Tree Physiology 22, 583–590
© 2002 Heron Publishing—Victoria, Canada
Age effects on Norway spruce (Picea abies) susceptibility to ozone
uptake: a novel approach relating stress avoidance to defense
GERHARD WIESER,1,2 KARIN TEGISCHER,1,3 MICHAEL TAUSZ,3 KARL-HEINZ
HÄBERLE,4 THORSTEN E. E. GRAMS4 and RAINER MATYSSEK4
1
Forstliche Bundesversuchsanstalt, Abt. Forstpflanzenphysiologie, Rennweg 1, A-6020 Innsbruck, Austria
2
Author to whom correspondence should be addressed ([email protected])
3
Institute of Plant Physiology, Karl-Franzens University Graz, Schubertstraße 51, A-8010 Graz, Austria
4
Department of Ecology/Forest Botany, Technische Universität München, Am Hochanger 13, D-85354 Freising, Germany
Received February 26, 2001; accepted November 2, 2001; published online May 1, 2002
Summary Cumulative ozone (O3) uptake and O3 flux were
related to physiological, morphological and biochemical characteristics of Norway spruce (Picea abies (L.) Karst.) trees of
different ages. Under ambient CO2 conditions, photosynthetic
capacity (Amax) declined in mature trees when cumulative O3
uptake into needles, which provides a measure of effective O3
dose, exceeded 21 mmol m –2 of total needle surface area. A
comparable decline in Amax of seedlings occurred when cumulative O3 uptake was only 4.5 mmol m –2. The threshold O3 flux
causing a significant decline in Amax ranged between 2.14 and
2.45 nmol m –2 s –1 in mature trees and seedlings subjected to
exposure periods of ≥ 70 and ≥ 23 days, respectively. The
greater O3 sensitivity of young trees compared with mature
trees was associated with needle morphology. Biomass of a
100-needle sample increased significantly with tree age,
whereas a negative correlation was found for specific leaf area,
these changes parallel those observed during differentiation
from shade-type to sun-type needles with tree ontogeny. Agedependent changes in leaf morphology were related to changes
in detoxification capacity, with area-based concentrations of
ascorbate increasing during tree ontogeny. These findings indicate that the extent of O3-induced injury is related to the ratio of
potentially available antioxidants to O3 influx. Because this ratio, when calculated for ascorbate, increased with tree age, we
conclude that the ratio may serve as an empirical basis for characterizing age-related differences in tree responses to O3.
Keywords: antioxidants, ascorbate, cumulative ozone uptake,
detoxification, needle morphology, ontogeny, ozone flux, scaling, tree age.
Introduction
Tropospheric ozone (O3) is one of the most detrimental air pollutants affecting forest trees (Lefohn 1991, Sandermann et al.
1997). Trees respond to O3-induced stress by mechanisms of
avoidance and defense (i.e., stress tolerance; Hogsett and
Andersen 1998) such as restriction of O3 uptake by stomatal
closure or detoxification through biochemical reactions in the
tissue. Much of the information on O3 effects on trees is based
on chamber studies conducted with seedlings (Reich 1987,
Pye 1988, Sandermann et al. 1997, Matyssek and Innes 1999).
Seedlings, however, are uncertain surrogates for mature trees
(Kelly et al. 1995, Kolb et al. 1997, Kolb and Matyssek 2001),
because mature trees and seedlings differ in morphological
and physiological characteristics that strongly influence plant
responses to O3 (Ryan et al. 1997, Bond 2000). Understanding
the influence of age on the response of trees to O3 is, therefore,
essential to a better interpretation of the available experimental data.
The effect of age on tree responses to O3 is poorly understood (Samuelson and Kelly 2001), because of the difficulty of
exposing large trees to O3 experimentally (Samuelson and Edwards 1993, Grulke and Miller 1994, Fredericksen et al. 1995,
1996, Werner and Fabian 2001). Limited data for Norway
spruce (Picea abies (L.) Karst.) trees indicate the potential for
large differences in physiological response to O3 among trees
of different ages and sizes (Wieser 1997). The magnitude of
tree responses to O3 depends on several factors, including exposure regime (Wieser et al. 1998), site conditions that influence stomatal aperture (Maurer et al. 1997, Kronfuß et al.
1998) and hence O3 influx (i.e., O3 uptake rate, defined as external O3 concentration times stomatal conductance) and cumulative O3 uptake (i.e., integrated flux during the exposure
period; Wieser et al. 2000), biochemical defense mechanisms
(Polle 1998) that vary with tree age, size (Kolb et al. 1997) and
genotype (Taylor 1994).
Ozone uptake is commonly estimated from assessments of
gas exchange. Differences in O3 sensitivity of trees differing in
age have been attributed to ontogenetic changes in stomatal
conductance (Kolb et al. 1997). However, differences in
stomatal conductance can only partially explain age-related
differences in O3 susceptibility. There is evidence linking O3
sensitivity to ontogenetic trends in leaf morphology (Pääkkönen et al. 1995, 1997, Ferdinand et al. 2000) and biochemistry (Taylor and Hanson 1992, Wellburn and Wellburn 1996).
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WIESER, TEGISCHER, TAUSZ, HÄBERLE, GRAMS AND MATYSSEK
To improve our ability to scale data from O3 sensitivity studies on seedlings to mature trees, we need information on O3
dose–response relationships in forest trees of different ages
and sizes. With the exception of studies by Baumgarten et al.
(2000) on beech (Fagus sylvatica L.) trees, no O3 uptake
thresholds for performance effects on either young or old trees
have been determined. Moreover, little information on age-related differences in needle morphology and O3 defense is
available. Our objectives were to: establish sensitivity thresholds for O3 influx and cumulative O3 uptake in mature Norway
spruce forest trees and seedlings; determine tree-age related
differences in needle morphology and antioxidative defense
capacity; and link stress avoidance (exclusion of O3 uptake)
and tolerance (detoxification) to O3 sensitivity in a conceptual
framework.
Materials and methods
Experimental design and ozone treatment
Calculation of ozone uptake
In field experiments, continuous measurements of microclimatic factors, O3 concentration and leaf gas exchange were
used to calculate O3 flux into needles according to Fick’s first
law:
FO3 = (Oa − Oi ) gO3 ,
(1)
where FO (nmol m –2 s –1) is flux or uptake rate of O3 into the
needles; Oa and Oi are molar concentrations of O3 (nl l –1) outside and inside the needles, respectively, and gO is stomatal
conductance for O3 (mmol m –2 s –1). We calculated gO by multiplying stomatal conductance of water vapor (gw) by 0.613,
i.e., the ratio of the diffusivities of water vapor and O3 (Nobel
1983), neglecting O3 flux through cuticles (cf. Kerstiens and
Lendzian 1989). The Oa approaches zero (Laisk et al. 1989,
Moldau et al. 1990) and the mesophyll resistance to O3 is typically small (cf. Runeckles 1992), indicating that O3 influx is
governed by both gO and Oa.
Stomatal conductance for water vapor (gw) in seedlings was
measured every second day with a portable gas exchange system (LCA3, ADC, Hoddesdon, U.K.) inside the growth chambers. To minimize disturbance of chamber conditions, these
measurements were performed twice a day (1000 to 1100 h
and 2200 to 2300 h). Occasional measurements of daily
courses showed that stomatal performance was representative
for light and dark periods and suitable for calculating mean
daily O3 uptake (Kronfuß et al. 1998, Wieser et al. 1998). Cumulative O3 uptake (regarded as the internal physiologically
effective O3 dose; mmol m –2) was calculated as the O3 flux integrated across the exposure period.
3
3
3
3
To characterize effects of long-term exposure to O3 on photosynthetic performance of current-year needles of Norway
spruce trees, we compared experimental data obtained from
seven O3 fumigation experiments with mature trees and seedlings (Table 1). Two exposure facilities were used, twig chambers and growth chambers. In field experiments, twigs from
the upper crown of 60- to 65-year-old trees were sealed in
transparent fumigation cuvettes (Havranek and Wieser 1990,
1994) throughout one or two growing seasons between 1986
and 1990. The O3 exposure regimes were charcoal-filtered air,
ambient air, and up to threefold ambient O3 concentrations
(Table 1). The system provided control of ambient climatic
conditions and O3 concentrations, and tracked their diurnal
and seasonal fluctuations inside the cuvettes, in addition to
controlling several replicates per treatment in parallel. In 1994
and 1995, 4- and 5-year-old seedlings were placed in growthchambers (Kronfuß et al. 1998) and exposed continuously
(day and night) to either charcoal-filtered air (control), or a
constant concentration of O3 (100 nl l –1) (1994), or O3 concentrations that increased weekly in steps of 25 nl l –1 from 0 to
100 nl l –1 (1995) (Table 1).
Ozone effects on photosynthesis
Photosynthetic capacity of needles in ambient CO2 concentration (i.e., maximum photosynthetic rate, Amax; Larcher 1994)
was measured in situ with a thermoelectrically climate-controlled chamber (Walz, Effeltrich, Germany) under standard
conditions (20 °C needle temperature, 9.2 Pa kPa –1 leaf to air
vapor pressure difference, 350 µmol mol –1 CO2 concentration,
2000 µmol m –2 s –1 photon flux density (artificial light source;
Table 1. Summary of the ozone response studies.
Year
Tree type
Experiment
Mean O3 concentration (nl l –1)
Reference
1986
1987
1988
1989
1990
1994
1995
Mature tree
Mature tree
Mature tree
Mature tree
Mature tree
Seedling
Seedling
Twig chamber
Twig chamber
Twig chamber
Twig chamber
Twig chamber
Growth chamber
Growth chamber
0 1, 65 2, 120 3
0 1, 64 2, 100 3
0 1, 47 2, 77 3, 107 3, 137 3
0 1, 37 2, 67 3, 97 3, 127 3
0 1, 30 2, 55 3, 90 3, 115 3
0 1, 100 4
0 1, 34 4, 75 4,100 4
Wieser and Havranek 1996
Wieser and Havranek 1996
Wieser and Havranek 1996
Wieser and Havranek 1996
Wieser and Havranek 1996
Kronfuß et al. 1998
Wieser et al. 1998
1
2
3
4
Charcoal-filtered air.
Ambient air.
Tracking ambient O3 concentration.
Constant (day and night) O3 concentration.
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ONTOGENY AND OZONE TOLERANCE IN NORWAY SPRUCE
Halo Star KLR 51, Osram, Germany)). Measurements were
made repeatedly throughout and at the end of each fumigation
period. All gas exchange parameters were calculated according to von Caemmerer and Farquhar (1981) and expressed on
the basis of total needle surface area, which was determined by
the glass-bead technique (Thompson and Leyton 1971).
At each measurement date, differences in Amax between O3treated plants and controls in charcoal-filtered air were examined by the Student’s t-test at P < 0.05. To account for the effects of age (Yoder et al. 1994), season (Kronfuß et al. 1998)
and year-to-year variation (Wieser and Havranek 1996) on
Amax and O3 exposure, photosynthetic rates obtained at each
date were expressed as percentages relative to the respective
rates of control twigs grown in charcoal-filtered air.
Data from the seedling and tree experiments were pooled to
determine dose–response functions for seedlings and mature
trees. The two data sets were then combined to determine incipient effects of cumulative O3 uptake on Amax of mature trees
and seedlings by boundary-line analysis. Because cumulative
O3 uptake is an integrated measure expressing the quantity but
not the time span of O3 exposure, we also determined O3 uptake rate on a short timescale.
585
concentrations of ascorbate allowed us to define O3 sensitivity
as the equilibrium between needle O3 uptake and detoxification processes.
Finally, dry mass of 100-needle samples and specific leaf
area (SLA; cm 2 leaf area per g dry weight) were determined
from twigs adjacent to twigs used for the biochemical analyses. Measurements of needle water potential, net photosynthesis and stomatal conductance in trees of different ages performed immediately before harvest indicated that there were
no significant age-related differences at either the Mt. Patscherkofel or the Kranzberg site. Means were –1.3 ± 0.15 MPa
for needle water potential, 1.2 ± 0.5 µmol CO2 m –2 s –1 for net
photosynthesis and 22.9 ± 3.1 mmol H2O m –2 s –1 for stomatal
conductance for trees on Mt. Patscherkofel. Corresponding
values for trees at the Kranzberg site were –1.6 ± 0.10 MPa,
3.2 ± 0.2 µmol CO2 m –2 s –1 and 57.7 ± 7.5 mmol H2O m –2 s –1,
respectively. Mean predawn needle water potentials assessed
in trees one day after harvest (0400–0500 h) were –0.7 ±
0.15 MPa at Mt. Patscherkofel and –1.2 ± 0.21 MPa at the
Kranzberg site.
Results and discussion
Biochemical analysis and morphological characteristics
We determined antioxidant concentrations in needles from 8to 60-year-old field-grown Norway spruce trees. All study
trees grew in a gap in a forest near the Austrian Timberline Research Station Klimahaus on Mt. Patscherkofel (1950 m a.s.l.,
47° N, 11° E), Innsbruck, Austria. Samples were taken from
exposed, southwest-facing branches in similar light conditions
(G. Wieser, unpublished data). In 1998, to account for microclimatic influences within a stand on needle antioxidant concentrations, we planted 4-year-old Norway spruce seedlings in
70-l containers (20 seedlings per container) filled with natural
forest soil. In spring 1999, five containers were transferred to
the upper sun crown (20 m high) of a 50-year-old Norway
spruce stand at the free-air ozone fumigation plot at Kranzberg
Forest (485 m a.s.l., 48° N, 11° E) near Munich, Germany (cf.
Pretsch et al. 1998, Häberle et al. 1999). The potted seedlings
were irrigated regularly throughout the growing season.
In September 1999, current-year needles from both mature
trees and seedlings were sampled under uniform light conditions (1200 to 1430 h, mean PPFD of 1500 µmol m –2 s –1) at
both study sites from five trees per age class (one seeding per
container). Needles were removed from the twigs, immediately frozen in liquid nitrogen and stored at –80 °C. The material was lyophilized, ground in a microdismembrator (Braun,
Melsungen, Germany) and stored in plastic vials at –80 °C until analyzed.
Reduced ascorbate and dehydroascorbate were quantified in
meta-phosphoric acid extracts after derivatization with
o-phenylenediamine. An isocratic reversed-phase chromatographic method using an ion-pairing reagent was employed
(Tausz et al. 1996). Ascorbate concentrations were expressed
on a needle dry mass basis, determined after 72 h at 80 °C and
on a total needle surface area, estimated as described by
Thompson and Leyton (1971). Determination of area-based
Quantification of tree response to cumulative O3 uptake and
O3 flux
In the field experiments with mature trees, O3 treatments
tracked ambient fluctuations, whereas seedlings in the chambers were exposed to a constant concentration of O3. Although
trees and seedlings were exposed to different O3 regimes, only
mean O3 concentrations above 100 nl l –1 induced a statistically
significant decline in Amax, by 20–40% in current-year needles
of mature trees (Wieser and Havranek 1996, Matyssek et al.
1997) and 20–30% in seedlings (Kronfuß et al.1998, Wieser et
al. 1998). The basic assumption underlying the use of branch
chambers on trees is that branches export but do not import
carbon during the growing season (Sprugel et al. 1991). It is
unclear, however, whether detoxification in individual
branches is supported by substrates translocated from other
tree parts (Matyssek et al. 1997). Nevertheless, dose–response
functions demonstrated seedlings were more sensitive to O3
than mature trees (Figure 1).
Thresholds based on cumulative O3 uptake can be derived
from the plot shown in Figure 2A for Amax. The boundary line
represents the threshold below which the combination of exposure period and cumulative O3 uptake did not cause statistically significant reductions in Amax relative to controls in O3free air. In mature trees, there was a significant reduction in
Amax when cumulative O3 uptake exceeded 21 mmol m –2 per
unit of total needle surface area (Figure 2A), which occurred
after 70 days of exposure to mean O3 concentrations persistently higher than 100 nl l –1. The derived threshold O3 uptake
was about 45% higher than that determined over one growing
season for evergreen conifers growing in ambient O3 in the
central European Alps (11.4 ± 1.7 to 14 mol m –2; Wieser et al.
2000). There was a comparable decline in Amax of seedlings
when cumulative O3 uptake was only 4.5 mmol m –2 (Fig-
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586
WIESER, TEGISCHER, TAUSZ, HÄBERLE, GRAMS AND MATYSSEK
Figure 1. Percent reduction in photosynthetic capacity at ambient CO2
concentration (Amax) of Norway spruce mature trees (䊉, 䊊) and seedlings (䊏, 䊐) after 11 to 253 days of exposure to mean O3 concentrations ranging from 0 to 137 nl l –1. Each value represents the mean of
4–20 twigs. Values were fitted by linear regression: mature trees: y =
–0.52x + 0.96, r 2 = 0.42; seedlings: y = –1.32x – 5.50, r 2 = 0.56. Open
symbols: not significantly different from O3-free controls (P > 0.05);
solid symbols: mean O3 concentrations eliciting significant reductions (P < 0.05) were as follows: mature trees (1) 137 nl l –1, (2) 115 nl
l –1, (3) 127 nl l –1, (4) 121 nl l –1, (5) 132 nl l –1, and seedlings: (6)
100 nl l –1 (modified from Wieser 1997).
ure 2A), which occurred after 23 days of exposure to an O3
concentration of 100 nl l –1.
Cumulative O3 uptake expresses the quantity rather than the
quality of exposure so that the delayed response to O3 in mature trees might be a result of age and size-related constraints
on stomatal conductance (Kolb et al. 1997). However, we
found that stomatal conductance for O3 was similar in mature
trees and seedlings (22.5 ± 2.1 and 23.1 ± 1.65 mmol O3 m –2
s –1, respectively). Therefore, we determined O3 flux, a measure of O3 exposure over a short timescale. Although the study
plants differed by O3 exposure, genotype and environmental
conditions, the threshold flux for inducing a significant reduction in Amax ranged between 2.14 to 2.45 nmol m 2 s –1 for both
mature trees and seedlings (Figure 2B). Again, the threshold
O3 flux exceeded the values determined in the field for mature
conifers (0.5 to 0.85 nmol m –2 s –1; Wieser et al. 2000). The
finding of a similar threshold O3 flux in mature Norway spruce
trees and seedlings in chambers (Figure 2B) is consistent with
studies on beech (Baumgarten et al. 2000). Based on a modeling approach, Emberson et al. (1998, 2000) determined that a
cumulative O3 uptake of 3.1 and 3.0 mmol m –2 was required to
induce visible leaf injury in mature beech trees and seedlings
in chambers, respectively. In both studies, the mature forest
trees exhibited a significant delay in O3 uptake (Figure 2).
The role of ontogeny in tree responses to O3 is unclear because of the shortcomings in scaling O3 effects from seedlings
in chambers to stands of mature trees in the field (Kelly et al.
1995, Hogsett and Andersen 1998, Kolb and Matyssek 2001).
Although the threshold values were similar in mature trees and
seedlings, the temporal variation in O3 flux differed. At night,
Figure 2. Boundary line analysis characterizing the relationship between minimum ozone exposure and incipient effects on photosynthetic capacity at ambient CO2 concentration (Amax) of Norway spruce
mature trees (䊉, 䊊) and seedlings (䊏, 䊐): (A) exposure time versus
cumulative O3 uptake eliciting significant reductions in Amax when
compared with O3-free controls (P < 0.05); (B) exposure time versus
average O3 flux eliciting significant reductions in Amax when compared with O3-free controls (P < 0.05). The boundary line defines the
threshold below which the combination of exposure time and cumulative O3 uptake and O3 flux, respectively, causes no statistically significant reduction in Amax (P > 0.05). Open and solid symbols, as well as
mean O3 concentrations of the numbered points are as indicated in
Figure 1. The boundary line in panel A was fit by linear regression: y =
0.25x – 1.42; r 2 = 0.96. Note that after 106 days of exposure the cumulative O3 uptake value eliciting significant reductions in Amax was
22.4 mmol m –2 in both mature trees and seedlings (* in panel A).
O3 flux into needles was reduced to 75 and 25% of daytime
values in seedlings (Kronfuß et al. 1998) and mature Norway
spruce trees (Wieser and Havranek 1993), respectively, as a result of high nighttime concentrations of O3 in mountainous regions (Wieser and Havranek 1995). Nocturnal O3 uptake may
promote susceptibility (cf. Musselman and Minnick 2000),
resulting in leaf injury in Betula pendula Roth., Populus × euramericana Dode. and Alnus glutinosa L. (Günthardt-Goerg et
al. 1997). Carbon allocation and plant productivity data also
indicate that birch is more sensitive to O3 at night than during
the day (Matyssek et al. 1995). This is probably because the
detoxification system requires photosynthesis to maintain antioxidants in a reduced (active) state (Esterbauer et al. 1980,
Schupp and Rennenberg 1988, Wieser et al. 1996, Wildi and
Lütz 1996). In addition, ontogenetic differences in O3 sensitivity might relate to contrasting allocation patterns. Young trees
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ONTOGENY AND OZONE TOLERANCE IN NORWAY SPRUCE
587
typically have a high proportion of total biomass in foliage and
thus a large surface area vulnerable to O3 attack. On the other
hand, the high proportion of non-green biomass in mature
trees may result in enhanced maintenance respiration. Such
age-dependent carbon demands may conflict with increases in
respiration induced by O3 defense (Matyssek and Innes 1999).
Net photosynthesis declined with increasing age in our trees
(data not shown), as has been observed in Pinus contorta var.
latifolia Engelm. and Pinus ponderosa Laws. (Yoder et al.
1994), indicating that, compared with mature trees, young
trees are more susceptible to O3 stress because of their high
metabolic activity (cf. Laurence et al. 1994). In the long term,
chronic O3 exposure may weaken stress tolerance because of
the persistently enhanced energy costs for defense (Langebartels et al. 1997).
Morphological, physiological and biochemical differences
among trees of different age
Tree responses to cumulative O3 uptake and O3 flux are linked
with morphological (Fredericksen et al. 1995, Ferdinand et al.
2000) as well as physiological and biochemical traits of leaves
(Taylor and Hanson 1992, Wellburn and Wellburn 1996). We
found a significant effect of tree age on morphology of current-year needles. Seedlings had thinner needles than mature
trees. In trees at the timberline, dry mass of 100-needle samples increased significantly with increasing tree age, whereas
specific leaf area (SLA) declined with tree age (Figure 3). This
trend was confirmed at low elevation (Kranzberg), where container-grown seedlings were placed in the sun crowns of mature trees. In the same light environment, needles of seedlings
had lower mass, but higher SLA, than needles of mature trees
(Figure 3). Needle differentiation during tree ontogeny resembled a change from shade-type toward a sun-type foliage, being consistent with findings by Perterer and Körner (1990) on
sun and shade needles in Norway spruce, and may account for
age-related differences in O3 sensitivity. Thin leaves, but not
thick leaves, of Betula pendula (Pääkkönen et al. 1995), Betula pubescens J. H. Ehrh. (Pääkkönen et al. 1997), Fraxinus
pennsylvanica Marsh. and Prunus serotina J. F. Ehrh. (Bennet
et al. 1992; but see Ferdinand et al. 2000) are O3-sensitive,
probably because of differences in the intercellular air space
(Evans and Miller 1972, Pääkkönen et al. 1995, 1997) and in
the diffusion pathway (Chappelka and Samuelson 1998) between thick and thin leaves. Data linking needle anatomy to
gas diffusion for Norway spruce trees of different ages or light
exposures are unavailable; however, we found no evidence
that differences in morphology between sun and shade needles
influenced detoxification capacity. There were no significant
differences in mass-based concentrations of needle ascorbate
among Norway spruce trees of different age (Figure 4A), and
the redox state of the ascorbate pool (reduced ascorbate expressed as % of reduced ascorbate + dehydroascorbate) was
about 70 ± 10% irrespective of tree age.
Relating avoidance to tolerance of O3 stress
Injury results from an imbalance between O3 uptake and detoxification (cf. Massman et al. 2000), the latter being deter-
Figure 3. Dry mass of 100-needle samples (A) and specific leaf area
(SLA; B) of current-year Norway Spruce needles in relation to tree
age at Mt. Patscherkofel (䊉) and in Kranzberg (䊊). Means ± SD; n =
5. Values were fitted by linear regression: 100-needle dry weight: y =
5.31x + 135.4, r 2 = 0.69; specific leaf area: y = –1.41x + 151.7, r 2 =
0.67.
mined by resource availability in the tree (Matyssek and Innes
1999). Although we did not measure detoxification rates directly in Norway spruce, we propose a measure that relates O3
flux to the antioxidant pool in the needles. As a preliminary
approach, we considered the area-based concentration of reduced needle ascorbate (ASCleaf) a central component in detoxification:
ASC leaf = ASC dwSLA − 1,
(2)
(ASCdw = mass-based concentration of ascorbate), because
ASCleaf (Figure 4B), but not ASCdw (Figure 4A), increased
with tree age. Use of needle surface area as the basis of expression of ascorbate concentration is appropriate because detoxification has to cope with the area-related flux density of O3
uptake. Hence, the potentially available amount of reduced
ascorbate in needles (AAASC, nmol ASCleaf nmol O3 –1 s –1) per
unit of O3 flux into needles can be expressed as:
AA ASC = ASC leaf / FO3.
(3)
This ratio may form an empirical basis for comparing agerelated differences in tree response to O3. Because significant
effects on photosynthesis occurred above an O3 flux threshold
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WIESER, TEGISCHER, TAUSZ, HÄBERLE, GRAMS AND MATYSSEK
Figure 5. The amount of potentially available reduced needle ascorbate per unit of O3 taken up at the threshold flux of 2.45 nmol m –2 s –1
eliciting significant effects on the photosynthetic capacity at ambient
CO2 concentration (Amax) in relation to tree age at Mt. Patscherkofel
(䊉) and in Kranzberg (䊊). Means ± SD; n = 5, except n = 1 in
50-year-old trees at Kranzberg. Values were fitted by linear regression: y = 3.28x + 416.6, r 2 = 0.57.
Figure 4. Reduced needle ascorbate concentration expressed on a
needle dry mass basis (A) and a total needle surface area basis (B) for
current-year Norway spruce needles in relation to tree age at Mt. Patscherkofel (䊉) and at Kranzberg (䊊). Means ± SD; n = 5, except n = 1
in 50-year-old trees at Kranzberg. Values were fitted by polynomial
and linear regression: dry weight basis: y = (0.007x – 0.54)(x + 19.8),
r 2 = 0.59; total needle surface area basis: y = 8.04x + 1020.6, r 2 = 0.57.
of 2.45 nmol m2 s –1 in both mature trees and seedlings (Figure
2B), the amount of reduced ascorbate in needles available under conditions of critical O3 uptake tended to increase with tree
age (Figure 5). A similar trend was observed for glutathione
(data not shown). These trends are consistent with higher O3
susceptibility in young trees than in mature trees. It is important to emphasize, however, that AAASC cannot reflect O3 effects on the antioxidant pool. There is evidence that, in Norway spruce seedlings, ascorbate concentrations increase
during continued O3 exposure and uptake (Kronfuß et al.
1998, Wieser et al. 1998). We determined that the apoplastic
ascorbate pool, which is depleted rapidly in response to O3
(Polle 1998, Polle et al. 2000), was no better as an indicator of
antioxidative capacity than the total ascorbate pool for two
reasons. First, only 5–10% of apoplastic ascorbate interacted
with the O3 taken up in Norway spruce needles (Polle et al.
1995). Second, it is known that ascorbate regeneration cannot
occur in the apoplast, but requires reentry of dehydroascorbate
into the cytoplasm where it is reduced through the ascorbate–glutathione cycle (Noctor and Foyer 1998). These studies together with the observation that O3 in plant cells promotes reactive oxygen species production, possibly depleting
the detoxification systems in chloroplasts (Sakaki 1998), led
us to conclude that use of the total leaf ascorbate pool to estimate overall detoxification capacity is justified. If Figure 5
reflects age-related control of tree responses to O3 through
both stomata-dependent regulation of O3 influx (stress avoidance) and antioxidant pools (stress tolerance), then determination of area-based antioxidative capacity is as important as
flux-related assessments of O3 doses in developing air quality
standards.
In conclusion, antioxidative capacity may provide a convenient means of assessing age-related differences in O3 susceptibility. This age-dependent relationship now needs to be
examined in mechanistic terms by direct experimental comparisons of antioxidant concentrations, leaf morphology, O3
uptake and physiological leaf responses across several ontogenetic stages. One approach may be to place seedlings in the
canopy of older trees of the same genotype (Kolb and Matyssek 2001, Samuelson and Kelly 2001), thereby ensuring similar exposure. The “free air” O3 canopy exposure system (Karnosky et al. 2001) that has recently begun operation at the
Kranzberg site can be used to validate our proposed approach
(Häberle et al. 1999, Werner and Fabian 2001).
Acknowledgments
The study was supported in part by the Deutsche Forschungsgemeinschaft (DFG) through the Sonderforschungsbereich 607 Growth and
Parasite Defense—Competition for Resources in Economic Plants
from Agronomy and Forestry and by Interreg II.
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