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Environmental Pollution 109 (2000) 501507

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Beech (Fagus sylvatica) response to ozone exposure assessed with a


chlorophyll a uorescence performance index
A.J. Clark a,b, W. Landolt a, J.B. Bucher a, R.J. Strasser b,*
a

Swiss Federal Institute for Forest, Snow and Landscape Research, CH-8903 Birmensdorf, Switzerland
b
Bioenergetics Laboratory, University of Geneva, CH-1254 Jussy Geneva, Switzerland
Received 30 June 1999; accepted 5 January 2000

``Capsule'': A chlorophyll a performance index obtained from uorescence analysis correlates to visual injury and biomass
loss caused by ozone, and thus could be used for the large-scale monitoring of tree vitality.
Abstract
This paper describes a relationship between ozone exposure, biomass, visual symptoms and a chlorophyll a uorescence performance index for young beech trees (Fagus sylvatica). The plants were exposed to four levels of ozone in open-top fumigation
chambers (50, 85, 100% of ambient, and 50% of ambient+30 nl l1 ozone) that uctuated in parallel with ambient ozone during a
single growing season. The trees were fumigated in the four treatments with ozone levels corresponding to an AOT40 (accumulated
exposure above a threshold of 40 nl l1) of 0.01, 3.35, 7.06 and 19.70 ml l1 h, respectively. Highly signicant dierences were found
between the 50% of ambient+30 nl l1 ozone treatment and all other treatments, with a 70.5% reduction in primary photosynthetic performance, as measured with the PI index. The reduction of the PI values demonstrated a high correlation with visual
symptom development (r2=0.98), and by the end of September with biomass loss (r2=0.99). A signicant ozone exposureresponse
relationship was found between AOT40 and primary photochemistry (r2=0.97). Thus, analysis of PI provides an alternative
method for regional monitoring of tree health within the context of the currently employed AOT40. # 2000 Elsevier Science Ltd.
All rights reserved.
Keywords: Fluorescence; Ozone; Biomass; Performance index; Visual symptoms

1. Introduction
Current ozone levels remain a risk to European forests (Skarby et al., 1998). In the determination of an
appropriate ozone critical level, a two-tier approach was
suggested at the 1996 workshop under the UN/ECE
convention on Long-Range Transboundary Air Pollution for abatement strategies to protect vegetation from
ozone injuries. The Level I approach prescribed biomass
loss to be the appropriate biological response parameter
and that beech would be the most appropriate species to
monitor (Karenlampi and Skarby, 1996; Fuhrer et al.,
1997). The Level II approach proposed several additional environmental parameters to also be included due
to their modifying inuence on tree ozone responses. It
was also stated that there was a scarcity of data upon
* Corresponding author. Tel.: +41-22-7591944; fax: +41-227591945.
E-mail address: strasser@uni2a.unige.ch (R.J. Strasser).

which to base an ozone doseresponse function, especially for Level II.


An important goal is the mapping of a physiologically
eective ozone concentration in order to monitor and
protect the European forests. An objective and simple
screening method that is sensitive and suitable for both
Level I and Level II assessments is required. Currently,
visual identication of ozone damage to leaves and
needles is the only simple method of diagnosis, which
often requires specialised knowledge because the environment and other stresses can modify the patterns of
ozone injury. The lack of suitable monitoring techniques makes regional mapping of actual ozone-induced
forest stress virtually impossible. A promising approach
could be the use of chlorophyll (Chl) a uorimetry
which can provide large amounts of accurate data with
a minimum of expertise and time and without injury to
the plants. The techniques could be cost eective,
objective, and the mapping of plant physiological condition a possibility.

0269-7491/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0269-7491(00)00053-1

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A.J. Clark et al. / Environmental Pollution 109 (2000) 501507

Photosynthesis is a core function in the physiology of


all plants. Its functional state has been considered an
ideal physiological activity to monitor when the health
and vitality of plants is under scrutiny. Measurements
of photosynthesis have often been used in the assessment of ozone injury (Heath, 1996), and although the
actual mechanisms by which ozone exposure alters
photosynthesis are not yet known, it is a sensitive diagnostic parameter (Saxe, 1991). The oxidative byproducts from reactions occurring at cell membranes
are believed to restrict carbon assimilation (Dann and
Pell, 1989; Reddy et al., 1993; Heath, 1996). Consequently, a change in the energy capture process around
photosystem II (PSII) and its concurrent uorescence
emissions occur (Farage et al., 1991; Mikkelsen, 1995).
It is unlikely that PSII is directly aected by ozone,
except possibly at very high concentrations. Chl a
uorescence emissions are, however, known to be sensitive to many of the environmental parameters chosen in
the Level II approach as well as ozone (Lichtenthaler
and Rinderle, 1988; Larcher, 1995; Mikkelsen, 1995;
Strasser and Strasser, 1995).
The aim of this paper was to test the sensitivity of a
newly described Chl a uorescence performance index
for plant screening (Strasser et al., 1999), and to analyse
the response of the performance index in relation to
biomass and visual symptom development for beech
trees growing in open-top chambers (OTCs).
2. Materials and methods
2.1. Plant material and ozone treatments
The experiment was performed in an OTC facility at
the Swiss Federal Institute for Forest, Snow and Landscaped Research (WSL), Birmensdorf, Switzerland. A
total of 20 chambers were divided between four ozone
treatments that were dependent upon prevalent ambient
levels of ozone in the 1997 growing season between the
beginning of April and the end of September (Table 1).
Ozone was produced by electrical discharge from pure
oxygen by an ozone generator (Model 502, Fischer,
Germany), and concentrations were measured with an

ozone monitor (Model 8810, Monitor Labs, USA).


Ozone concentrations within the chambers were regulated to oscillate in parallel with ambient ozone concentrations. At high ambient light levels (in excess of
600 mmol m2 s1) partial shading was automatically
provided for the plants with the use of a translucent
shading canopy which covered all chambers to maintain
the chamber temperatures within an optimal range.
Fagus sylvatica from two Swiss provenances, Hirschthal and Aarburg, were used in the experiment. All
saplings were potted in a standard soil mixture (50%
sandy soil, 25% brown earth, 25% peat, 2 g l1 fertiliser
(Osmocote plus) that was kept moist throughout the
experiment. There was a single sapling from each provenance in each chamber, with ve replicate chambers
within each treatment. Visual symptom development
was assessed each week from two leaves per tree per
chamber in accordance with Gunthardt-Goerg et al.
(1993). The biomass data were calculated from sets of
approximately 150 seedlings grown in the same OTCs,
as described by Landolt et al. (2000). Parallel uorescence measurements were taken from both the seedlings
and saplings.
2.2. Chl a uorescence measurements
Chl a uorescence transients were measured at ambient temperatures within the chambers using a portable
uorimeter (Model PEA, Hansatech Inst., UK). All fast
uorescence transients were recorded up to 2 s with 12bit resolution and a data acquisition rate of 10 ms in the
time span from 10 ms to 2 ms, while after 2 ms and 1 s
the instrument automatically switches to slower digitisation rates (for details see Strasser et al., 1995). The
uorescence emission was induced by an homogenous
illumination on a 4-mm-diameter area of the leaf samples by red light (peak at 650 nm) of 3000 mmol m2 s1
provided by an array of six light-emitting diodes. Chl a
uorescence transients (1600) were recorded between
23:00 and 03:00 from leaves that had been in the dark
for a minimum of 3 h to attain a fully dark-adapted
state of all samples. Four primary uorescence values
parameters were retained. The uorescence intensities at
50, 300 ms, and 2 ms, were denoted as F50 ms, F300 ms, and

Table 1
The four ozone exposure treatments (IIV) used in the fumigation of beech trees in open-top chambers, their derivations in relation to the ambient
level of ozone, and measured ozone concentrations and cumulative ozone exposure (AOT40) from April to September 1997
Treatment

I
II
III
IV
a

Treatment type

Pre-industrial level
Reduced level
Level at low altitudes (400700 m a.s.l.)
Level at high altitudes (16001800 m a.s.l.)
S.D., 1 standard deviation.

Ozone level relative


to ambient air

Ozone concentration (nl l1)


Mean

S.D.a

Minimum

Maximum

50%
85%
100%
50%+30 nl l1

15.3
23.5
27.4
44.0

8.2
12.6
15.1
13.0

0.5
1.0
0.9
3.3

48.7
79.9
102.6
93.5

AOT40
(ml l1 h)
0.01
3.35
7.06
19.70

A.J. Clark et al. / Environmental Pollution 109 (2000) 501507

F2 ms respectively, and FM is the maximum uorescence


intensity. These values were used to calculate the performance index, denoted as PI (Clark et al., 1999;
Strasser et al., 1999) which has been dened as the ratio
of two recently described StructureFunction-Indexes
(SFI). The rst, SFIP (Tsimilli-Michael et al., 1998)
responds to structural and functional PSII events leading to electron transport within photosynthesis. The
second, SFIN (Strasser et al., 1999), refers to the energy
that is dissipated or lost from photosynthetic electron
transport. It has been dened as:
SFIP ChlRC =Chltot  'Po  0

SFIN 1 ChlRC =Chltot


 1 'Po  1 0

SFIP
ChlRC =Chltot
'Po
0



SFIN 1 ChlRC =Chltot 1 'Po 1 0
ChlRC
'Po
0



Chlantenna 1 'Po 1 0

PI

503

The index 0 refers to the state at the onset of illumination. The PI dened here refers to photochemical
events, an analogous expression denoted PIN can be
dened for non-photochemical events where:
PIN 1=PI:

2.3. Statistical analysis


The distribution of all Chl a uorescence data was
tested for non-normality and heterocedasticity to ensure
the assumptions were met for parametric statistics. The
treatment eect was tested with a univariate analysis of
variance (ANOVA), designed as a mixed model threestage nested using a type III sum of squares calculation.
The highest level of the model was the treatment level
with ve chambers nested in each treatment, and subgroups of provenances and trees, with subsubgroups of
leaves within trees. Tests of signicance were made at
the 95% condence level using a Tukey test. The SAS
system for windows (Release 6.12, SAS Institute Inc.,
NC, USA) was used for all statistical analyses.

or in experimental terms:

3. Results

RC FV 1 VJ


;
PI
VJ
ABS F0
where Chltot is the total quantity of Chl a, and
Chltot Chlantenna ChlRC . The ratio ChlRC/Chlantenna
can be replaced by the ratio RC/ABS, where RC is the
number of active PSII reaction centres, and ABS is
the quantity of light absorbed by the antenna. RC/ABS,
'Po and 0 can be calculated according to the JIP-test
using the experimentally collected parameters (Strasser
and Strasser, 1995; Strasser et al., 1995).
Maximum quantum yield of primary photochemistry:
'Po 1 F0 =FM TR0 =ABS

Density of reaction centres per chlorophyll:


RC=ABS 'Po  VJ =M0 ;

where VJ is the relative variable uorescence at 2 ms of


the uorescence rise, VJ F2m s F0 =FM F0 , and
M0 is the slope at the origin of the relative variable
uorescence M0 F300 s F0 =FM F0 . The value
at 50 ms was used for F0.
The probability of 0 that an electron is transported
(ET) beyond Q
A is:
0 1 VJ ET0 =TR0 :

The ozone fumigation resulted in cumulative exposures (AOT40) of 0.01, 3.35, 7.06 and 19.7 ml l1 h for
the treatments IIV, respectively. AOT40 was calculated
for daylight hours (>50 Wm2) using 1-h mean concentrations in excess of 40 nl l1 ozone, in accordance
with the UN/ECE denition (Karenlampi and Skarby,
1996) (Table 1). Daily levels of hourly mean ozone
concentration in the 50% of ambient+50 nl l1 were
representative of those found at higher altitudes in the
Swiss Alps.
The repeated measures ANOVA calculated the eect
of ozone on the PI between the 50% ambient+50 nl l1
treatment and the other three treatments to be highly
signicant over the entire season (P>0.0026) (Table 2).
The PI values recorded from all plants remained indistinguishable until Day of Year 190, at which time the
50% ambient+30 nl l1 ozone treatment PI values
became increasingly reduced over time compared to the
three other treatments (Fig. 1). The treatment PI means
became signicantly dierent by the fourth measuring
event (P>0.009) (Day of Year 226), and remained so
for the rest of the season. By September, when the
plants had received close to the complete seasons exposure, mean PI was reduced by an average of 70.5, 15.3,
and 1% relative to the control (50% of ambient level).
The development of visual damage was scored from 1,
no visual symptom, to 5, large necrotic areas. The rst
visual symptoms developed at about the same time
as the mean PI value for trees in the 50% of ambient+
30 nl l1 treatment started to diverge from the other

504

A.J. Clark et al. / Environmental Pollution 109 (2000) 501507

Table 2
The results of a repeated measures ANOVA, testing the between and within treatment eects of four ozone treatments on beech trees from two
provenances using the primary photosynthetic performance index (PI) are showna
df
Between-treatment eects
Treatment (error = *)
Provenance (error = **)
Treatmentprovenance**
*Replication (Treatment) (error = **)
Tree (Treatmentreplication rovenance)
**Leaf
Within-treatment effects
Day of Year (DY)
DYtreatment
DYprovenance
DYtreatmentprovenance
DYreplication (Treatment)
DYtree (Treatmentreplicationprovenance)
Leaf (DY) error
Huynh-Feldt "

Mean square

P>F

3
1
3
15
15
150

21.586
0.106
1.041
2.855
2.53
0.082

0.0026
0.2577
0.0001
0.0001
0.0001

7
21
7
21
105
105
1050

21.223
2.187
0.759
0.166
0.44
0.372
0.086

0.0001
0.0001
0.0001
0.0069
0.0001
0.0001

1.0947

Replications refer to ve open-top chambers within each treatment. The degrees of freedom (df) and the mean square data are shown from
which the F-test probabilities were calculated (P>F ). Asterisks indicate the specic error terms used at each level of the model.

Fig. 1. Performance index (PI) through the growing season (Day of


Year). Four treatments are 50% ambient (!), 85% ambient (~),
100% ambient (*) and 50% of ambient+30 nl l1 ozone (&). Error
bars are calculated as 1 S.E.

treatments shown in Fig. 1. By Day of Year 210, some


of the plants in the 50% of ambient+30 nl l1 treatment were showing slight stippling on the leaf surfaces.
The leaf symptoms became steadily more apparent
throughout the season, until late August, when there
were large necrotic areas. The mean PIN values demonstrated highly signicant correlation with the development of leaf damage (P>0.0001) (Fig. 2), with a linear
regression r2 of 0.977. The visual symptoms can be
viewed in terms of cumulative AOT40 exposure by

identifying the PIN values in Fig. 2 and reading from the


corresponding values in Fig. 4 the AOT40 value.
At the end of the season the mean weights of the
seedlings were 1.91, 2.08, 1.81, and 1.30 g for the four
treatments, respectively, as reported in full by Landolt
et al. (2000). When compared to the control seedlings
mean biomass per treatment was equivalent to a slight,
but insignicant stimulation of 9% in the 85% of
ambient ozone, an insignicant retardation of 5% in
the ambient ozone and a highly signicant 32% reduction in the 50% ambient+30 nl l1 ozone. Seedling
weight was found to be highly correlated with mean PI
values of the last 3 days measurements in September
(Days 270, 273 and 279; Fig. 1) (Fig. 3). The signicant
regression (r2=0.966) lacks biomass data between PI
values of 0.3 and 0.8 because seedlings were harvested
and biomass measured only at the end of the season. PI
values between 0.3 and 0.8 are shown in Fig. 4 and
would have been likely to t along the regression line
were biomass data to have been collected throughout
the season. PI demonstrates an amplied sensitivity
to the ozone exposure, and a degree of data noise, which
is largely due to observed environmental dierences
between the three measuring days (data not shown).
The exposure response of beech primary photosynthesis performance is plotted in Fig. 4. PI and PIN are
identical data sets and correspond with data previously
illustrated in Figs. 13. The graph illustrates the eect
of ozone independently of the large seasonal variation;
all measuring dates were normalised by measuring
treatment dierences relative to the control. The cumulative AOT40 ozone exposure the plants received at
each successive measuring event versus the normalised

A.J. Clark et al. / Environmental Pollution 109 (2000) 501507

Fig. 2. The inverse of the mean performance index (PIN) versus mean
visual symptom score. PIN means were repeatedly measured during the
season as visual symptoms developed. Error bars are calculated as 1
S.E.

505

Fig. 4. Performance index (PI) and the inverse of the mean performance index (PIN) versus cumulative AOT40 ozone exposure. Data
points represent means repeatedly recorded during the 1997 season as
the trees exposure to the seasons cumulative ozone increased. All
ozone treatments are included. Error bars are calculated as 1 S.E.

Each point in Fig. 4 refers to the mean of 50 measured


samples collected repeatedly throughout the growing
season. For all treatments, a portion of the variance of
the means was due to the signicant interaction that the
tree provenances had with the Day of Year (Table 2).
This is likely to be an adaptation to the local environmental conditions from which the groups of trees came.
There was a highly signicant degree of variation
between the individual trees (DYTree, Table 2). This
probable genetic variation between individuals heavily
outweighed the consistency recorded between leaves on
the same tree. The stability of the error term at the leaf
level resulted in a balanced data set, indicated by the
Huynh-Feldt e. The broad variance between trees largely accounted for the broad variance apparent between
OTC replicates because the number of tree replicates
per OTC was only two.
Fig. 3. End of season mean seedling biomass plotted versus performance index (PI), data have been normalised in respect to the 50% of
ambient ozone treatment. Gridlines highlight control PI value of 1.
Error bars are calculated as 1 S.E.

performance indices exposes the underlying response of


primary photosynthesis (Fig. 4). PI and PIN can, with
reference to the Figs. 1 and 2, be used to nd the visual
symptoms induced and relative biomass changes correlated to a particular AOT40 exposure the trees received
during the 1997 growing season (Fig. 4).

4. Discussion
Exposure to ozone signicantly reduces the photosynthetic performance of beech over a single growing
season, as measured by the PI index. This response
demonstrated that the energy transduction process
around PSII lost performance, conrming previous
research using Chl a uorimetry (Mikkelsen, 1995;
Mikkelsen and Heide-Jorgensen, 1996; Clark et al.,
1998) and carbon dioxide gas exchange methods

506

A.J. Clark et al. / Environmental Pollution 109 (2000) 501507

(Matyssek et al., 1991; Ruth and Weisel, 1993; Braun


and Fluckiger, 1995; Kellomaki and Wang, 1997; Wieser, 1997; Gunthardt-Goerg et al., 1999).
Higher values of PIN, the index for non-photochemical events, were highly correlated with the progressive development of visual symptoms of ozone
damage reected the increase in symptomatic energy
dissipation through thermal losses. The divergence of
PIN in the 50% ambient+30 nl l1 ozone treatment
from the other treatments occurred at the same time as
the appearance of visual symptoms in July.
The reduction of PI in the 50% ambient+30 nl l1
ozone treatment relative to the other three treatments
resulted in a reproducible correlation with the biomass
of seedlings at the end of the season. Biomass increment
and photosynthetic performance are likely to be constrained by similar physiological limitations and both
can be considered measures of energy capture eciency.
Cumulative stresses of ozone exposure were apparent in
both relative biomass loss and the uorescence measurements. The uorescence emissions represent the
active energetics occurring within seconds at the leaf
tissue level while biomass is accumulated over months.
More research will be needed to explain the reason for
this correlation.
The threshold value for the detection of the eect of
ozone exposure on photosynthetic performance lay
above the cumulative ambient values recorded in 1997.
There were visible symptoms in the ambient treatment
late in the season, but only in the 50%+30 nl l1 treatment were they found to be signicant.
In the attempt to determine an accurate method of
ozone damage diagnosis that can be used regionally, the
experimental results found signicant correlations
between ozone exposure and photosynthetic performance index, PI. In the absence of a cost-eective ozone
dose measurement, PI provides an alternative method
for regional monitoring of tree health within the context
of the currently employed AOT40.
Once the PI, in combination with meteorological and
ecological parameters, is more fully understood, detection and monitoring of forest areas at risk will be an
achievable goal. Inclusion of the modifying inuence of
the environment is central to modelling the damage due
to ozone. The nature of leaf coupling to the atmosphere,
stomatal control, water relations and ozone interception
are all severely aected by the experimental situation
(Wieser, 1997; Manseld, 1998), and have specic eects
on the performance of photosynthesis (Grunhage and
Jager, 1994).
The high statistical signicance of the data show Chl a
uorescence measurements enable many samples to be
screened in a short time. This method could be used in
both Level I and Level II approaches to mapping critical levels of ozone as set out by Karenlampi and
Skarby (1996).

Acknowledgement
This work was supported by the Swiss National
Science Foundation.

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