COST Action 837. WG2 toxic metals Workshop 2002 – INRA Bordeaux-Aquitaine, France, 25. – 27. 4. 2002.
Cellular injury, heavy metal uptake and growth of poplar, willow and spruce
influenced by heavy metals and soil acidity.
M.S. Günthardt-Goerg and P. Vollenweider
Swiss Federal Research Institute WSL, Zürcherstrasse 111, CH-8903 Birmensdorf, Switzerland
Email goerg@wsl.ch, vollenwe@wsl.ch
Introduction
Areas moderately contaminated by heavy metals (HM), but at a level exceeding the critical
levels for food production are widespread in Europe and could be utilised for fibre production. They
may also be afforested for landscape improvement and an environmentally sound and cost effective
soil reclamation using indigenous tree species. Before considering genetically modified organisms,
more should be known about the potential of our native trees. Small quantities of mobile heavy
metals (Cu, Zn) are essential for plants, because they are cofactors of important enzymes. In higher
concentrations however, mobile as well as less mobile non-essential heavy metals (Cd, Pb) can
induce acute tissue and cell injury. At the cellular level, HM enhance the formation of free radicals
and reactive oxygen species resulting in oxidative stress (Dietz et al. 1999). Phenols, both in the cell
wall and in the cell vacuole, seem to play an important but still unclear role in detoxification
reacting as antioxidants and in several cases also as phytochelators forming complexes with heavy
metal ions (Lichtenberger and Neumann 1997; Moran et al. 1997). The framework 'From Cell to
Tree' was designed to study heavy metal uptake and allocation relative to cellular defence reactions
and tree growth in near-natural model ecosystems with different combinations of soil and rain
acidity.
Materials and methods
In 16 large (6 m2) open-top chambers (OTCs), model ecosystems have been established in
April 2000 with different tree and understory species. Preliminary results, obtained after the first 6
months of treatment are given here for poplar (Populus tremula L.), willow (Salix viminalis L.),
both grown from cuttings, and spruce (Picea abies (Karst.) L.) grown from seed. Trees grow under
competition together with understory plants on natural forest soils split in an acidic pH 4.2 (acid
soil) and neutral pH 7.4 (neutral soil) subsoil compartment, depth 15 - 150 cm. The OTCs (with
roofs which close automatically to exclude rain), are arranged in a Latin square. Four treatments are
applied: control with rain-like buffered irrigation pH 5.5 (CO), heavy metals (HM), acidic irrigation
pH 3.5 established with HCl (AR), and their combination (HMAR), with four replicates each. The
treatment with HM dust was applied manually in the topsoil layer (15 cm, pH 6.4) resulting in Zn
2700mg/kg, Cu 385mg/kg, Pb 63mg/kg and Cd 10mg/kg which represent a moderately
contaminated soil, as exists for example around metal processing industries. The experiment is
repeated in the field (field plots = FPs) with acidic subsoil and irrigation only during dry periods.
Above ground tree foliage and wood biomass is determined yearly at the end of the vegetation
period by coppicing the deciduous trees and estimating the tree biomass for spruce applying a model
which calculates the relationship between harvested branches from the second whirl and the whole
tree current shoot mass; this model was determined on another set of trees of the same age. Heavy
metal content is determined in HNO3 extracts by ICP AES in aliquots taken from the entire mixed
foliage and wood per tree. The significance of the treatments was calculated using ANOVA.
1
Results and discussion
Visible leaf symptoms were induced in poplar leaves by the HM treatment. They appeared as
light-green stipples on the adaxial side of leaf and were first recognised in July 2000, 70 days after
the start of treatment. Later, during the vegetation season, leaf edge necroses developed allowing us
to apply a simple symptom rating scale (Fig. 1). Symptom expression increased with leaf age. The
same symptoms were observed in the OTCs and FPs, however, symptom intensity was lower in the
FPs, probably following the reduced biomass production in these plots (see legend to Fig. 8). In the
OTCs, leaf injury was more severe on the acidic soil and in the mountainous poplar provenance
Orvin-BE (960 m a.s.l.) than in the provenance Birmensdorf-ZH (550 m a.s.l.) (Fig. 2). Leaf
morphology was still juvenile because of the plant propagation by cuttings.
Most consistent and specific microscopical symptom of HM stress was the extended
necrosis of lower epidermis, which could be the determining factor for the observed leaf edge
necroses. Cell wall reactions observed along the pathway of HM from the veins through the leaf
blade included cell wall thickenings, inlays of polyphenolics and wart-like protrusions, all indicative
of extra cellular oxidative stress. Inside the cells, nuclei were more condensed, chloroplast size was
apparently reduced and starch accumulated. Some cells were necrotic and collapsed. These changes
suggested an acceleration of cell senescence. The visible stipples resulted from discrete groups of
necrotic cells in the assimilative tissue surrounded by degenerating cells filled with antioxidative
compounds including proanthocyanidines (Fig. 3). They indicated the induction of the plant
defences resulting in a frequent plant reaction called the 'hypersensitive-like response' [HR-like]
(Sandermann 1996; Vollenweider et al. 2002). However, compared to the effects of ozone
(Günthardt-Goerg et al. 2000, Vollenweider et al. 2002), the levels of defence reaction to HM stress
were generally low in poplar leaves suggesting that poplar, on a tree level, followed the strategy of
pioneer species which replace rather than repair injured leaves. Cell injuries suggested that the HM
impaired gas exchange in the leaf and photosynthesis.
Zn appeared to be responsible for visible symptoms since the analyses showed an important
Zn accumulation in poplar leaves. Moreover, in parallel to the observed leaf injury symptoms, Zn
was significantly more accumulated in the Orvin-BE provenance and on acidic soil than in the
Birmensdorf-ZH provenance and on neutral soil (Fig. 4). Zn accumulation was also significant in
willow foliage (particularly on acidic soil), and was increased by the acid rain in the combined
treatment (HMAR) in the FP. In spruce needles Zn allocation to the foliage was significant
independent of the soil acidity (Fig. 5). When data was pooled over the soil compartments and
provenances Zn reached, an average concentration of 1050 mg/kg foliage dry mass in poplar vs. 280
mg/kg in the CO. In the HM treatment the poplar foliage Zn concentration therefore was not
hyperaccumulated but still reached 39 % of the topsoil concentration (2700 mg Zn/kg soil dry
mass). In willow Zn uptake reached 760 mg/kg in the HM treatment vs. 430 in the CO, and in
spruce 110 vs. 50 mg/kg. Cd uptake in the poplar foliage (Fig. 6) behaved similarily to that of Zn. In
contrast, there was no difference in Cd uptake between the provenances. Cd in poplar foliage
reached an average concentration of 9.5 mg/kg in the HM vs. 1.5 mg/kg in the CO treatment.
Again poplar showed not to be a Cd hyperaccumulator, but reached 95 % of the topsoil
concentration (10 mg/kg). In the willow, after HM treatment Cd reached 5.6 vs. 2.4 mg/kg in the
CO. Cd remained below the detection limit of 0.6 mg/kg in spruce. With on average 11.7 mg/kg
(only 1.5 % of the upper soil Cu concentration) vs. 9.1 mg/kg in the control, Cu content in the
poplar foliage was significantly increased (Fig. 7), independent of soil or provenance. In willow Cu
was significantly increased by HM on both soil types (on average 9.4 vs. 8.2 mg/kg in the CO). In
spruce (on average 4.5 vs. 3.5 mg/kg), the difference was only significant on acidic soil. Pb
concentration was often (in spruce always) below the detection limit of 3 mg/kg.
2
Regarding HM uptake from the soil (phytoremediation), biomass production plays an
equivalent role to the accumulation capacity of the different species. Consequently, and in respect to
the phytoremediation efficiency, the species rating was: poplar > willow >> spruce. Total HM
concentration in the foliage was increased by the HM treatment, depending on the soil type, but
similarly in the FPs and OTCs. However, when also considering the biomass effect, more HM were
taken up in the OTCs than in the FPs (Fig. 8), and AR became a significantly modifying factor for
HM uptake in poplar for the whole tree, foliage and wood mass, and in spruce for the wood
biomass. Zn uptake summed up for the current year foliage and wood mass of 4 poplar, 2 willow
and 6 spruce plants per soil compartment (Fig. 9) show that a considerable amount of almost 500
mg has been extracted from the acidic soil by these very young native plants within a 6 months
period and without significant reduction of tree growth.
The nutrient elements of the foliage were only slightly modified by the treatments, except
phosphorous, which was significantly decreased through HM by more than 10 % (not shown).
0
no abiotic symptoms
2
1 + beginning leaf
edge necrosis
Fig. 1: Visible symptom rating scale for poplar leaves.
3
2
1
0
CO
AR
3
1 + whole leaf edge
necrotic
4
OTC, acidic subsoil
Symptom class
Symptom class
4
1
adaxial stippling
HM
HMAR
4
3 + developing
interveinal necroses
OTC, calcareous subsoil
3
2
1
0
CO
AR
HM
HMAR
Fig. 2: Symptom intensity (class 0 - 4 Fig. 1) on 19.11.2000 (mean values + SE). Poplar provenance Orvin, 960 m a.s.l.
(grey) and Birmensdorf 550 m a.s.l. (hatched). Significant factors: HM (0.0001); provenance (0.0001); interaction
HM*provenance (0.0001).
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Fig. 3. Microscopical symptoms of Zn intoxication. A, C from HM treated samples vs. B, D from CO. A. Transversal
view through a stipple. The central palisade cells are dead (*) as shown by their disrupted cell content (no organelles,
e.g. nucleus or chloroplast, visible). Surrounding palisade cells have a degenerated cell structure with more condensed
nuclei (n) and apparently smaller and more condensed chloroplasts (ch) with larger starch grains (white dots). Phenolics
(p), including proanthocyanidins, are accumulated. This stipple structure is typical of an HR-like reaction. C. Tissue and
cell changes on the apoplastic transport pathway. After transport as solutes in water, Zn came first into contact with
lower epidermis (le) and spongy parenchyma. Spongy cells reacted by thickening their cell walls (arrowheads) and
developing pectinaceous droplets impregnated with polyphenols (arrows), which indicate apoplastic oxidative bursts.
Cell content degenerated, as shown by apparently smaller chloroplast (ch) with larger starch grains and condensed nuclei
(n). In the end cells can die and collapse (*) especially in the lower epidermis (le), a site of HM accumulation. Bar = 6
µm.
4
Zn (mg/kg)
1500 Field plots, acidic subsoil
1500
1000
1000
1000
500
500
500
0
CO
AR
HM
0
HMAR
1500
OTC, acidic subsoil
CO
AR
HM
OTC, calcareous subsoil
0
HMAR
CO
AR
HM
HMAR
Zn (mg/kg dry mass)
Fig. 4: Foliar Zn concentration in poplar (mean values  SE). Poplar provenance Orvin, 960 m a.s.l. (grey) and
Birmensdorf 550 m a.s.l. (hatched). Significant factors in OTCs: HM (0.0001); soil acidity (0.0213); interaction M*soil
(0.0257); provenance (0.0133); interaction HM*prov. (0.0133); in FPs: HM (0.0001).
1500
1500
Field Plots, acidic subsoil
Poplar
Willow
Spruce
1000
500
0
1000
1000
500
500
0
CO
AR
HM
1500
OTC, acidic subsoil
HMAR
CO
AR
HM
0
HMAR
OTC, calcareous subsoil
CO
AR
HM
HMAR
Cd (mg/kg dry mass)
Fig. 5: Foliar Zn concentration in poplar, willow and spruce (mean values  SE).
Significant factors in OTCs: poplar HM (0.0001), soil acidity (0.0213), interaction M*soil (0.0257); willow HM (0.0001),
soil acidity (0.0093), interaction M*soil (0.001); spruce HM (0.0001).
Significant factors in FPs: poplar HM (0.0001); willow HM (0.0001), interaction HM*AR (0.0131); spruce HM (0.0001).
15
15
Field Plots, acidic subsoil
10
Poplar
Willow
5
0
10
10
5
5
0
CO
AR
HM
HMAR
15
OTC, acidic subsoil
CO
AR
HM
HMAR
0
OTC, calcareous subsoil
CO
AR
HM
AHMAR
Fig. 6: Foliar Cd concentration in poplar, willow and spruce (mean values + SE).
Significant factors in OTCs: poplar HM (0.0001), soil acidity (0.0001), interaction M*soil (0.0028); willow HM
(0.0001), soil acidity (0.0001), interaction M*soil (0.0001); spruce below the detection limit of 1 mg/kg.
Significant factors in FPs: poplar HM (0.0001); willow HM (0.0001), interaction HM*AR (0.0435); spruce below
detection limit.
5
Cu (mg/kg)
20 Field Plots, acidic subsoil
Poplar
15
Willow
Spruce
10
5
0
CO
AR
HM
HMAR
20 OTC, acidic subsoil
20
15
15
10
10
5
5
0
CO
AR
HM
0
HMAR
OTC, calcareous subsoil
CO
AR
HM
AHMAR
Fig. 7: Foliar Cu concentration in poplar, willow and spruce (mean values  SE).
Significant factors in OTCs: poplar HM (0.0082); willow HM (0.0066), AR (0.001), soil acidity (0.0001); spruce HM
0.0405), interaction HM*soil (0.0119).
Significant factors in FPs: poplar HM (0.0025); willow HM (0.0085); spruce HM (0.0079).
Zn per tree (mg)
100
80
Poplar
Willow
Spruce
60
40
Foliage
Wood
20
0
80
80
60
60
40
40
20
20
0
CO
AR
HM
100 OTC, neutral subsoil
100 OTC, acidic subsoil
Field plots, acidic subsoil
HMAR
0
CO
AR
HM
HMAR
CO
AR
HM
HMAR
Fig. 8: Zn uptake in current year foliage and wood mass per tree in poplar, willow and spruce (stalked mean values +
SE). Poplar mean current year foliage mass per tree in OTCs 50, in FPs 16 g. Poplar wood mass in OTC 99 in FPs 23 g.
Willow mean current year foliage mass per tree in OTCs 27, in FPs 9 g. Willow wood mass in OTC 59 in FPs 28 g.
Spruce mean current year needle mass per tree in OTCs 70, in FPs 33 g. Spruce wood mass in OTC 20 in FPs 9 g.
Significant factors in OTCs: poplar foliage mass HM (0.0002), soil acidity (0.0221), interaction HM*soil (0.0004),
interaction AR*soil (0.003); Poplar wood mass mass HM (0.002), interaction HM*soil (0.0002), interaction AR*soil
(0.0296); willow foliage mass HM (0.0033), soil acidity (0.0151); willow wood mass HM (0.0406); spruce needle mass
HM (0.0001); spruce wood mass HM (0.0001), AR (0.0004).
Significant factors in FPs: poplar foliage mass HM (0.0001); poplar wood mass HM (0.0097); willow foliage mass HM
(0.0085); willow wood mass HM (0.0358); spruce needle mass HM (0.0007); spruce wood mass (0.0002).
Zn (mg)
500
500
Field plots, acidic subsoil
500
OTC, acidic subsoil
400
400
400
300
300
300
200
200
200
100
100
100
0
CO
AR
HM
HMAR
0
CO
AR
HM
HMAR
0
OTC neutral subsoil
CO
AR
HM
HMAR
Fig. 9: Summed up mean Zn uptake in current year foliage + wood mass of 4 poplar, 2 willow and 6 spruce trees per
soil compartment.
6
Conclusions:
HM dust, dispersed in the top-soil, was taken up and allocated to the foliage of young
indigenous trees growing in near-natural model ecosystems. Phytoremediation, in respect to the tree
species, was most efficient in poplar followed by willow and then spruce. Although the growth was
reduced in the field plots relative to the OTCs and although the three tree species differed much in
their biomass production, the HM were similarly allocated to the foliage and wood whatever the
species and soil type. In contrast to the acidic soil which favoured HM uptake, acidic irrigation in
the OTCs did not contribute directly to the uptake, but modified the HM content in the above
ground tree mass by changing the biomass production and interacting with soil acidity.
Microscopical results confirmed the specificity of HM symptoms in poplar and gave important
insights in leaf and tree response to HM stress. They confirmed that oxidative stress and plant
defense are induced by zinc in the leaf. However, in contrast to another abiotic stress (ozone), Zninduced reactions are gradually located along the water pathway through the leaf. As the provenance
factor proved to be less important than the HM effect, the two poplar provenances can now be used
as bioindicators (using the visible symptom rating scale) to compare treatment effects and to follow
up the evolution of the model ecosystems during the next years. Other projects of the framework
indicate that particularly spruce stabilised the HM in its rhizosphere. Consequently, when by
coppicing HM are removed in a large biomass with HM concentrations giving no cause of concern,
deciduous trees remediate the HM pollution in the model ecosystems, whereas spruce might
stabilise it. However, further analyses should allow us to better characterise plant strategies
(allocation of HM in the above ground biomass versus HM stabilisation in the rhizosphere) of
different tree species.
References
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plants. In: Heavy metal stress in plants. Prasad M. N. V., Hagemeyer J. (eds), Springer, Berlin: 73-97.
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deciduous tree species related to current critical ozone levels. Environmental Pollution 109, 489-500.
Lichtenberger O., Neumann D.:1997, Analytical electron microscopy as a powerful tool in plant cell biology: Examples
using electron energy loss spectroscopy and X-ray microanalysis. European Journal Of Cell Biology. Aug, 73(4):
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Moran J. F., Klucas R. V., Grayer R. J., Abian J., Becana M.:1997, Complexes of iron with phenolic compounds from
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Acknowledgements: Collaboration of Peter Bleuler, Ueli Bühlmann, Ivano Brunner, Toni Burkart, Sandra Hermle,
Michael Lautenschläger, Jörg Luster, Terry Menard, Véronique Michellod, Daniele Pezzotta at WSL, and Bernd
Nowack, Susanne Scheid and Rainer Schulin at the Swiss Federal Institute of Technology Zürich, Institute of Terrestrial
Ecology, and financial contribution to COST-Action 837 by the Swiss Federal Office for Education and Science is
gratefully acknowledged.
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