289
For. Snow Landsc. Res. 80, 3: 289–304 (2006)
Influence of heavy metals and acid rain on enzymatic activities
in the mycorrhizosphere of model forest ecosystems
Karin Pritsch1*, Madeleine S. Günthardt-Goerg2, Jean Charles Munch1,
Schloter3
3
and Michael
1
Chair of Soil Ecology (TUM), GSF-National Research Center for Environment and Health,
München-Neuherberg, Germany. [email protected]
2 Swiss Federal Institute of Forest, Snow and Landscape Research (WSL), Birmensdorf, Switzerland.
[email protected]
3 Institute of Soil Ecology, GSF-National Research Center for Environment and Health, MünchenNeuherberg, Germany. [email protected], [email protected]
* Corresponding author
Abstract
The influence of a moderate contamination with heavy metals (Cu, Zn, Cd, Pb /640, 3000, 10, 90
mg kg–1 of HNO3-extractable metals), acid rain (pH 3.5), and the combined treatment was studied
in model forest ecosystems. Extracellular enzymatic activities were measured in soil samples and
on absorbing fine roots of seven different tree species (Betula pendula, Fagus sylvatica, Populus
tremula, Picea abies, Salix viminalis, Alnus incana, Acer pseudoplatanus). The five studied enzymes
represent the carbon, phosphorus and nitrogen cycle, and include phosphatase, β-glucosidase,
cellobiohydrolase, chitinase, and xylosidase.
Acid rain alone had no effect on soil enzyme activities, while heavy metals strongly reduced
activities of all enzymes. Surprisingly, this reduction was less pronounced in the combined treatments with HM and acid rain. The absorbing fine roots of the seven different tree species exhibited
a broad range of reactions to both stressors. Overall, the results suggest that studies on heavy
metal reactions in woody plants should consider species effects as well as other environmental
factors such as acid rain.
Keywords: mycorrhiza, stress response, forest trees, extracellular enzymes
1
Introduction
Anthropogenic heavy metal (HM) pollution of soils originates from mines, landfills,
atmospheric deposition (smelters, waste and fossil fuel combustion), military activities,
applications of sewage sludge, urban composts and HM containing pesticides (ALLOWAY
1995b). In Western Europe, an estimated 1.4 million sites are HM contaminated, with areas
and loads still increasing (MCGRATH et al. 2001). Since HM can not be degraded, HM
pollution of soils is a very long-term matter (ALLOWAY 1995b). Currently, two strategies are
followed to deal with HM contaminated soil: 1) physico-chemical and biological remedial
technologies, with the disadvantage of being expensive, labour intensive, and resulting in
extensive changes to the physical, chemical, and biological characteristics of the treated soil;
2) phytoremediation using plants which either accumulate HM in their tissues and thus
remove HM from soils or plants which tolerate HM and stabilise the contaminants in the
soil (KHAN 2005). In many cases of moderately contaminated sites, the stabilisation with a
cover of tolerant plants is the most feasible approach.
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Karin Pritsch et al.
In contrast to most agricultural soils, a major concern in HM polluted forest ecosystems
and afforestation sites is the low pH of forest soils, facilitating the mobilisation of most HM
(STREIT and STUMM 1993). This may not only lead to an increased risk of metal leaching
into ground and surface waters but also to higher ecotoxicity and impairment of ecosystem
functions.
HM at higher concentrations are toxic to living organisms primarily because of their
protein binding capacity and hence to their ability to inhibit enzymes (SPEIR and ROSS
2002). In soils, cycling of the major nutrients almost entirely depends upon microbial
processes that are mediated by enzymes. It has been demonstrated that microbial biomass
and enzyme activities decrease with increasing heavy metal pollution (KANDELER et al.
1996; KANDELER et al. 2000; KUPERMAN and CARREIRO 1997) and, therefore heavy metal
contaminated soils loose very common biochemical properties which are necessary for the
functioning of the ecosystem (KANDELER et al. 1996).
The extent to which enzyme activities are affected depends on HM availability as
influenced by soil acidity and base saturation, amounts and properties of soil organic matter
and clay minerals, as well as interactions with other inorganic constituents, including other
metal ions (TYLER 1981). In addition, plant roots influence physicochemical soil conditions
that determine mobility and bioavailability of HM by changing pH, taking up ions and
releasing low molecular weight organic compounds (ALLOWAY 1995a). Different plant
species, cultivars and local populations of wild types react very differently towards high HM
concentrations in soils (BROADLEY et al. 2001). Roots of HM hyperaccumulator plants show
stimulated growth towards higher HM concentration (MCGRATH et al. 2001) while plants
that are not especially adapted to tolerate unusual HM concentrations in soil show reduced
root growth (COSIO et al. 2006), root respiration (MASAROVIKOVA et al. 2004) and nutrient
uptake (ARDUINI et al. 1998). Altered plant root reactions together with direct HM effects
may directly influence composition and functions of microbial populations in the
rhizosphere. This may also apply in forest ecosystems, where almost all roots form
mycorrhizae, although not much is known about functional changes in the mycorrhizosphere of trees challenged by HM.
Within the frame of the project “From Cell To Tree” (http://www.wsl.ch/forschung/
forschungsprojekte/von_zelle_zu_baum), a model forest ecosystem was established in field
plots, that simulated afforestation of a moderately HM polluted soil. Seven tree species were
grown together in field plots with HM contaminated topsoil under ambient or acid
irrigation, and the influence of these factors as well as plant competition were studied. The
present study focussed on below ground processes at the plant soil interface by measuring
potential enzymatic activities on the surface of absorbing fine roots and in mycorrhizosphere soil samples. Our hypotheses were that 1) in addition to the known reduction of
enzyme activities in soil under HM stress, HM reduce activity of hydrolytic enzymes at the
root surface, 2) roots of different plant species react specifically to HM and acid rain (AR),
and 3) higher HM availability due to AR increases the HM effect.
2
Materials and methods
2.1
Experimental set-up
The experimental field is located at the Swiss Federal Research Institute WSL, Birmensdorf,
Switzerland and consists of 20 circular plots with 2 m in diameter and a depth of 1.5 m. In
autumn 1999, each plot was filled with 0.45 m acidic subsoil, pH [0.01 M CaCl2] = 4.2, from a
For. Snow Landsc. Res. 80, 3 (2006)
291
riverside at Eiken, Switzerland (without confinement, above the natural subsoil), and
covered by a layer of 15 cm of a silty loam pH [0.01 M CaCl2] = 6.55, from an inherently
agricultural site near Birr, Switzerland. Soil chemical and physical parameters of the acidic
subsoil and the topsoil are given in detail by MENON et al. (2005). In 10 plots, filter dust from
a non-ferrous smelter was manually mixed with the topsoil to give an average amount of
HNO3-extractable metals in the contaminated top soil of Cu / Zn / Cd / Pb = 640 / 3000 / 10 /
90 mg kg–1, the original soil contained Cu / Zn / Cd / Pb = 28 / 97 / 0.1 / 37 mg kg–1. In a parallel lysimeter study with the same top and sub soil, soil solution concentrations of Cu, Zn, and
Cd were determined as Cu 0.2-0.6 µmol L–1, Zn < 0.05 µmol L–1, and Cd < 0.2 nmol L–1 in the
control and Cu 1.0–1.6, Zn 14–25, and Cd 0.02–0.04 µmol L–1 in the contaminated soil
(NOWACK et al. 2006). Five of the HM-treated and five of the not HM enriched plots were
additionally irrigated with acidic rain (AR), pH = 3.5, while the other plots were irrigated
with water of the same chemical composition as ambient rain at pH = 5.5, each pH
established with HCl. Irrigation was applied during dry periods whenever it was necessary to
prevent water stress of the plants. The resulting four treatments were control (CO), heavy
metal (HM), acid rain (AR), and the combination of both (HMAR), each with 5 replicate
plots. Each plot was planted with the same seven tree species and a number of understorey
plants, arranged in a randomized pattern. In total, each plot contained 14 trees, namely, Picea
abies (L.) Karst. (3 specimen), Fagus sylvatica L., Betula pendula Roth, Populus tremula L.,
Alnus incana Medik., Salix viminalis L. (2 specimen each), and Acer pseudoplatanus L.
(1 specimen) and understorey plants (Tanacetum vulgare L., Carex sylvatica Huds., Allium
ursinum L.). In the present study, we focussed on the tree species and one specimen of each
of seven tree species at each plot was studied.
At the time of harvest (spring 2005), soil pH [0.01M CaCl2] was: CO 5.3+/–0.2, AR
5.2+/–0.1, HM 5.9+/–0.1, and HMAR 5.9+/–0.1.
2.2
Harvest
Prior to root harvest, the above ground parts had been removed in autumn 2004 (deciduous
trees, by coppicing as in the previous years) and during winter (spruce). At each of two
sampling dates (April 25 and May 09, 2005), 10 plots (harvest1: 3 HMAR, 2 HM, 3 AR,
2 CO; harvest 2: 2 HMAR, 3 HM, 2 AR, 3 CO) were sampled and roots of one specimen of
each tree species at each plot were collected. Because of the patchy and irregular distribution of roots, a targeted sampling was performed, i.e. tracing of roots from the stem base,
to increase the probability to sample vital roots of the desired species. Cut off root systems
were collected together with surrounding soil, placed in plastic bags, stored at 6 °C, and
processed within the two weeks following each harvest.
2.3
Soil enzyme activities
Because of intermingled root growth, the soil collected together with each root sample could
not be attributed to roots of one species and, therefore, only mixed samples of each plot
were used to determine enzyme activities. 1 g of each of the seven individual soil samples at
each plot were combined after sieving (< 2 mm) thoroughly mixed and analysed individually.
400 mg of this mixed sample were suspended in 40 mL of sterile distilled water by shaking
and ultrasonification as described by PRITSCH et al. (2005).
The following five enzyme substrates were used in enzyme assays based on the release of
4-methylumbelliferone (MU): MU-phosphate (MU-P) / acid phosphatase (EC 3.1.3.2),
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Karin Pritsch et al.
MU-β-1,4-glucopyranoside (MU-G) / β-glucosidase (EC 3.2.1.21); MU-cellobiohydrofurane
(MU-C) / 1,4-β-cellobiohydrolase (EC 3.2.1.91), MU-β-1,4-N-acetylglucosaminide (MUNAG) / 1,4-β-poly-N-acetylglucosaminidase (chitinase) (EC 3.2.1.14), and MU-β-D-xyloside
(MU-X) / Xylan 1,4-β-xylosidase (EC 3.2.1.37). All chemicals were derived from SigmaAldrich Chemicals (Germany) and dilutions of substrates were prepared from 5 mM stock
solutions in 2-methoxy ethanol as previously described (PRITSCH et al. 2004). The substrate
concentrations in the incubation mix and the required incubation times were: MU-P 800 µM
20 min, MU-β-G 500 µM 60 min, MU-NAG 500 µM 40 min, MU-C 400 µM 60 min, and
MU-X 400 µM 60 min. The incubation mix contained 50 µL each of soil suspension,
substrate, and sterile distilled water. Controls for autofluorescence of the substrate
contained 100 µL of sterile distilled water and 50 µL of substrate, calibration wells contained
50 µL of each soil suspension, sterile distilled water and calibration solutions (0, 100, 200,
300, 400, 500 pmol MU in 50 µL). The reaction was stopped with Tris 2.5 M pH 10-11 (3/1
v/v). Prior to fluorescence measurements, microplates were centrifuged for 5 min at 750 x g.
Fluorescence was measured at an excitation wavelength of 360 nm and an emission wavelength of 450 nm, both at slit widths of 5 nm with a Cary Eclipse Fluorescence
Spectrophotometer with a microplate reader (Varian, Australia).
From the calibration curves, the concentration of released MU was calculated and
enzyme activities were expressed as MU release in nmol per g soil dry weight and hour
[nmol g–1 h–1].
2.4
Enzyme activities of individual root tips
Prior to enzyme analysis, roots were studied under a stereomicroscope to verify the identity
of the tree species and to confirm the vitality of roots. According to their typical morphology,
all tree roots could be distinguished, at least in direct comparison with each other. Only tips
with an intact meristem, a turgescent whitish cortex and an intact hyphal mantle (latter only
in the case of ectomycorrhizae, EM) were considered for subsequent enzyme assays.
Different types of ectomycorrhizae were present at the root systems of the known ectomycorrhizal (EM) species B. pendula, F. sylvatica, P. tremula, and P. abies. S. viminalis and
A. incana which regularly have both, ecto- and arbuscular mycorrhizae, formed EM on less
than 5 % of their absorbing roots, and A. pseudoplatanus a mainly arbuscular mycorrhizal
(AM) and only rarely EM-forming species did not form ectomycorrhizae. The root tips were
not further differentiated in different types i.e. ectomycorrhizal morphotypes, arbuscular
mycorrhizal and non-mycorrhizal roots since detailed studies on the mycorrhizal status of
the investigated species were beyond the scope of this study.
The selected root tips were cut to a length of 4 to 5 mm and processed as previously
described in detail (COURTY et al. 2005; PRITSCH et al. 2004). In brief, individual root tips
were placed in small sieves which allowed the subsequent incubation in different enzyme
substrates. For each species of each plot, 14 individual root tips were assayed resulting in 70
roots per species and treatment, and 1960 roots in total.
For enzyme assays on individual fine roots, the same five enzyme substrates and concentrations were used as for soil samples with incubation times appropriate for mycorrhizal
roots: MU-P 10 min, MU-NAG and MU-G 20 min, MU-Cel 40 min, MU-X 60 min. Assays
were run under buffered conditions using 100 µL buffer (75 mM maleic acid to which 75 mM
Tris was added to give a pH of 4.5) and 50 µL of substrate. After the enzyme assays, all root
tips were scanned and their projection area was determined (WinRhizo 2003b, Regent
Instruments. Inc., Canada). Enzyme activities of root tips are given as MU release in pmol
mm–2 min–1.
For. Snow Landsc. Res. 80, 3 (2006)
2.5
293
Statistical analysis
Taking into account possible biases due to patchy root distribution, two calculations on root
enzyme data were made. Firstly, the mean was calculated from 14 roots of one species at one
plot, and these mean values were further analysed in the statistical analysis resulting in n = 5
per treatment and species. Additionally, in a second calculation, data of all individual roots
of one species and treatment were combined (n = 70) and statistically analyzed, thus
overcoming the possible influence of a patchy distribution of vital roots, morphotypes or
heterogeneous soil conditions.
All statistical analyses were performed on log-transformed data. To explain the effects of
treatments and interactions of the two stresses imposed, a two-way ANOVA was performed
for each species separately including heavy metal and acid rain as factors.
Means of each enzyme were compared between different species by a one way ANOVA
followed by Duncan’s multiple range test for pairwise comparison of means.
Statistical significance was set at p<0.05 if not mentioned otherwise. SPSS 12.0 for
Windows (SPSS, USA) was used for all statistical analyses.
3
Results
3.1
Soil enzyme activities
Activities of all five enzymes were strongly reduced in soil samples of both heavy metal
treatments (HM, HMAR), but not in the AR treatment (Fig. 1). The remaining enzyme
activity in the HM treatment relative to the control was (in average) phosphatase (35 %),
chitinase (37 %), xylosidase (39 %), cellobiohydrolase (53 %), and β-glucosidase (57 %). A
slight but not significantly reduced activity was observed in the AR treatment for
phosphatase (77 %) and chitinase (79 %) while activities of xylosidase, cellobiohydrolase, βglucosidase remained nearly unchanged (97–98 %). The remaining enzyme activity in the
combined treatment (HMAR) was higher compared to the HM treatment with activity rates
compared to the control of phosphatase (49 %), chitinase (45 %), xylosidase (50 %),
cellobiohydrolase (73 %), and β-glucosidase (67 %), but the combined treatment caused
only a significant effect for phosphatase (Table 1), indicating that acid irrigation in this case
significantly reduced the HM effect.
Table 1. p and F values of ANOVA on soil enzyme activities based on n = 5 replicate plots. Significant
results p<0.05 bold, abbreviations see Figure 1.
phosphatase
F
p
HM
38.4842 0.0000
AR
0.2282 0.6393
HM *AR 4.9287 0.0412
chitinase
F
p
12.8257 0.0025
0.3026 0.5898
1.2841 0.2738
β-glucosidase cellobiohydrolase
xylosidase
F
p
F
p
F
p
79.1836 0.0000 23.7824 0.0002 209.9090 0.0000
0.7451 0.4008
1.0517 0.3204
1.1568 0.2981
2.3127 0.1478
3.0060 0.1022
2.7897 0.1143
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Karin Pritsch et al.
chitinase
phosphatase
1200
MU release [nmol g-1 h-1]
MU release [nmol g-1 h-1]
1400
1000
800
600
400
800
*23
600
400
37
200
200
CO
70
90
cellobiohydrolase
61
*72
60
50
68
40
30
20
HM
AR
treatment
HMAR
xylosidase
80
70
60
97
50
40
30
20
CO
600
MU release [nmol g-1 h-1]
CO
HMAR
MU release [nmol g-1 h-1]
MU release [nmol g-1 h-1]
80
HM
AR
treatment
HM
AR
treatment
HMAR
CO
HM
AR
treatment
HMAR
β-glucosidase
*43
500
400
41
300
200
CO
HM
AR
treatment
HMAR
Fig. 1. Activity of soil enzymes as influenced by heavy metal (HM), acid rain (AR), and combined treatment (HMAR) compared to the control (CO).
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For. Snow Landsc. Res. 80, 3 (2006)
3.2
Enzyme activities of absorbing fine roots
Enzyme activities of fine roots strongly depended upon the tree species. In the statistical
model including the factors HM, AR and species, the tree species was the significant variable
for most enzymes even when the mean values of all plots were considered (Table 2). Similar
to soil enzymes, those of absorbing fine roots in average showed a strong reaction towards
the HM treatment. The reduction of activity was significant for phosphatase and xylosidase
when mean values of each plot were considered (n = 5) and significant for all enzymes when
analyses were based on values of individual roots (n = 70) (Table 2). The influence of AR
was significant only for chitinase (n = 70). In the combined treatment, an interaction of both
factors (HM and AR) independent from the tree species was found for chitinase (p =
0.0508) and cellobiohydrolase (P = 0.0008) (n = 70).
A comparison of enzyme patterns of the 7 tree species revealed species differences at the
level of activity, the reaction towards the stresses and the specific response according to each
enzyme (Fig. 2, Table 4 ).
In the control treatment, the phosphatase activity was highest for B. pendula and F. sylvatica, intermediate for A. incana, P. abies and P. tremula and lowest for A. pseudoplatanus
and S. viminalis (Fig. 2). Chitinase activities in the controls were especially high in B. pendula
and F. sylvatica, around average in P. abies and P. tremula, and below average in A. pseudoplatanus, A. incana, and S. viminalis. Species showed similar activity patterns for the two
cellulose degrading enzymes (cellobiohydrolase, β-glucosidase) with B. pendula, F. sylvatica,
P. abies above average, P. tremula, A. pseudoplatanus around average and A. incana,
S. viminalis below average activities. Xylosidase activities were more similar among the tree
species, but again A. incana and S. viminalis showed the lowest activity compared to the
other species.
The acid rain treatment had no significant effect on phosphatase activity of S. viminalis,
A. pseudoplatanus, and P. tremula. Due to strongly decreased phosphatase activity, effects
were observed for A. incana (p = 0.0000) and B. pendula (p = 0.0753), while an increased
activity was recorded for P. abies (p = 0.0717) and F. sylvatica (0.0316). Chitinase activity was
almost double compared to the control in B. pendula but the increase was not significant
(Table 3). The slight increase of chitinase activity for F. sylvatica, and P. abies and the
decrease for A. pseudoplatanus, S. viminalis, and A. incana did not cause a significant AR
effect. For xylosidase and β-glucosidase, no AR-effect was found, although B. pendula
showed highly increased β-glucosidase activity compared to the control. Cellobiohydrolase
activity showed a significant AR effect for B. pendula and F. sylvatica, both with higher
activities than the control and no significant effects for the other species.
Table 2. p values from ANOVA on all data of root enzyme activities of each plot (variables: HM, AR,
species). Bold significant (p≤0.05) for n = 70 single root measurements per treatment and species,
underlined significant (p≤0.05) for n = 5 mean values per treatment and species, abbreviations see
Figure 1.
HM
AR
species
HM*AR
phosphatase
0.0000
0.1675
0.0000
0.1931
chitinase
0.0068
0.0010
0.0000
0.0508
β-glucosidase
0.0000
0.7193
0.0000
0.2993
cellobiohydrolase
0.0000
0.2069
0.0000
0.0008
xylosidase
0.0000
0.6070
0.0000
0.3617
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Karin Pritsch et al.
phosphatase
chitinase
Bpen
250
Bpen
250
200
Ainc
200
Fsyl
150
Ainc
100
100
50
50
0
0
Svim
Pabi
Apse
cellobiohydrolase
Pabi
Svim
Apse
Ptre
xylosidase
Bpen
250
200
Fsyl
150
Ainc
100
50
50
0
0
Pabi
Apse
Bpen
250
Fsyl
150
Pabi
Ptre
CO
AR
HM
HMAR
200
Ainc
Svim
Apse
Ptre
Fsyl
150
100
Svim
β-glucosidase
Ptre
Bpen
250
200
Ainc
Fsyl
150
100
50
0
Svim
Pabi
Apse
Ptre
Fig. 2. Relative enzyme activities [%] of
absorbing fine roots of seven tree species. The
values of each species and each treatment are
expressed relative to the average control value
which was calculated as the mean of n = 7
species for each enzyme separately. Values
>100 indicate activities above, and <100 activities below community average. Abbreviations:
Bpen-Betula pendula, Fsyl-Fagus sylvatica,
Pabi-Picea abies, Ptre-Populus tremula, ApseAcer pseudoplatanus, Svim-Salix viminalis,
Ainc-Alnus incana, treatments see Fig. 1.
For. Snow Landsc. Res. 80, 3 (2006)
297
The HM treatment reduced phosphatase activity of all species. However, significant effects
were only observed for F. sylvatica, P. abies, and P. tremula (Table 3). Chitinase activity was
significantly decreased in B. pendula and A. incana, slightly decreased or unchanged in
P. abies, S. viminalis, A. pseudoplatanus, and F. sylvatica, while some effect (p = 0.0521) was
observed in P. tremula caused by an increased activity. HM-effects on cellobiohydrolase and
β-glucosidase were either a reduction for S. viminalis and A. incana (both not significant),
A. pseudoplatanus (significant for β-glucosidase), P. abies and B. pendula (significant for
cellobiohydrolase and β-glucosidase) or an increase for F. sylvatica and P. tremula (not
significant). A significant HM-effect due to reduced xylosidase activity was observed for all
species except P. tremula which showed no effect.
A significant interaction of HM × AR was observed in few cases. For some plant/enzyme
combinations, the combined treatment resulted in lower or similar values than the HM
treatment while the AR treatment alone stimulated enzyme acitivities: B. pendula /
phosphatase, chitinase, cellobiohydrolase, β-glucosidase and F. sylvatica / xylosidase. In other
significant interactions, the HMAR treatment stimulated enzyme activities whereas both
single treatments (HM and AR) reduced enzyme activities: P. abies / cellobiohydrolase; S.
viminalis / chitinase, cellobiohydrolase, β-glucosidase; A. incana / β-glucosidase, xylosidase;
A. pseudoplatanus /, β-glucosidase, cellobiohydrolase. In one case, (A. incana, phosphatase)
the decrease of enzyme activity in the combined (HMAR) and the AR treatment was
stronger than in the HM treatment.
A comparison of means between the species of one community under each treatment by
one-way ANOVA followed by Duncan’s multiple range test revealed that treatments caused
shifts in enzyme patterns of the species. In the CO treatment, distinct patterns were
observed among all seven species (Table 4 a). AR caused an even greater differentiation
except for S. viminalis and A. incana which became more similar to each other than in
the CO treatment (Table 4 a). The HM treatment grouped B. pendula, F. sylvatica, and
P. tremula together, while S. viminalis and A. incana formed a separate group, P. abies and
A. pseudoplatanus were in between these two groups (Table 4 b). The HMAR treatment
reduced differences of the enzyme activity patterns which was particularly distinct for the
xylosidase activity (Table 4 b).
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Karin Pritsch et al.
Table 3. p-values from ANOVA, reflecting the influence of HM and AR on individual species, bold significant (p≤0.05) for n = 70 single root measurements per treatment and species, underlined significant
(p≤0.05) for n = 5 mean values per treatment and species.
B. pendula
F. sylvatica
P. abies
P. tremula
A. pseudoplatanus
S. viminalis
A. incana
HM
AR
HM * AR
HM
AR
HM * AR
HM
AR
HM * AR
HM
AR
HM * AR
HM
AR
HM * AR
HM
AR
HM * AR
HM
AR
HM * AR
phosphatase
chitinase
0.6638
0.0753
0.0750
0.0007
0.0316
0.5331
0.0000
0.0717
0.5580
0.0030
0.0202
0.9278
0.2425
0.0891
0.7082
0.0877
0.1590
0.6801
0.0768
0.0000
0.0440
0.0000
0.2103
0.0028
0.0521
0.1853
0.3443
0.1137
0.8524
0.8446
0.0376
0.2430
0.4591
0.1155
0.0438
0.8299
0.9238
0.0007
0.0000
0.0207
0.0000
0.3884
βglucosidase
0.0000
0.0658
0.0059
0.6825
0.1782
0.1301
0.0005
0.7723
0.3663
0.1577
0.5099
0.3638
0.0184
0.6738
0.0063
0.6570
0.2757
0.0035
0.1631
0.3623
0.0814
cellobiohydrolase
0.0000
0.0135
0.0441
0.1648
0.0278
0.6003
0.0014
0.5970
0.0932
0.3517
0.6668
0.6747
0.6876
0.7062
0.0005
0.6401
0.1976
0.0000
0.2560
0.7964
0.1269
xylosidase
0.0000
0.2307
0.8002
0.0000
0.5776
0.0011
0.0000
0.6949
0.6020
0.1620
0.5793
0.3200
0.0001
0.8940
0.5760
0.0013
0.2799
0.7354
0.0304
0.3917
0.0009
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Table 4. Comparison of enzyme activity patterns between species a) under CO (above grey fields) and
AR treatments (below grey fields), b) under HM (above grey fields) and HMAR treatments (below
grey fields); summarised from comparison of one-way ANOVA followed by Duncan’s multiple range
test, n = 70, p≤0.05. Abbreviations: pho, phosphatase; chi, chitinase; gls, β-glucosidase; cel, cellobiohydrolase; xyl, xylosidase; species abbreviations see Figure 2.
a)
CO
AR
Bpen
Bpen
Fsyl
.
.
.
.
xyl
pho
.
.
.
.
.
.
.
.
xyl
.
.
.
.
.
.
.
.
.
.
pho
.
.
.
.
Ptre
Pabi
Apse
Svim
Ainc
Fsyl
pho
chi
.
.
xyl
.
.
.
.
.
.
.
.
.
xyl
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Ptre
.
.
.
.
.
.
.
gls
cel
xyl
.
chi
gls
cel
.
.
.
gls
.
xyl
.
.
.
.
.
pho
.
.
.
.
Pabi
pho
.
gls
.
xyl
.
.
gls
.
xyl
.
chi
gls
.
.
.
.
gls
.
.
.
.
.
.
.
.
.
.
.
.
Apse
.
.
gls
.
xyl
.
.
gls
cel
xyl
.
.
gls
cel
xyl
.
.
gls
.
.
.
chi
.
cel
xyl
.
chi
.
cel
.
Svim
.
.
.
.
.
.
.
.
.
.
.
chi
.
.
.
.
chi
.
.
.
pho
.
.
cel
.
.
chi
gls
cel
xyl
Ainc
pho
.
.
.
.
pho
.
.
.
.
.
.
.
.
.
pho
.
.
.
.
.
chi
.
.
.
.
.
gls
cel
.
300
Karin Pritsch et al.
b) HM
HMAR
Bpen
Bpen
Fsyl
pho
.
gls
cel
xyl
.
chi
gls
cel
xyl
.
chi
gls
cel
xyl
.
.
gls
cel
xyl
.
.
.
cel
.
.
.
.
.
.
Ptre
Pabi
Apse
Svim
Ainc
Fsyl
pho
chi
gls
cel
xyl
.
.
gls
cel
xyl
.
.
.
cel
xyl
.
.
gls
.
xyl
.
.
.
.
xyl
.
.
.
.
xyl
Ptre
.
chi
gls
cel
.
.
chi
gls
cel
xyl
pho
.
gls
cel
xyl
pho
.
gls
.
xyl
.
.
.
.
.
pho
.
.
.
xyl
Pabi
.
.
.
cel
xyl
.
.
.
.
.
pho
.
.
.
.
.
.
gls
cel
xyl
.
.
.
cel
xyl
pho
.
.
.
xyl
Apse
.
.
.
.
xyl
.
.
.
.
.
pho
.
.
.
.
pho
.
gls
.
xyl
pho
.
.
cel
xyl
.
chi
.
.
xyl
Svim
.
.
.
.
.
.
.
.
.
.
pho
.
.
.
.
pho
.
.
.
.
pho
chi
.
cel
.
.
.
gls
cel
xyl
Ainc
pho
.
.
.
.
pho
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
chi
.
cel
.
.
chi
gls
cel
xyl
For. Snow Landsc. Res. 80, 3 (2006)
4
301
Discussion
Enzyme activities measured on absorbing roots of common forest trees were shown to be
sensitive indicators for reactions of individual plant species as well as for their reactions to
different stresses. We could show, that each of seven tree species showed specific levels of
activity, which changed under the stress conditions. Thus we demonstrated for the first time
that under HM exposure, enzymatic reactions involved in nutrient turnover in the mycorrhizosphere can be specifically affected in different plant species. In soil samples, we could
confirm the detrimental effect of HM on biochemical functions in soils, however, an
unexpected result was that the HMAR treatment caused a less severe reduction of enzyme
activity than the HM treatment.
These results support our initial hypotheses 2, while hypothesis 1 was confirmed for soil
but not for roots, and hypothesis 3 was rejected.
Hypotheses 1 that HM reduce activity of hydrolytic enzymes was confirmed for soil but
not for enzymes at the root surface. The result that heavy metals reduce soil enzyme
activities is in accordance with a number of studies with HM contaminated soil. KANDELER
et al. (1996) observed an especially strong decrease in phosphatase and arylsulfatase activity,
while the reduction of enzyme activities involved in C-cycling was less pronounced, which
was similar in the present study except for xylosidase which was as strongly affected as
phosphatase and chitinase activity. KANDELER et al. (1996), however, studied unplanted soil,
so carbon cycling may have been different in our system.
The same hypothesis was rejected for root samples. Several plant/enzyme combinations
indicated stimulation rather than reduction of enzyme activities in the HM treatments.
While most enzymes revealed no general patterns and were stimulated and/or reduced in
different species, phosphatase showed a reduction but never an increase in activity under
HM influence. A possible consequence of heavy metal pollution may be an imbalance of
nutrient uptake on the long run. This may be of less importance for species such as P. abies
with comparatively low nutrient demands and good growth on acidic soils but may negatively
influence fast growing pioneer species such as A. incana and S. viminalis. A stimulation of
enzyme activities on the other hand can indicate stress reactions as has been discussed for
reactions in the mycorrhizosphere of spruce and beech challenged by elevated ozone and
pathogen stress (PRITSCH et al. 2005). In this case higher phosphatase activities compared to
the control were attributed to higher energy demands of stressed plants, and higher chitinase
activities were interpreted as unspecific stress reactions (PRITSCH et al. 2005), indicating root
damage and/or an efficient defence by the roots under HM stress. Higher activities of
cellulose and hemicellulose degrading enzymes could point towards higher amounts of dead
plant material in soil. It is evident that more data are needed from complex systems and
multiple stress conditions, to better interpret combined treatment effects.
Hypothesis 2 was supported since roots of different plant species reacted specifically to
HM and AR. Plant roots influence their soil environment by several processes summarised
as the ‘rhizosphere’ effect which results in higher microbial activity than in the bulk soil
further distant to the root. In the present study, this has been taken into account by
measuring not only soil enzymes but also enzymes at the surface of absorbing roots. Enzyme
activities at the root surface which is colonised by mainly bacteria and fungi sum up the
activities of all of these components. In a previous study, mycorrhizosphere soil samples of
spruce and beech exhibited species specific enzyme activity patterns under ozone and
pathogen stress which could be related to biomass parameters (PRITSCH et al. 2005). In the
present study which demonstrated that enzyme activities of different tree species showed a
wide range of species reactions, these enzyme activities have to be related to HM exposure,
uptake and resulting performance of individual species as well as to plant physiological
302
Karin Pritsch et al.
parameters. Data on these parameters are not yet available from the same experiment but
from a parallel lysimeter study under similar conditions which revealed species specific
patterns for heavy metal allocation, growth and physiological behaviour of P. abies, S. viminalis, B. pendula and P. tremula (HERMLE et al. 2006). In this study, Picea abies showed the
lowest HM uptake and no growth reduction, followed by S. viminalis and B. pendula, whereas
toxic Zn concentrations and growth reductions were detected particularly in the foliage of
P. tremula. In the present study, S. viminalis showed significantly lower activities for most
enzymes than HM sensitive B. pendula and P. tremula. P. abies was in between these
extremes. From this it would be interesting to see if F. sylvatica with high enzyme activities in
HM stress conditions belongs to the sensitive, while A. incana with low activities under HM
stress belongs to the tolerant species, and A. pseudoplatanus may be a moderately tolerant
species. If this is the case, enzyme activities may serve as very sensitive indicators for HM
sensitivity in different plant species and would allow a rapid assessment of the differences in
the belowground stress level among species of one site.
Hypothesis 3 that higher HM availability due to acid rain increases the HM effect was
rejected for soil samples. In the HMAR treatment, we did not observe a further depression
of enzyme activities as was hypothesised due to an expected higher mobilisation of HM.
Conversely, a slightly but not significantly higher activity of all studied enzymes was
observed compared to the HM treatment. Similarly unexpected amelioration effects were
observed in a parallel experiment of the project “From Cell to Tree” run in open-top
chambers with the same treatments, tree species and soil conditions. In this study, acid
irrigation tended to increase evapotranspiration of plants grown in HM contaminated soils
(MENON et al. 2005). The authors discussed that the acid rain treatment exerted some
additional effect other than increasing metal solubility compensating for such mobilisation
(MENON et al. 2005). However, the mechanism of this effect is unclear and the result clearly
demonstrates that multiple stress scenarios are needed in further studies on effects of heavy
metal polluted soils on plants.
The present study points towards several aspects which should be addressed in future
investigations. The high variation of enzyme activities in individual roots of one species can
be attributed to 1) local heterogeneity in HM distribution due to the mode of HM
application which could have caused heterogeneous exposure (RAIS 2005); 2) colonisation
by different fungal symbionts which can have distinct enzyme patterns and variations in
activities as has been shown for several ectomycorrhizal types (COURTY et al. 2005; PRITSCH
et al. 2004). The first aspect should have been overcome, theoretically, by the large sample
number, but cannot be ruled out completely. The second aspect points towards the fungal
symbionts which deserve further attention. HM tolerance of plants may be significantly
influenced by the fungal symbiont as has been shown for a Zn tolerant and sensitive isolate
of Suillus bovinus and Scots pine (ADRIAENSEN et al. 2004). Tolerance acquired by metallotolerant fungal mycobionts may play an important role when replanting of forest trees is
considered on HM contaminated sites (HARTLEY et al. 1997). Therefore, in future studies, it
would be worth to study enzyme activities of mycobionts more specifically with the aim to
integrate HM tolerant mycorrhizal inoculum for revegetation of HM polluted sites. In
addition, seasonal shifts should be considered to verify how species specific reactions appear
at different times of the vegetation period. Furthermore, it is evident that more data are
needed from complex systems and multiple stress conditions, to enable interpretation of
combined treatment effects.
For. Snow Landsc. Res. 80, 3 (2006)
303
Acknowledgements
We thank Michael Lautenschläger (WSL) for his help during the harvest, Clara Tschiersch and
Gudrun Hufnagel (GSF, TUM) for excellent lab assistance.
5
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