Download Plant responses to abiotic stresses: heavy metal

Journal of Experimental Botany, Vol. 53, No. 372,
Antioxidants and Reactive Oxygen Species in Plants Special Issue,
pp. 1351–1365, May 2002
Plant responses to abiotic stresses: heavy metal-induced
oxidative stress and protection by mycorrhization
Andres Schützendübel and Andrea Polle1
Forstbotanisches Institut, Abteilung I, Forstbotanik und Baumphysiologie, Georg August Universität Göttingen,
Büsgenweg 2, 37077 Göttingen, Germany
Received 3 August 2001; Accepted 2 December 2001
Abstract
The aim of this review is to assess the mode of action
and role of antioxidants as protection from heavy
metal stress in roots, mycorrhizal fungi and mycorrhizae. Based on their chemical and physical properties three different molecular mechanisms of heavy
metal toxicity can be distinguished: (a) production of
reactive oxygen species by autoxidation and Fenton
reaction; this reaction is typical for transition metals
such as iron or copper, (b) blocking of essential
functional groups in biomolecules, this reaction
has mainly been reported for non-redox-reactive
heavy metals such as cadmium and mercury, (c) displacement of essential metal ions from biomolecules;
the latter reaction occurs with different kinds of heavy
metals. Transition metals cause oxidative injury in
plant tissue, but a literature survey did not provide
evidence that this stress could be alleviated by
increased levels of antioxidative systems. The reason
may be that transition metals initiate hydroxyl radical
production, which can not be controlled by antioxidants. Exposure of plants to non-redox reactive
metals also resulted in oxidative stress as indicated
by lipid peroxidation, H2O2 accumulation, and an
oxidative burst. Cadmium and some other metals
caused a transient depletion of GSH and an inhibition
of antioxidative enzymes, especially of glutathione
reductase. Assessment of antioxidative capacities
by metabolic modelling suggested that the reported
diminution of antioxidants was sufficient to cause
H2O2 accumulation. The depletion of GSH is apparently a critical step in cadmium sensitivity since
plants with improved capacities for GSH synthesis
displayed higher Cd tolerance. Available data suggest that cadmium, when not detoxified rapidly
enough, may trigger, via the disturbance of the redox
1
control of the cell, a sequence of reactions leading
to growth inhibition, stimulation of secondary metabolism, lignification, and finally cell death. This view
is in contrast to the idea that cadmium results in
unspecific necrosis. Plants in certain mycorrhizal
associations are less sensitive to cadmium stress
than non-mycorrhizal plants. Data about antioxidative
systems in mycorrhizal fungi in pure culture and in
symbiosis are scarce. The present results indicate
that mycorrhization stimulated the phenolic defence
system in the Paxillus–Pinus mycorrhizal symbiosis.
Cadmium-induced changes in mycorrhizal roots
were absent or smaller than those in non-mycorrhizal
roots. These observations suggest that although
changes in rhizospheric conditions were perceived by
the root part of the symbiosis, the typical Cd-induced
stress responses of phenolics were buffered. It is
not known whether mycorrhization protected roots
from Cd-induced injury by preventing access of
cadmium to sensitive extra- or intracellular sites, or
by excreted or intrinsic metal-chelators, or by other
defence systems. It is possible that mycorrhizal fungi
provide protection via GSH since higher concentrations of this thiol were found in pure cultures of
the fungi than in bare roots. The development of
stress-tolerant plant-mycorrhizal associations may
be a promising new strategy for phytoremediation
and soil amelioration measures.
Key words: Antioxidant systems, heavy metals, mycorrhiza,
oxidative stress.
Introduction
To date an unprecedented, rapid change in environmental
conditions is observed, which is likely to override the
To whom correspondence should be addressed: Fax: [ 49 551 39 2705. E-mail: [email protected]
ß Society for Experimental Biology 2002
1352
Schützendübel and Polle
adaptive potential of plants, especially that of tree species
with their long reproductive cycles. These environmental
changes mainly originate from anthropogenic activities,
which have caused air and soil pollution, acid precipitation, soil degradation, salinity, increasing UV-B radiation, climate change, etc. In addition, plants are exposed
to natural climatic or edaphic stresses, for example, high
irradiation, heat, chilling, late frost, drought, flooding,
and nutrient imbalances. Some of these stress factors
may fluctuate significantly in intensity and duration on
time scales of hours, days, seasons, or years; others
may change slowly and gradually affect plant growth
conditions. Since plants are sessile organisms and have
only limited mechanisms for stress avoidance, they need
flexible means for acclimation to changing environmental
conditions. In order to improve a plant’s protection, it
is important to understand the mechanisms contributing
to stress tolerance.
A common consequence of most abiotic and biotic
stresses is that they result, at some stage of stress
exposure, in an increased production of reactive oxygen
species (Polle and Rennenberg, 1993). The successive
reduction of molecular oxygen to H2O yields the
intermediates O2Y , HO and H2O2, which are potentially
toxic, because they are relatively reactive compared with
O2. Reactive oxygen species may lead to the unspecific
oxidation of proteins and membrane lipids or may cause
DNA injury. As a consequence, tissues injured by
oxidative stress generally contain increased concentrations of carbonylated proteins and malondialdehyde and
show an increased production of ethylene (Dean et al.,
1993; Ames et al., 1993).
For a long time reactive oxygen species have been
considered mainly as dangerous molecules, whose levels
need to be kept as low as possible. Now this opinion is
changing rapidly. It has been realized that reactive oxygen
species play important roles in the plant’s defence system
against pathogens (‘oxidative burst’, Alvarez and Lamb,
1997; Doke, 1997; Bolwell et al., 2002), mark certain
developmental stages such as tracheary element formation, lignification and other cross-linking processes in
the cell wall (‘programmed cell death’, Jacobson, 1996;
Teichmann, 2001; Fath et al., 2002) and act as intermediate signalling molecules to regulate the expression of
genes (May et al., 1998; Karpinski et al., 1999; Neill et al.,
2002; Vranova et al., 2002). Because of these multiple
functions of activated oxygen, it is necessary for cells to
control the level of reactive oxygen molecules tightly, but
not to eliminate them completely.
The control of oxidant levels is achieved by antioxidative systems. These defence systems are composed
of metabolites such as ascorbate, glutathione, tocopherol,
etc., and enzymatic scavengers of activated oxygen such
as SODs, peroxidases and catalases (Noctor and Foyer,
1998; Asada, 1999). The maintenance of ascorbate in its
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reduced form is achieved by monodehydroascorbate
radical reductase (MDAR) and NAD(P)H or ferredoxin
as reductant or by the operation of the ascorbate–
glutathione pathway (Foyer and Halliwell, 1976;
Borraccino et al., 1986; Miyake and Asada, 1994). In
the latter pathway the reduction of dehydroascorbate is
coupled to the oxidation of glutathione (GSH), which,
in turn, is reduced by glutathione reductase by oxidation
of NADPH (Foyer and Halliwell, 1976). Antioxidant
systems and their significance for the acclimation of
plants to air pollution and climatic stresses have been
reviewed frequently with emphasis on the responses of
leaves (Smirnoff and Pallanca, 1996; Polle, 1996, 1997,
1998; Smirnoff, 1996; Noctor and Foyer, 1998; Asada,
1999). Less attention has been paid to soil-borne stresses
and their effects in roots.
In soils influenced by human activities a range of
different problems such as overexploitation, salinity,
acidification, and contamination by various pollutants
have been reported. Increasing emissions of heavy metals
are dangerous because they may get into the food chain
with risks for human health (Lantsy and Mackenzie,
1979; Galloway et al., 1982; Angelone and Bini, 1992).
For the recultivation of degraded soils and the reclamation of industrial sites, stress-tolerant plants are required.
Biotechnological efforts are underway to improve plant
stress tolerance and the ability to extract pollutants from
the soil with the aim of using plants for soil clean-up
(Salt et al., 1995). In order to devise new strategies for
phytoremediation and improved tolerance, it is important to understand the basic principles as to how the
pollutants are taken up and act at the cellular and tissue
level. In the present study the occurrence and mode of
action of metal pollutants will be briefly reviewed, and the
role of antioxidants as defence systems will be discussed.
By applying metabolic modelling, oxidant fluxes will be
calculated as an estimate of oxidative stress levels and for
the prediction of efficient compensation mechanisms in
roots. A further question that will be addressed is whether
there is evidence that mycorrhizal symbionts improve
plant performance under heavy metal stress through
increased antioxidative systems.
Occurrence, chemical and physical properties
of heavy metals and their mode of action
Heavy metals are defined as metals with a density higher
than 5 g cm Y 3. 53 of the 90 naturally occurring elements
are heavy metals (Weast, 1984), but not all of them are of
biological importance. Based on their solubility under
physiological conditions, 17 heavy metals may be available for living cells and of importance for organism and
ecosystems (Weast, 1984). Among these metals, Fe, Mo
and Mn are important as micronutrients. Zn, Ni, Cu, V,
Co, W, and Cr are toxic elements with high or low
Plant responses to abiotic stresses
importance as trace elements. As, Hg, Ag, Sb, Cd, Pb,
and U have no known function as nutrients and seem to
be more or less toxic to plants and micro-organisms
(Godbold and Hüttermann, 1985; Breckle, 1991; Nies,
1999).
In most terrestrial ecosystems, there are two main
sources of heavy metals: the underlying parent material
and the atmosphere. The concentrations of heavy metals
in soils depend on the weathering of the bedrock and
on atmospheric inputs of metals. Natural sources are
volcanoes and continental dusts. Anthropogenic activities
like mining, combustion of fossil fuels, metal-working
industries, phosphate fertilizers, etc., lead to the emission
of heavy metals and the accumulation of these compounds in ecosystems (Lantsy and Mackensie, 1979;
Galloway et al., 1982; Angelone and Bini, 1992). It has
been estimated that, for example, the anthropogenic
emissions of Cd are in the range of 30 000 t per year
(di Toppi et al., 1999). In unpolluted soil Cd is present at
concentrations of 0.1–0.5 mg kg Y 1, but in Great Britain,
in heavily polluted soils of sewage sludge, concentrations
of up to 150 mg kg Y 1 have been found (Jackson and
Alloway, 1991). In the soil, mobile and immobilized
fractions have to be distinguished since heavy metals
bind to inorganic and organic soil compounds and to the
humus. The solubility and mobility of metals is affected
by adsorption, desorption, and complexation processes,
which in turn are dependent on the soil type.
The availability of heavy metals to plants and, thus,
their toxicity depends on complex rhizospheric reactions
involving not only exchange processes between soil
and plants but also microbial activities. In this respect,
mycorrhizal fungi appear to play a central modulating
role (see below). Access of heavy metals to bare roots is
confined to the first few millimetres of the root tip. Within
the cortex the metals are transported in the apoplastic
space according to their concentration gradient and also
accumulate in the cell walls (Arduini et al., 1996). Toxic
effects are exerted at the plasma membrane and within
the cell. Two different uptake routes have been reported:
(a) passive uptake, only driven by the concentration gradient across the membrane and (b) inducible substratespecific and energy-dependent uptake (Nies, 1999;
Williams et al., 2000). A common transmembrane transporter was found for Cd, Cu, and Ni (Clarkson and
Lüttge, 1989). The uptake of these compounds was
competitively inhibited by K, Ca, and Mg (Clarkson
and Lüttge, 1989). Active and passive transport systems
have also been reported for Cd and Ni in roots of spruce
and soybean (Cataldo et al., 1978, 1981; Godbold, 1991).
Measurements in the authors’ laboratory indicated that
the phase of net accumulation of Cd in the root tip was
only short in pine (24 h) suggesting that a steady-state
flux between import and export rates was acquired
relatively quickly (Schützendübel et al., 2001).
1353
To understand the mode of action leading to heavy
metal toxicity in living cells, their chemical properties
have to be considered. Most of the heavy metals are
transition metals with an incompletely filled d-orbital
present as cations under physiological conditions. The
physiological redox range of aerobic cells stretches from
Y420 mV to [ 800 mV. Therefore, heavy metals of
biological significance can be divided into two groups of
redox active and inactive metals. Metals with lower redox
potentials than those of biological molecules can not
participate in biological redox reactions (Table 1).
Autoxidation of redox active metals such as Fe2 [ or
Cu [ results in O2Y formation and subsequently in H2O2
and OH production via Fenton-type reactions. Cellular
injury by this type of mechanism is well-documented for
iron (Halliwell and Gutteridge, 1986; Imlay et al., 1988),
copper (Li and Trush, 1993a, b) as well as other metals
(Jones et al., 1991; Lund et al., 1991, 1993; Shi and Dalal,
1993; Shi et al., 1993).
Another important mechanism of heavy metal toxicity
is their ability to bind strongly to oxygen, nitrogen
and sulphur atoms (Nieboer and Richardson, 1980). This
binding affinity is related to free enthalpy of the
formation of the product of metal and ligand. Table 2
shows a range of heavy metal cations with increasing
affinity for sulphides and the low solubility of these
products. Because of these features, heavy metals can
inactivate enzymes by binding to cysteine residues. Direct
effects of cadmium on the sulphydryl homeostasis of cells
and inhibition of enzymes have been reported for
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Table 1. Electrochemical potentials (mV) of heavy metals in
aqueous media (pH 7, 25 C, after Weast, 1984)
Metal cation
Redox potential (mV)
Zn2 [
Cd2 [
Ni2 [
Pb2 [
Cu2 [
Fe2 [
Hg2 [
Ag2 [
Y 1.18
Y 0.82
Y 0.65
Y 0.55
Y 0.26
[ 0.35
[ 0.43
[ 1.57
Table 2. Free energy of formation of metal-sulphides (DFf )
from free metals in Joules at 25 C and their solubility (Ksp)
(after Weast, 1984)
Compound
Ksp
DFf Ag2S
HgS
CuS
CoS
CdS
ZnS
MnS
PbS
NiS
6.7 Z 10 Y50
1.6 Z 10 Y52
6.3 Z 10 Y36
2.0 Z 10 Y25
8.0 Z 10 Y27
1.6 Z 10 Y23
2.5 Z 10 Y13
8.0 Z 10 Y28
1.0 Z 10 Y24
Y 9.3
Y 11.6
Y 11.7
Y 19.8
Y 33.6
Y 47.4
Y 49.9
Y 62.2
Y 184.9
1354
Schützendübel and Polle
mammalian and animal cells (Canesi et al., 1998;
Chrestensen et al., 2000).
Many enzymes contain metals in positions important
for their activity. The displacement of one metal by
another will normally also lead to inhibition or loss of
enzyme activities. Divalent cations such as Co2 [ , Ni2 [ ,
and Zn2 [ were found to displace Mg2 [ in ribulose1,5-bisphosphate-carboxylaseuoxygenase and resulted in
loss of activity (Wildner and Henkel, 1979; van Assche
and Clijsters, 1986). Displacement of Ca2 [ by Cd2 [ in
the protein calmodulin, important in cellular signalling, led to an inhibition in the calmodulin-dependent
phosphodiesterase activity in radish (Rivetta et al., 1997).
These examples show that, according to their chemical
and physical properties, three different molecular mechanisms of metal toxicity can be distinguished: (a) production of reactive oxygen species by autoxidation and
Fenton reaction, (b) blocking of essential functional
groups in biomolecules, and (c) displacement of essential
metal ions from biomolecules.
Heavy metals and antioxidative defences
There is ample evidence that exposure of plants to excess
concentrations of redox active heavy metals such as Fe
and Cu results in oxidative injury (De Vos et al., 1992;
Gallego et al., 1996; Weckx and Clijsters, 1996; Mazhoudi
et al., 1997; Yamamoto et al., 1997). The ability of
plants to increase antioxidative protection to combat
negative consequences of heavy metal stress appears to be
limited since many studies showed that exposure to
elevated concentrations of redox reactive metals resulted
in decreased and not in increased activities of antioxidative enzymes (Table 3). Growth with excess Fe
resulted in increased O2Y and HO -production (Caro and
Puntarulo, 1996). Autoxidation and Fenton reaction may
cause the oxidative loss of defence enzymes. For example,
catalase activity is directly inhibited by O2Y (Kono and
Fridovich, 1982). Cu-Zn-superoxide dismutase is fragmented by HO -radicals (Casano et al., 1997). If uptake of
excess Fe2 [ or Cu [ preferentially drives the formation
of HO -radicals, protection mediated by antioxidative
enzymes is unlikely (Polle, 1997). The authors of the
present paper have not been able to find literature data
providing evidence that elevated levels of antioxidant
enzymes protect from excess copper or iron, whereas
there are reports that overexpression of iron- or copperchelators, for example, of metallothioneins and ferritin,
protect against metal-induced oxidative injury (Fabisiak
et al., 1999).
Interestingly, the occurrence of activated oxygen and
symptoms of oxidative injury have also been observed in
plants exposed to heavy metals, which do not belong
to the group of transition metals (Cd: Gallego et al., 1996;
Lozano-Rodriguez et al., 1997; Chaoui et al., 1997; Cho
$
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$
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and Park, 1999; Piqueras et al., 1999; Romero-Puertas
et al., 1999; Schützendübel et al., 2001; Zn: Weckx and
Clijsters, 1997; Prasad et al., 1999; Rao and Sresty, 2000;
Ni: Baccouch et al., 1998; Rao and Sresty, 2000). Since
these metals do not interfere directly with cellular oxygen
metabolism, the question arises as to the reasons of the
observed oxidative stress. Exposure to heavy metals also
provoked pronounced responses of antioxidative systems,
but the direction of the response was dependent on the
plant species and tissue analysed, the metal used for
the treatment and the intensity of the stress (Table 3).
However, some common reaction patterns can be found.
In most cases, exposure to heavy metals initially resulted
in a severe depletion of GSH (Cd: Rauvolfia serpentina:
Grill et al., 1987; pine: Schützendübel et al., 2001; carrot:
di Toppi et al., 1999; tobacco: Vögeli-Lange and Wagner,
1996; Cu: Silene cucubalus: de Vos et al., 1992; Cu or Cd:
Arabidopsis: Xiang and Oliver, 1998; Ni and Zn:
pigeonpea: Rao and Sresty, 2000; Fe, Cu or Cd: sunflower
leaves: Gallego et al., 1996). This is a common response to
Cd caused by an increased consumption of glutathione
for phytochelatin production (Zenk, 1996; Mehra and
Tripathi, 1999). The significance of phytochelatins for
protection from heavy metals has frequently been
reviewed (Rauser, 1995; Zenk, 1996; Mehra and Tripathi,
1999) and, therefore, will be summarized here only briefly.
Phytochelatins sequester heavy metals. For Cd, the
formation of Cd–thiolate (Cd–S) complexes in phytochelatins has been shown (Strasdeit et al., 1991). The
chelated metals are transported to the tonoplast, taken up
by active transport systems, and deposited in the vacuole
(Tommasini et al., 1998; Rea, 1999). This mechanism
contributes to the protection from heavy metal toxicity in
several plant species and in some fungi as well (Ishikawa
et al., 1997). In pine, which is a relatively Cd-sensitive
species, the vacuolar Cd-concentrations in cells of root
tips were as high as 20 mM, even though the exposure
medium contained only 50 mM Cd (Fritz, unpublished
data). In general, the glutathione pool recovered after
prolonged Cd-exposure, frequently to levels above those
of controls (Vögeli-Lange and Wagner, 1996; Xiang and
Oliver, 1998; Arisi et al., 2000; Schützendübel et al.,
2001). The ability to synthesize glutathione appears to
be crucial for protection from cadmium, as shown by
the increased tolerance of plants with elevated levels of
GSH as well as a decreased tolerance in plants with
diminished levels of GSH (Howden et al., 1995; Zhu et al.,
1999a, b). However, the threshold required to enhance
protection seems to be plant-specific since the amelioration of growth under cadmium stress by elevated GSH
has not been observed in all cases (Arisi et al., 2000).
Since glutathione is also an important component for
the redox balance of the cell, as it is involved in the
regulation of the cell cycle, the detoxification of oxidants,
and acts as a transport form of reduced sulphur
Plant responses to abiotic stresses
1355
Table 3. Relative activities of antioxidative enzymes in different plant species exposed to heavy metals
Enzyme activities of controls were set as 100%. When it was not possible to determine relative enzyme activity, the trend is given ([ \ increase).
Enzyme
Compound
Superoxide
dismutase
Hg
Concentration (mM)
10
10
Cd
10
Cd
50
Cd
500
Cd
5000
Fe
900
Cu
500
Cu
630
Zn
5000
Zn
5000
Ascorbate
peroxidase
Cd
5
Cd
5
Cd
50
Cd
500
Cd
5000
Fe
900
Cu
15
Cu
Speciesuorgan
Lycopersicon esculentum
Roots
Leaves
Hordeum vulgare
Leaves
Helianthus annuus
Leaves
Helianthus annuus
Leaves
Nicotiana tabacum
BY2 cell culture
Phaseolus vulgaris
Leaves
Helianthus annuus
Leaves
Phaseolus vulgaris
Leaves
Cajanus cajan
Leaves
Brassica juncea
Shoots
Phaseolus vulgaris
Roots
Phaseolus vulgaris
Leaves
Helianthus annuus
Leaves
Helianthus annuus
Leaves
Nicotiana tabacum
BY2 cell culture
Phaseolus vulgaris
Leaves
Phaseolus vulgaris
Roots
Lemna minor
Relative
enzyme
activity (%)
50
Cu
500
Zn
612
Zn
100
Zn
5000
Catalase
Hg
5
Hg
10
Cd
5
Cd
5
Cd
50
Cd
100
Cd
500
Lycopersicon esculentum
Roots
Leaves
Helianthus annuus
Leaves
Phaseolus vulgaris
Roots
Phaseolus vulgaris
Leaves
Brassica juncea
Shoots
Phaseolus aureus
Leaves
Roots
Lycopersicon esculentum
Leaves
Roots
Phaseolus vulgaris
Roots
Phaseolus vulgaris
Leaves
Hordeum vulgare
Leaves
Helianthus annuus
Leaves
Helianthus annuus
Leaves
Reference
Cho and Park, 2000
120
150
240
240
Patra and Panda, 1998
48
[
Gallego et al., 1999
100
96
77
12
Gallego et al., 1996
Piqueras et al., 1999
180
0.08
Shainberg et al., 2000
24
[
Chaoui et al., 1997
118
12
83
48
230
144
220
240
100
96
210
96
117
96
75
12
Weckx and Clijsters, 1996
Rao and Sresty, 2000
Prasad et al., 1999
Chaoui et al., 1997
Chaoui et al., 1997
Gallego et al., 1999
Gallego et al., 1996
Piqueras et al., 1999
170
0.16
Shainberg et al., 2000
480
24
130
48
71
24
100
38
168
50
12
Gupta et al., 1999
Teisseire and Guy, 2000
10
Cu
Exposure
time (h)
Mazhoudi et al., 1997
Gallego et al., 1996
Gallego et al., 1999
48
[
Chaoui et al., 1997
144
96
260
240
113
0
48
48
100
140
240
240
75
96
75
96
120
96
Prasad et al., 1999
Shaw, 1995
Cho and Park, 2000
Chaoui et al., 1997
Chaoui et al., 1997
Gallego et al., 1999
Patra and Panda, 1998
48
[
Gallego et al., 1996
70
12
1356
Schützendübel and Polle
Table 3. (Continued)
Enzyme
Compound
Concentration (mM)
Cd
Catalase
5000
Cu
Speciesuorgan
Nicotiana tabacum
BY2 cell culture
Lemna minor
10
Cu
50
Cu
500
Cu
10 000
Zn
5000
Zn
5000
Glutathione
reductase
Cd
5
Cd
50
Cd
500
Fe
900
Cu
Lycopersicon esculentum
Roots
Leaves
Helianthus annuus
Leaves
Oryza sativa
Leaves
Cajanus cajan
Leaves
Brassica juncea
Shoots
Phaseolus vulgaris
Leaves
Helianthus annuus
Leaves
Helianthus annuus
Leaves
Phaseolus vulgaris
Leaves
Lemna minor
10
Cu
15
Cu
500
Cu
10 000
MDHAR
Zn
100
Zn
5000
DHAR
Cd
15
Cd
5000
Cd
50
Cd
500
Cu
500
Zn
5000
POD
Hg
5
Hg
10
Cd
50
Cd
5000
Cu
50
Cu
Phaseolus vulgaris
Roots
Helianthus annuus
Leaves
Oryza sativa
Leaves
Phaseolus vulgaris
Leaves
Brassica juncea
Shoots
Phaseolus vulgaris
Roots
Brassica juncea
Shoots
Helianthus annuus
Leaves
Helianthus annuus
Leaves
Helianthus annuus
Leaves
Brassica juncea
Shoots
Phaseolus aureus
Leaves
Lycopersicon esculentum
Roots
Leaves
Helianthus annuus
Leaves
Nicotiana tabacum
BY2 cell culture
Lycopersicon esculentum
Roots
Leaves
Lemna minor
10
Zn
100
Zn
5000
Zn
5000
Phaseolus vulgaris
Leaves
Brassica juncea
Shoots
Cajanus cajan
Leaves
Relative
enzyme
activity (%)
Exposure
time (h)
Reference
Piqueras et al., 1999
75
0.08
Teisseire and Guy, 2000
347
24
76
100
168
33
12
46
24
75
144
1200
240
80
96
120
96
80
12
155
24
68
24
119
24
54
12
33
24
150
96
258
240
120
96
280
240
120
96
75
12
50
12
570
240
210
24
Mazhoudi et al., 1997
Gallego et al., 1996
Chen and Kao, 1999
Rao and Sresty, 2000
Prasad et al., 1999
Chaoui et al., 1997
Gallego et al., 1999
Patra and Panda, 1998
Shainberg et al., 2000
Teisseire and Guy, 2000
Gupta et al., 1999
Gallego et al., 1996
Chen and Kao, 1999
Chaoui et al., 1997
Prasad et al., 1999
Gupta et al., 1999
Prasad et al., 1999
Gallego et al., 1999
Gallego et al., 1996
Gallego et al., 1996
Prasad et al., 1999
Shaw, 1995
Cho and Park, 2000
2.5
100
240
240
Gallego et al., 1999
116
96
Piqueras et al., 1999
100
0.16
Mazhoudi et al., 1997
130
100
168
166
24
110
96
400
240
156
144
Teisseire and Guy, 2000
Chaoui et al., 1997
Prasad et al., 1999
Rao and Sresty, 2000
Plant responses to abiotic stresses
(Bergmann and Rennenberg, 1993; May et al., 1998;
Noctor and Foyer, 1998; Vernoux et al., 2000), it may be
suspected that a short-term lack of GSH may favour the
accumulation of reactive oxygen and disturb developmental processes. The idea, that Cd and perhaps also
other toxic metals, act in cells through a depletion of
antioxidative defences is further supported by the
observation that glutathione reductase, ascorbate peroxidase and catalase activities were inhibited at time scales
similar to those found for the depletion of GSH (Fig. 1).
Heavy metal-induced loss in glutathione reductase has
frequently been observed: in pea by Zn, Cu and Fe
(Bielawski and Joy, 1986), in sunflower by Fe, Cu and Cd
(Gallego et al., 1996), in Lemna minor by Cu (Teisseire
and Guy, 2000). Glutathione reductase contains a highly
conserved disulphide bridge between Cys76 and Cys81
(Creissen et al., 1992; Lee et al., 1998), which may
undergo cleavage by heavy metals. The sensitivity of
Fig. 1. Antioxidative enzymes and antioxidants in pine roots (Pinus
sylvestris). (A) Mean volume activities of enzyme and concentrations
of antioxidants were calculated on the basis of the water content of
the roots (93%) of control plants. Cat, catalase; SOD*, superoxide
dismutase (indicated as concentration); APX, ascorbate peroxidase;
MDAR, monodehydroascorbate radical reductase; DAR, dehydroascorbate reductase; GR, glutathione reductase; ASC, ascorbate
(black) [ dehydroascorbate (grey); GSH, GSH (black) [ GSSG (grey).
Data are means of five replicates. Nd, not detected. (B) Changes in
enzyme activities and antioxidant concentrations relative to controls
after 6 h (B) and 96 h (C) exposure to 50 mM cadmium.
1357
glutathione reductase to direct inhibition by Cd was
shown in in vitro assays (Fig. 2). If EDTA, a chelator
of divalent cations, was added, glutathione reductase
activity was recovered (data not shown). Currently, it
is unknown whether roots exposed to Cd accumulate
sufficiently high free concentrations of this compound
for direct interaction with glutathione reductase in situ.
However, it is tempting to speculate that the initial
decrease in glutathione reductase activity may have been
caused by Cd-binding when the concentrations of GSH
were severely diminished (Fig. 1B). The activities of
defence enzymes recovered after prolonged Cd-exposure
(Fig. 1C). The observed increase of thiol concentrations
above those of controls (Fig. 1C) may be necessary to
protect sensitive enzymes.
‘Unspecific’ peroxidases, i.e. enzymes oxidizing phenolic substrates such as guaiacol, were also affected by
exposure to cadmium (Table 3). In pine roots, ‘unspecific’
peroxidases were not inhibited by Cd, but increased
slowly with a time pattern clearly distinct from that
observed for the constituents of the SOD–ascorbate–
glutathione pathway (Schützendübel et al., 2001). ‘Unspecific’ peroxidase activities were elevated in root tips,
which showed increased concentrations of phenolics and
lignification in response to Cd (Schützendübel et al.,
2001). The final result of Cd in roots resembles that of
plant tissues exposed to pathogens. During pathogenic
attack, plant cells display an increased production of
reactive oxygen species (oxidative burst) followed by
secondary defence reactions (Alvarez and Lamb, 1997).
These responses lead to mechanical strengthening of cell
walls including lignification to prevent intrusion of the
pathogen (Alvarez and Lamb, 1997). The scheme that
Cd induces defence pathways resulting in cell wall rigidification is also consistent with the observation that
root growth stops or is significantly inhibited after
Fig. 2. Inhibition of glutathione reductase by cadmium. Glutathione
reductase (EC 1.6.4.2) from bakers yeast was incubated with different
concentrations of Cd for 30 min and then tested. Data are means of
three replicates (] SD). Different letters indicate significant differences at
PF0.05 as determined by ANOVA followed by a multiple range test
(LSD).
1358
Schützendübel and Polle
Fig. 3. Growth inhibition in pine seedlings (Pinus sylvestris L.) exposed
to different Cd-concentrations. Pine seedlings were grown in sandculture under a light regime of 17 h and 200 mE at 20 C. Nine-week-old
plants were exposed for 21 d to different concentrations of CdSO4 in the
nutrient solution. Data are means of six replicates (] SD). Different
letters indicate significant differences at PF0.05 as determined by
ANOVA followed by a multiple range test (LSD).
Fig. 4. Simulated changes in the steady-state concentrations of GSSG
Y
by increasing O2 production rates. The black line indicates a steadyY
state O2 production rate, which results in the initial GSSG concentration. The calculations are based on the model SHAG-ENZ (Polle, 2001),
which was enlarged by a component for catalase. The activities of
antioxidant enzymes and concentrations of antioxidants shown in Fig.
1A were used for the model calculations. It was assumed that the
measured enzyme activities reflected Vmax. The concentration of SOD
was calculated by conversion of the measured units to a concentration
on the basis of the relationship: 500 units nmol Y 1 of enzyme protein. It
was assumed that the supply of NAD(P)H was not limiting.
$
$
Cd-exposure (Punz and Sieghardt, 1993; Kahle, 1993). It
may be suspected that, as a result of Cd-induced defence
reactions, lignified root tips have also lost their capacity
for nutrient uptake, and, thus, their ability to sustain
plant growth. This would lead to growth retardation at
the whole-plant level (Fig. 3).
Metabolic modelling as a means to
predict changes in oxidant levels from
measured Cd-induced changes in
‘antioxidative capacities’
An intriguing question is whether Cd induces an
‘oxidative burst’ similar to that reported for pathogens
or whether the concentrations of reactive oxygen increase
because of the initial depletion of GSH and inhibition
of protective enzymes. To find out whether the observed
decreases in antioxidative defences would be sufficient
to explain H2O2-accumulation, quantitative estimates of
the ‘antioxidative capacity’ are necessary. As a first step
towards an assessment of the oxidant scavenging efficiency, mean concentrations of antioxidants and volumerelated activities of defence enzymes in healthy root tips
were calculated (Fig. 1A). By contrast to needles, pine
roots contained relatively low concentrations of soluble
antioxidants. Even more striking was that the redox
status of the antioxidant pool was also very low, which
means that the concentrations of reduced antioxidants
(ascorbate and GSH) were low relative to their oxidized
counterparts (dehydroascorbate and GSSG).
A metabolic model, which can be used to calculate
oxidant scavenging activity (Polle, 2001), suggested that
the ratio of GSHuGSSG was especially sensitive to
changes in oxidative stress and, thus, reflects the steadystate flux of oxidants and reductants under normal
conditions. Given this presumption, the measured concentrations of antioxidants and activities of protective
enzymes and their known biochemical properties (Polle,
2001) can be used to provide a semi-quantitative estimate
of the ‘antioxidative capacity’ and intrinsic oxidative
stress under ‘normal’ conditions. To find the stress rate,
which would result in the measured mean GSSHuGSH
ratio, an increasing production rate of O2Y radicals was
simulated (as in Polle, 2001). This condition, indicating
mean intrinsic stress in ‘normal’ root tips, was fulfilled
at a O2Y production rate of 93 mM s Y 1 (Fig. 4). Currently, this model is coarse because it describes only
an average situation not taking into account the subcellular distribution of antioxidant systems and possible
differences in intrinsic stress exposure. For comparison,
O2Y production rates of 120–250 mM s Y 1 were estimated
in chloroplasts under ‘normal’ conditions and up to
720 mM s Y 1 under stress (Asada, 1999). These considerations show that the above model provides an estimate in
the appropriate range.
In a second modelling step, a situation was envisaged
where the ‘antioxidative capacity’ was severely diminished
after Cd-exposure (as indicated for 6 h of Cd-treatment
in Fig. 1B). It was assumed that the production rate of
O2Y remained unchanged (93 mM s Y 1), i.e. no ‘oxidative
burst’. When the model was run under these conditions, a
significant accumulation of H2O2 was predicted (Fig. 5C),
while the steady-state concentrations of O2Y and monodehydroascorbate radicals were somewhat decreased
(Fig. 5A, B) because of increased activities of superoxide
dismutase and monodehydroascorbate radical reductase
(Fig. 1B). In fact, accumulation of H2O2 has been
$
$
$
$
$
Plant responses to abiotic stresses
1359
Fig. 6. Hypothetical view of cadmium action on the cellular redox
control; for further explanations, see text.
Fig. 5. Simulated oxidant concentrations in the absence and presence of
50 mM cadmium. (A) Superoxide radials, (B) monodehydroascorbate
radical, (C) hydrogen peroxide. The calculations were performed as
described in Fig. 4 using activities of enzymes and concentrations of
antioxidants in controls or those obtained after 6 h of cadmium
exposure.
observed in Cd-exposed roots (Schützendübel et al., 2001)
and in Cd-exposed tobacco suspension cultures (Piqueras
et al., 1999). It was suggested that Cd triggered an
‘oxidative burst’ as in pathogenesis because they detected
H2O2 in the culture medium (Piqueras et al., 1999).
However, they also found a significant inhibition of
catalase and since H2O2 is membrane permeable, the site
of H2O2 generation in response to Cd may not be totally
clear.
In conclusion, the above estimates suggest that a significant intracellular H2O2 accumulation can be expected
after Cd exposure, simply because of the Cd-induced
depletion of GSH and inhibition of antioxidative
enzymes. In pine roots, H2O2 was detected at an early
stage (6 h after Cd addition), when the roots still
appeared visibly fully viable and no lipid peroxidation
was found (Schützendübel et al., 2001). H2O2 disappeared
within some hours but, thereafter, the differentiation
of protoxylem elements became apparent in unusual
places of the previous elongation zone of root tips
(Schützendübel et al., 2001). Taking all these observations
together, the following hypothetical framework may
be suggested (Fig. 6): Cd induces a transient loss in
‘antioxidative capacity’, perhaps accompanied by a stimulation of oxidant producing enzymes, which results in
intrinsic H2O2 accumulation. H2O2, then, would act as a
signalling molecule triggering secondary defences. These,
in turn, would cause an untimely cell wall rigidification
and lignification, thereby, decreasing cellular viability
and finally resulting in cell death (Fig. 6). This view would
be clearly distinct from the alternative idea that Cd results
in unspecific necrosis and is also supported by the
observation that Cd-exposed cells show a distinct pattern
of DNA-fragmentation typical for programmed cell death
(Fojtova and Kovarik, 2000).
Heavy metals and stress responses in
mycorrhizal symbiosis
Under natural conditions, roots of many plant species,
especially those of trees are associated with mycorrhizal
symbionts. This modifies the response of plants to heavy
metals significantly. Several studies have dealt with a
possible alleviation of metal toxicity by mycorrhization,
but only a few presented direct evidence for such effects
(Hartley et al., 1997; Leyval et al., 1997; Jentschke and
Godbold, 2000). In the present study non-mycorrhizal
pine seedlings in the presence of Cd-concentrations
G15 mM showed 35% diminished biomass as compared
to controls (Fig. 3). By contrast, a remarkable protection
of plant performance against the negative effects of Cd,
1360
Schützendübel and Polle
i.e. no biomass reduction was observed, when the seedlings were 80% associated with a strain of the mycorrhizal
fungus Paxillus involutus isolated from a heavy metalpolluted site (Fig. 7). It is still unclear whether the
observed alleviation is a consequence of better nutrition,
a fungal influence on the physiological stress reaction of
the plant or simply hindered access of heavy metals to
the root surface caused by the fungal sheath around the
root surface (Jentschke and Godbold, 2000). In Hebeloma
crustuliniforme the latter suggestion has some support
(Frey et al., 2000), whereas Paxillus involutus also shows
significant Cd accumulation in the vacuole (Blaudez et al.,
2000).
Little is known about the heavy metal-induced stress
responses of mycorrhiza-building basidiomycetes in pure
cultures and in association with their hosts. In most fungi,
metallothioneins are induced to detoxify the metals in
a reaction similar to that found in animal cells (Mehra
and Winge, 1991). In yeast, heavy metals also caused
oxidative stress (Mannazu et al., 2000). Some scarce data
also suggest that heavy metals affect antioxidative
systems in mycorrhizal fungi. For example, an inhibition
of Mn-SOD was found in pure cultures of Rhizopogon
roseolus treated with 300 mM Cd (Miszalski et al., 1996).
By contrast, an increase in Mn-SOD activity was found in
Cd-treated Paxillus involutus cultures (Jacob et al., 2001).
The antioxidative systems of mycorrhizal fungi revealed
important differences in comparison with plant tissues.
For example, in pure cultures of Laccaria laccata, Suillus
bovinus, and Paxillus involutus typical ‘unspecific’ peroxidase activities were not detected (Münzenberger et al.,
1997; Schützendübel et al., 2001) neither was ascorbate
Fig. 7. Mycorrhization frequency of root tips (A) and fresh mass (B) of
14-week-old P. sylvestris–Paxillus involutus associations treated for
2 weeks with different concentrations of CdSO4 in the nutrient solution.
For growth conditions, see Fig. 3. Mycorrhizal root tips and nonmycorrhizal root tips of each plant were counted under a binocular
microscope and the degree of mycorrhization was estimated as number
of mycorrhizal root tips per total number of root tips Z 100. Data are
means of six replicates (] SD). The asterix indicates a significant
difference at PF0.05 as determined by ANOVA followed by a multiple
range test (LSD).
peroxidase activity or ascorbate as a potential substrate
(T Ott and A Schützendübel, unpublished data). Much
higher concentrations of glutathione were found in pure
cultures of Suillus bovinus and Paxillus involutus than in
pine roots (2–10 mmol g Y 1 fresh weight compared with
0.2–1 mmol g Y 1 fresh weight plant tissues, Schützendübel
et al., 2001).
The question arises as to whether the wholemycorrhizal association and each individual partner
(Paxillus–Pinus) exposed to Cd at concentrations,
which did not result in a loss of the degree of mycorrhization and only in small growth reduction (Fig. 7),
show stress reactions similar to those found in bare roots
(Fig. 1B). Initially ‘total’ SOD activities in mycorrhizal
roots were similar to those of non-mycorrhizal roots and
remained unaffected by Cd. In the mycorrhizal roots
SOD activities increased with time, whereas in the
non-mycorrhizal roots and in Cd-treated roots
(] mycorrhiza) SOD activities remained low after
prolonged Cd-exposure (Fig. 8B). The activities of
‘unspecific’ peroxidases, which can be used as a marker
Fig. 8. POD (EC 1.11.1.7) activity (A) and SOD (EC 1.15.1.1) activity
(B) in mycorrhizal root tips of 14-week-old P. sylvestris–Paxillus
involutus mycorrhiza (myc) and non-mycorrhizal pine roots (nm) of
control seedlings or presence of 50 mM of CdSO4 in the nutrient solution
for 1 d and 14 d (myccd, mycorrhizal seedlings [ cadmium; nmcd, nonmycorrhizal seedlings [ cadmium). For growth conditions see Fig. 3.
Data are means of six replicates (] SD). Different letters indicate
significant differences at PF0.05 as determined by ANOVA followed by
a multiple range test (LSD).
Plant responses to abiotic stresses
for the root-specific response in the mycorrhizal association, initially was neither affected by the fungal
symbiont nor by Cd-exposure (Fig. 8A). However, after
14 d, POD activities were increased in non-mycorrhizal
Cd-exposed roots (as observed previously in hydroponically grown bare roots) but not in mycorrhizal
Cd-exposed roots (Fig. 8A). This observation suggests
that the stress reaction is diminished or perhaps the stress
not perceived in mycorrhizal roots.
Analysis of the effects of mycorrhization and
Cd-exposure on soluble and cell wall-bound phenolics
in the Paxillus–Pinus symbiosis supports this idea
(Fig. 9). Cd-treatment resulted in increased concentrations of ‘total’ soluble phenolics only in nonmycorrhizal roots but not in mycorrhizal roots (Fig. 9).
However, mycorrhizal roots generally contained elevated
concentrations of soluble phenolics as compared with
non-mycorrhizal roots (Fig. 9). A pattern similar to that
of ‘total’ soluble phenolics was found for kaempherol3-glucoside (Fig. 10A), whereas the major plant phenolic
acid, catechin, remained unaffected by both Cd-exposure
and the presence or absence of the mycorrhizal symbiont
(Fig. 10A). Increases in free p-coumaric acid were found
in response to mycorrhization as well as in response to
Cd (Fig. 10A). Interestingly, in non-mycorrhizal roots
free ferulic acid was not detected either in the presence or
absence of Cd (Fig. 10A). By contrast, mycorrhizal roots
contained low, but consistently detectable concentrations
of free ferulic acid, which were slightly increased after
exposure to Cd (Fig. 10A). Since none of the phenolics
were found in pure cultures of Paxillus, these compounds
mark root-specific differential responses to mycorrhization and Cd. The observed increases in ferulic acid and
p-coumaric acid are particularly interesting in this respect
because they indicate that the root part in the mycorrhizal
association displayed a specific new response to Cd, which
was not found in non-mycorrhizal Cd-exposed roots.
Both ferulic acid and p-coumaric acid play roles in cell
wall rigidification because they may become cross-linked
with lignins, proteins or carbohydrate residues decreasing
the extensibility of walls (Fry, 1986). Major wall-bound
phenolics were 3,4-dihydroxybenzoic acid, p-coumaric
acid and ferulic acid (Fig. 10B). 3,4-dihydroxybenzoic
acid appeared at elevated concentrations in mycorrhizal
roots (Fig. 10B). The concentration of wall-bound ferulic
acid was neither affected by mycorrhization nor by Cd
(Fig. 10B), whereas bound p-coumaric acid increased
specifically in mycorrhizal Cd-exposed roots (Fig. 10B).
Taken together, these results show that Paxillus–Pinus
mycorrhizal associations contained higher concentrations of secondary metabolites than non-mycorrhizal
roots. This suggests that mycorrhization stimulated the
defence systems. However, the Cd-induced changes in
mycorrhizal roots were absent or smaller than those in
non-mycorrhizal roots. These observations suggest that
1361
Fig. 9. Soluble phenolics in mycorrhizal root tips of 14-week-old
P. sylvestris–Paxillus involutus mycorrhizal (myc) and non-mycorrhizal
pine roots (nm) of control seedlings or presence of 50 mM of CdSO4 in
the nutrient solution for 1 d and 14 d (myccd, mycorrhizal seedlings [
cadmium; nmcd, non-mycorrhizal seedlings [ cadmium). For growth
conditions see Fig. 3. Data are means of six replicates (] SD). Different
letters indicate significant differences at PF0.05 as determined by
ANOVA followed by a multiple range test (LSD).
Fig. 10. Soluble (A) and cell wall-bound (B) phenolic compounds in
mycorrhizal root tips of 14-week-old P. sylvestris–Paxillus involutus
mycorrhizal (myc) and non-mycorrhizal pine roots (nm) of control
seedlings or presence of 50 mM of CdSO4 in the nutrient solution
for 1 d and 14 d (myccd, mycorrhizal seedlings [ cadmium, nmcd
non-mycorrhizal seedlings [ cadmium). Abbreviations: Cat, catechin;
Kä, kaempferol-3-glucoside; Ca, p-coumaric acid; Fa, ferulic acid;
DHB, 3,4-dihydroxybenzoic acid. For growth conditions see Fig. 3.
Data are means of six replicates (] SD); nd; not detected. Different
letters indicate significant differences at PF0.05 as determined by
ANOVA followed by a multiple range test (LSD).
although changes in rhizospheric conditions are perceived
by the root part of the symbiosis, the typical Cd-induced
stress response was significantly buffered. The mechanism
1362
Schützendübel and Polle
by which mycorrhization protects from Cd is unclear.
Metallothioneins may be involved, but this has not
been investigated in mycorrhiza-building basidiomycetes.
Other chelators of heavy metals such as excreted organic
acids etc. as well as binding Cd in the Hartig net, may also
be involved as a protective mechanism. Since much higher
concentrations of glutathione were found in the mycorrhizal fungi Suillus bovinus and Paxillus involutus than
in bare pine roots, it is also possible that roots in
mycorrhizal associations are ‘armed’ with a powerful
physiological defence against Cd. The use of stresstolerant mycorrhizal fungi may be a promising strategy
to develop tools for soil reclamation and amelioration.
Acknowledgements
We are grateful to Dr E Fritz and T Ott (Forest Botanical
Institute, University of Göttingen) for communicating unpublished data, to D Godbold (School of Biological Sciences,
University of Bangor) for helpful discussions and to C Kettner
and C Rudolf for technical assistance. We acknowledge financial
support by the European Community.
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