast (c) shape is less lobate and thylakoids (t) are
regularly arranged into parallel stacks. i9600. C, Detail of the cystoplasm of algal cell showing mitochondria (m) and lipid droplets (l).
Plasmalemma (arrowhead). i15 000. D, View of an autosporangium (asp) where autospores (a) begin to dissociate into single vegetative cells.
mw, Disrupted mother cell wall. i7000. E, Fungal cell in the algal layer showing well developed cell wall (fw) with melanized outer layer. The
plasmalemma is invaginated (arrow) and cytoplasm contains lipid droplets (l), mitochondria with intact cristae (m) and concentric bodies
(cb). i16 000.
Tarhanen—Effect of Pollution on Bryoria Ultrastructure
T     3. Percentage (%) (meanps.d.) of dead algal cells in
the thallus of Bryoria fuscescens after different treatments in
the study years 1992 and 1993
Treatment
IC
pH 3
CuNi1
CuNi1jpH 3
CuNi2
CuNi2jpH 3
1992
1993
21n1p9n8a*
23n8p10n7a*
47n1p30n2a*
33n5p23n2a*
45n4p28n3a†
56n3p22n1a†
25n9p12n9a†
38n9p16n9a†
30n3p22n7a†
29n5p18n0a†
32n5p17n1a†
63n0p30n8b*
Values in each vertical column followed by the same superscript do
not differ significantly at P 0n05 by Duncan’s multiple range test.
* n l 4 ; † n l 5. IC, Irrigated control ; pH 3, acid rain treatment of
pH 3 ; CuNi1, medium Cu and Ni treatment ; CuNi1jpH 3, combined
medium Cu and Ni and acid rain treatment ; CuNi2, high Cu and Ni
treatment ; CuNi2jpH 3, combined high Cu and Ni and acid rain
treatment.
mitochondria, lipid droplets (Fig. 1 C) and the nucleus (Fig.
1 A) were present in the cytoplasm. Besides young and
mature cells, thalli contained some dividing (Fig. 1 D),
senescing and degenerate algal cells. The proportion of dead
algal cells observed by light microscopy was approx. 21 to
26 % in the control lichens (Table 3).
Fungal cells in the algal layer showed well-developed cell
walls with an electron-opaque (assumed to be melanized)
upper hyphal layer. Invaginated plasmalemma enveloped
the cytoplasm which contained ribosomes, often large
vacuoles, and varying amounts of other cell organelles, i.e.
lipid droplets, mitochondria and concentric bodies (Fig.
1 E).
Lichens treated with simulated pollutants
Two months of treatment caused almost no change in the
size of cell compartments in the algal cells (data not shown).
Although some cells showed strong plasmolysis, the mean
volume of cells (2–5 %) varied little between the different
treatments. However, the year significantly (P l 0n004)
affected plasmolysis of the algal cells. In all the treatments,
the plasmolysis volume was generally 25 % higher in 1993
than 1992. There were also some other differences between
study years which are presented below.
Effects of acidity
Chloroplasts of algal cells were noticeably lobate in the
acid rain treatment (Table 2). Thylakoid swelling occurred
in some cells in 1992 (Fig. 2 A, Table 2). In many mature
and senescing algal cells in 1993, numbers of dilated mitochondrial cristae were significantly increased by acidity
(Table 2, Fig. 2 B). In the main, the fungal ultrastructure
in the acid rain treatments resembled that of the control
lichens, although the vacuole matrices of some fungal cells
were dark (Fig. 2 C) and contained electron-opaque vacuolar
deposits (Fig. 2 D).
739
Effects of heaŠy metals
The proportion of young algal cells was significantly
greater in the heavy metal treatments (CuNi1 and CuNi2) ;
this was particularly noticeable in 1992 (Table 2). Some
young algal cells showed strong plasmolysis and shrunken
protoplasts (Fig. 3 A) at medium metal concentrations
(CuNi1), otherwise there were few ultrastructural changes
in the algal and fungal cells in the combined Cu and Ni
treatments in 1992. In contrast, in 1993, severely swollen
mitochondrial crista were found in young algal cells in both
medium (CuNi1) (Fig. 3 B) and high concentrations of heavy
metals (CuNi2) (Table 2). Swelling of the mitochondrial
cristae was related to increased Cu and Ni concentrations of
treatments (Table 2). Electron-opaque vacuolar deposits
(Fig. 3 C) and small polyphosphate granules (Fig. 3 D)
occurred in both the algal and fungal cells (Fig. 3 E). At high
metal concentrations (CuNi2), fungal cell contents were
often totally disrupted (Fig. 3 F), while in algae, some cell
structure was preserved (Fig. 3 C).
Effects of heaŠy metal and acid
The proportion of dead algal cells was significantly
increased (P l 0n017) to over 50 % following treatment with
metals in combination with acidity (CuNi2jpH 3) in both
years (Table 3). Differences between treated and control
(IC) lichens were only significant (P 0n05) in 1993
(Table 3).
Most algal cells showed strongly swollen and degenerate
chloroplast thylakoids in the mixed pollutant treatment at
medium metal concentrations (CuNi1jpH 3) (Table 2, Fig.
4 A) in 1992. A significant interaction (P l 0n026) between
year, acid and metal treatment was identified for stroma
volume, which was significantly increased in 1992. The
percentage of stroma in chloroplasts was 5n6p1n6 % in the
treated thalli and 3n3p1n9 % in controls. Swollen thylakoids
were also present in the high metal treatment (CuNi2j
pH 3), although in smaller numbers than in the medium
metal concentration. In 1993, early stage degradation and
senescence of the algal cells was substantial in the
CuNi1jpH 3 treatment. This was seen as swollen mitochondrial cristae (Fig. 4 B), vacuoles containing electronopaque deposits (Fig. 4 C), darkened chloroplast stroma
and mitochondrial matrix, and degenerate pyrenoglobuli
(Fig. 4 D).
Most fungal cells had unaltered ultrastructure in the
mixed pollutant treatments irrespective of metal concentration (CuNi1jpH 3 and CuNi2jpH 3), although electron-opaque vacuolar deposits were found in the fungal
cells of both the cortex and algal layer (Fig. 4 E).
The ultrastructure of soredia
Sorediate lichen thalli were present in all treatments. The
soredia were formed predominantly by new dissociated
autospores (Fig. 5 A) and young algal cells (Fig. 5 B)
enclosed by hyphae. No ultrastructural differences were
740
Tarhanen—Effect of Pollution on Bryoria Ultrastructure
F. 2. Effects of sulphuric acid treatment at pH 3 (pH 3) on B. fuscescens. A, pH 3, 1992. View of a chloroplast (c) with swollen thylakoid
membranes (arrow). i16 000. B, pH 3, 1993. A senescing algal cell showing swelling of thylakoids (arrow) in the chloroplast (c) and mitochondrial
cristae (m). Nucleus (n). i16 000. C, pH 3, 1992. Fungal cell displaying dark appearance of the vacuole matrix (v). Lipid droplet (l), mitochondria
(m). i17 000. D, pH 3, 1993. Fungal cell showing electron-opaque deposits (d) in the vacuole (v). i24 000.
observed in the diaspores between control and acid rain
(pH 3) or heavy metal (CuNi1 and CuNi2) treatments
applied singly. In contrast, in the mixed acid rain and heavy
metal treatment (CuNijpH 3), many algal cells within
soredia showed either early or advanced stages of degeneration (Fig. 5 C), or a totally disintegrated ultrastructure
(Fig. 5 D). The ultrastructure of fungal cells in the soredia
did not show any detectable response to the different
treatments.
DISCUSSION
Results from this study show that acidity and heavy metal
treatment, applied both alone and together, induce ultrastructural changes in algal and fungal cells of the lichen B.
Tarhanen—Effect of Pollution on Bryoria Ultrastructure
741
F. 3. Effects of combined copper and nickel treatments (CuNi1 and CuNi2) on B. fuscescens. A, CuNi1, 1992. Young algal cell showing shrunken
protoplast caused by strong plasmolysis. Algal cell wall (aw), plasmalemma (arrowheads) i9500. B, CuNi1, 1993. Detail of swollen mitochondria
(m) in the algal cell. i17 000. CkF, CuNi2, 1993. C, Degenerating algal cell showing the darkened chloroplast (c) with starch grains, thylakoids
are weakly discernible. Osmiophilic deposits (d) are visible in the vacuoles. i17 000. D, Degenerated algal cell showing numerous polyphosphate
granules (arrows) restricted to the vacuoles and cytoplasm. Chloroplast (c). i16 000. E, Degenerated fungal cell showing polyphosphate granule
(arrow) in the vacuole (v). i16 000. F, Cytoplasmic content of the fungal cell is totally disrupted, only remnants of lipid droplets (l) are visible.
Fungal cell wall (fw). i14 000.
fuscescens. Their response varied depending on the pollutant, metal concentration, and exposure year (i.e. climate).
These ultrastructural injuries were mainly consistent with
those previously described in lichens growing in industrial
environments (Holopainen, 1983, 1984 a, b) and in laboratory experiments employing SO pollution (Holopainen
#
and Ka$ renlampi, 1984 ; Eversman and Sigal, 1987). The
photobiont was the most sensitive component, developing
ultrastructural injuries very quickly when thallus metal
concentrations exceeded 58–76 µg g−" for Cu and 7–
20 µg g−" for Ni in combination with acidity (see also
Tarhanen et al., 1998). Although the algal component has a
greater capacity for metal accumulation, most of the metal
ions are sequestered by the fungus, due to the greater
contribution of fungal symbionts to total thallus weight
(Goyal and Seaward, 1982). The main ultrastructural
changes in algal cells following acid rain and heavy metal
treatment were located in the chloroplasts and mitochondria, which could explain the loss of photosynthetic function
seen in a similar study (Roy-Arcand et al., 1989) and
742
Tarhanen—Effect of Pollution on Bryoria Ultrastructure
F. 4. Effects of combined heavy metal and sulphuric acid treatments (CuNi1jpH 3, CuNi2jpH 3) on B. fuscescens. A, CuNi1jpH 3, 1992.
An algal cell showing fewer thylakoid membranes with strong swelling (arrow) and increased stroma area (s) in the chloroplast. i9500. B–D,
CuNi1jpH 3, 1993. B, Senescing algal cell showing strong swelling of mitochondrial cristae (m). i8500. C, Senescing algal cell showing electronopaque deposits (d) in the vacuoles (v). Note the mitochondria (m) with darkened matrix and swollen cristae, and low electron density of
pyrenoglobuli in the pyrenoid (p). i17 000. D, Degenerating algal cell showing the darkened chloroplast stroma (c) and mitochondria (m).
Contrast of pyrenoglobuli (p) is lost, thylakoid membranes (arrowhead) are still visible. i16 000. E, CuNijpH 3, 1992. Fungal cell displaying
intact cell structure and electron-opaque deposits (d) in the vacuoles. i18 000.
changes in respiratory activity of mitochondria and ATP
synthesis (Ciamporova! and Mistrı! k, 1993).
The increased number of senescent and dead algal cells in
lichen thalli following treatment with acid rain and heavy
metal combinations suggested that in addition to causing
harmful effects on its own, acidity may exacerbate metal
toxicity in the photobiont. Acidity and Cu can both increase
free-radical formation and intracellular oxidizing conditions
leading to lipid peroxidation in plant cell membranes and
ensuing injury to thylakoids (Sandmann and Bo$ ger, 1980 ;
Tarhanen—Effect of Pollution on Bryoria Ultrastructure
743
F. 5. Effects of combined heavy metal and sulphuric acid treatments at pH 3 (CuNi1jpH 3, CuNi2jpH 3) on soredia of B. fuscescens. A,
Control lichen, 1992. View of a soredia where three young algal cell (a) are enveloped by dense hyphal cells (f). i5000. B, CuNi1jpH 3, 1993.
Young algal cell in the soralia of the thallus. Note round chloroplast (c) and small lipid droplets (arrow). Mitochondria (m), nucleus (n). i10 000.
C, CuNi1jpH 3, 1992. An autosporangium (asp) in the soralia of the thallus showing a totally degenerated cell structure of autospores (a).
i7000. D, CuNi2jpH 3, 1992. View of a soralia in the thallus showing totally degenerated algal cells and fungal cells with intact cell structure.
i7400.
Chia, Mayfield and Thompson, 1984 ; Foyer, Lelandais and
Kunert, 1994), and accelerating senescence of the cells
(Mehta et al., 1992). Swelling of cytoplasmic organelles,
especially mitochondria, is indicative of the initial stages of
cell death caused by toxic environmental conditions and
substances (Schwarzman and Cidlowski, 1993). In plant
cells, thylakoid swelling is non-specific, being observed in a
variety of stress situations including metal toxicity (Stoya-
nova and Tchakalova, 1993), acidity (Wulff, Sheppard and
Leith, 1994) and ion deficiencies (e.g. Fe and Ca) (HechtBuchholz, 1983 ; Fink, 1989). Swelling of the mitochondrial
cristae and increased density of the matrix are also associated
with more than one stress factor including oxygen and ion
deficiencies (e.g. K, B and Ca), salt stress and drought
(Ciamporova! and Mistrı! k, 1993 ; Ouzounidou et al., 1995 ;
Alscher, Donahue and Cramer, 1997). These ultrastructural
744
Tarhanen—Effect of Pollution on Bryoria Ultrastructure
changes are a response to the loss of control of selective
permeability of the plasma membranes which may occur
either directly, as a result of membrane damage, or
secondarily due to cellular energy depletion (Schwartzman
and Cidlowski, 1993). As ions move across the membrane
along concentration gradients, the accompanying fluid shifts
cause cellular swelling (Schwartzman and Cidlowski, 1993).
In this study, swelling of the mitochondrial cristae as well
as the increase in plasmolysis in algal cells may partly reflect
the loss of K+ ions observed in the acid and metal treated
lichens in 1993 (Tarhanen et al., 1998). These ultrastructural
alterations as well as changes in the thallus K+ concentration
(Tarhanen et al., 1998) were less apparent during the rainy
summer in 1992. This suggests that K+ ion concentrations
of the ambient rain water and stemflow may have been
sufficient to compensate metal stress-mediated K+ deficiency
and osmoregulatory changes in the algal cells at low
pollutant levels. During the summer months (June–August),
the sum of K+ ion concentration in the ambient rain water
was 50 % higher (9n7 mg m−# K+) in 1992 than 1993
(4n9 mg m−# K+) (Leinonen, 1993, 1994). Although Cu and
Ni concentrations in the high metal treatment were
approx. 20 % higher in 1993 than in 1992, the medium
concentrations, which also caused ultrastructural injuries,
were same in both years.
Degeneration of fungal ultrastructure at high metal
concentrations was consistent with the observed loss of
membrane integrity of the lichen (i.e. enhanced K+ leakage
and loss of ergosterol) when thallus metal concentrations
exceeded 400 µg g−" Cu and 100 µg g−" Ni (Tarhanen et al.,
1998). Such damage was not seen at medium metal
concentrations, where ultrastructural injury was restricted
to the algal partner. Metal treatments, in the presence of
acidity, caused less ultrastructural injury to the fungal than
the algal partner suggesting that acidity might alleviate the
metal toxicity in the mycobiont (Gadd, 1993). This
observation is compatible with the reduced accumulation of
Ni and Cu in the lichen thalli observed in mixed pollutant
treatments (Tarhanen et al., 1998). Competition between H+
and free metal cations for cellular binding sites can reduce
metal uptake by the fungus (Gadd, 1993).
Electron-opaque deposits, particularly in the vacuoles of
fungal cells, appeared in more or less all the pollutant
treatments. Vacuolar deposits in both algal and fungal cells
are often associated with the occurrence of N compounds
(Ascaso and Fortun, 1981 ; Holopainen and Ka$ renlampi,
1985). Although the chemical composition of these vacuolar
deposits in lichens is still unknown, they could have a
detoxification function similar to that suggested for other
microalgae and fungi (Ballan-Dufrançais et al., 1991 ; Nassiri
et al., 1997 ; Turnau, Kottke and Oberwinkler, 1993). The
vacuole plays an important role in the regulation of cytosolic
metabolism and concentrations of ions (Gadd, 1993).
Sequestering metal ions in the vacuole is a method of
maintaining low cytosolic concentrations of ions. Incapacity
of metal ion transport mechanisms into the vacuole may
lead to cell damage (Ballan-Dufrançais et al., 1991). Joho et
al. (1993) reported that the difference in the Ni-resistance
between yeast strains was due to the different Ni-sensitivity
of the vacuolar membrane H+-ATPase enzyme which allows
the cell to transport nickel into the vacuole without any
inhibition of cytosolic metabolism.
In this study, polyphosphate granules were most abundant
in the vacuoles and cytoplasm of degenerating algal and
fungal cells treated with high concentrations of heavy
metals. Acid phosphatase, involved in the synthesis of
polyphosphates, is normally absent from the cytosol of
healthy cells, but has been shown to increase in the cytoplasm
of degenerating cells in metal-stressed mycorrhizal fungal
cells (Turnau and Dexheimer, 1995). The high metal
concentration did not necessarily cause degeneration of the
total lichen tissue. This is consistent with findings for metalstressed higher plants, where the same tissue can consist of
both cells with completely damaged cytoplasm and well
preserved cell components or cells (Ouzounidou et al.,
1995). Increased phosphatase activity of degenerating cells
may provide a detoxification mechanism for the remaining
healthy lichen tissue by immobilizing the toxic metal ions as
insoluble metal phosphatases in the dead cells (Turnau and
Dexheimer, 1995).
The increased frequency of immature algal cells in lichen
thalli, particularly in heavy metal treatments during 1992,
suggest that in the absence of acidity Cu and Ni may
stimulate metabolic activity and division of vegetative cells.
It is known that climatic factors, especially rainfall, can
affect metabolic activity and growth of lichens (Armstrong,
1988) and some pollutants such as SO #− (at pH 4 and 5)
%
may have a fertilizing effect on the photobiont (Roy-Arcand
et al., 1989). Holopainen (1983, 1984 a, b) also observed an
increase in the thickness of the algal layer and in the number
of dividing algal cells of Bryoria capillaris and Hypogymnia
physodes growing near a fertilizer plant and pulp mill. The
reproductive capacity of algal cells may also be improved
due to the negative effects of Cu and Ni on the fungus,
which may interfere with the mycobiont–photobiont interaction leading to an imbalance between them and possibly
breakdown of the symbiosis (Holopainen, 1984 a).
The acidic metal treatments also injured the algal cells of
soredia indicating that contaminated vegetative propagules
may be unable to form new young thalli. Diaspores are
assumed to be more effective dispersal agents than ascospores, the sexual propagules of lichen fungi, which must
contact compatible algae in order to initiate a lichen thallus
(Ott, 1987 ; Marshall, 1996). It is unknown whether the
soredia are more important than sexual reproduction in
lichens growing in boreal-arctic environments, but the
dominance of soredia over ascospores in Antarctic fellfield
lichens emphasizes the importance of vegetative mechanisms
in barren environments (Marshall, 1996).
It may be concluded that the algal and fungal components
of the lichen B. fuscescens exhibit different pH-optima with
respect to metal stress. Ultrastructural results show that
metal toxicity combined with acidity can appear in algal
cells long before injuries in fungal cells become apparent.
For algal cells, the critical metal concentration in the thallus
of B. fuscescens was above 50 µg g−" for Cu and 7 µg g−" for
Ni in the presence of acidity, and 20 µg g−" for Ni in the
absence of acidity. Degeneration of fungal cells increases
above 400 µg g−" for Cu and 100 µg g−" d. wt for Ni supplied
together. These values are relatively consistent with a study
Tarhanen—Effect of Pollution on Bryoria Ultrastructure
of epiphytic lichens growing near a brass foundry (Folkeson
and Andersson-Bringmark, 1988). They found the first signs
of reduction in epiphytic lichen cover on pine trunks and
twigs when concentrations of Cu in H. physodes exceeded
130 µg g−". Ultrastructural observations of injured algal
cells in lichen thalli and soredia suggest that the lichen’s life
span can be reduced in industrial environments where they
are simultaneously exposed to acidity and metal stress.
A C K N O W L E D G E M E N TS
I thank Drs Toini Holopainen, Jari Oksanen and Lucy
Sheppard for their invaluable comments on the manuscript.
I also thank the staff of the Kevo Subarctic Research
Station of the University of Turku for co-operation, and Ms
Minna Kittila$ and Mr Jarkko Utriainen for technical
assistance. This work was supported by the Ministry of
Agriculture and Forestry, the Academy of Finland, and the
Foundation of Jenny and Antti Wihuri.
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