Tree Physiology 29, 1349–1365

doi:10.1093/treephys/tpp066

Within-canopy and ozone fumigation effects on d13C and D18O

in adult beech (Fagus sylvatica) trees: relation to meteorological
and gas exchange parameters

ARTHUR GESSLER,1 MARKUS LÖW,2 CHRISTIAN HEERDT,3

MAARTEN OP DE BEECK,4 JOHANNES SCHUMACHER,5 THORSTEN E.E. GRAMS,7
GÜNTHER BAHNWEG,8 REINHART CEULEMANS,4 HERBERT WERNER,3
RAINER MATYSSEK,7 HEINZ RENNENBERG5 and KRISTINE HABERER5,6,*
1
Centre for Systems Biology (ZBSA), Core Facility Metabolomics, Albert-Ludwigs-University, Habsburgerstraße 49, 79104 Freiburg, Germany
2
Centre for Plant and Food Science, University of Western Sydney, Locked Bag 1797, Penrith South DC NSW 1797, Australia
3
Bioclimatology and Air Pollution Research, Department of Ecology, Technische Universität München, Life Science Center Weihenstephan,
Am Hochanger 13, 85354 Freising, Germany
4
Research Group Plant and Vegetation Ecology, Department of Biology, University of Antwerpen (CDE), Universiteitsplein 1,
2610 Wilrijk/Antwerpen, Belgium
5
Institute of Forest Botany and Tree Physiology, Chair of Tree Physiology, Albert-Ludwigs-University, Georges-Köhler-Allee 053/054,
D-79110 Freiburg, Germany
6
Corresponding author (Kristine.Haberer@biologie.uni-freiburg.de)
7
Ecophysiology of Plants, Technische Universität München, Am Hochanger 13, 85354 Freising, Germany
8
Helmholtz Zentrum München-German Research Centre for Environment and Health, Institute of Biochemical Plant Pathology,
Ingolstädter Landstraße 1, 85764 Neuherberg, Germany

Received December 8, 2008; accepted August 2, 2009; published online 4 September 2009

Summary In this study, the effects of different light
intensities either in direct sunlight or in the shade crown
of adult beech (Fagus sylvatica L.) trees on d13C and D18O
were determined under ambient (1 · O3) and twice-
ambient (2 · O3) atmospheric ozone concentrations dur-
ing two consecutive years (2003 and 2004). We analysed
the isotopic composition in leaf bulk, leaf cellulose,
phloem and xylem material and related the results to (a)
meteorological data (air temperature, T and relative
humidity, RH), (b) leaf gas exchange measurements
(stomatal conductance, gs; transpiration rate, E; and
maximum photosynthetic activity, Amax) and (c) the
outcome of a steady-state evaporative enrichment model.
d13C was significantly lower in the shade than in the sun
crown in all plant materials, whilst D18O was increased
significantly in the shade than in the sun crown in bulk
material and cellulose. Elevated ozone had no effect on
d13C, although D18O was influenced by ozone to varied
degrees during single months. We observed significant
seasonal changes for both parameters, especially in 2004,
and also significant differences between the study years.
Relating the findings to meteorological data and gas
*
Present address: Institute of Biology II-Botany, Albert-Lud-
wigs-University, Schänzlestraße 1, 70104 Freiburg, Germany.
exchange parameters, we conclude that the differences in
D18O between the sun and the shade crown were
predominantly caused by the Péclet effect. This assump-
tion was supported by the modelled D18O values for leaf
cellulose. It was demonstrated that independent of RH,
light-dependent reduction of stomatal conductance (and
thus transpiration) and of Amax can drive the pattern of
D18O increase with the concomitant decrease of d13C in
the shade crown. The effect of doubling ozone levels on
time-integrated stomatal conductance and transpiration
as indicated by the combined analysis of D18O and d13C
was much lower than the influence caused by the light
exposure.
Keywords: evaporative enrichment, light exposure, Pe´clet
effect, seasonal and inter-annual differences.
Introduction
The interaction of plants with their environment is predom-
inantly influenced by their requirement for resources that are
necessary for growth, survival and defence. Nitrogen, water
and carbon are crucial resources that influence plant growth
and development. Photosynthetic activity (A) resulting in

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1350 GESSLER ET AL.
carbon incorporation depends on the atmospheric CO2 con-
centration, the amount and activity of ribulose-1,5-bisphos-
phate carboxylase/oxygenase (Rubisco) and on the stomatal
conductance (gs) (Cowan 1982). As a consequence, A and gs
are important parameters for assessing the amount of carbon
acquired in relation to water loss and are both influenced by
environmental constraints such as light intensity, tempera-
ture and water availability. Owing to the complex structure
of the canopy in forest ecosystems, carbon assimilation and
stomatal opening differ between the upper and lower canopy
(Niinemets et al. 2004, Eensalu et al. 2008). The structure of
the canopy has an effect by generating gradients of light
intensity through self-shading; it also affects leaf water avail-
ability and causes differences in leaf-to-air water vapour pres-
sure. In European beech forests, intra-canopy light gradients
are particularly large as beech trees suppress the competing
species by intense shading. Besides the above-mentioned fac-
tors, air pollutants, e.g., reactive nitrogen compounds (Van
Hove et al. 1992) or tropospheric ozone (O3) (Löw et al.
2007), are known to influence A and gs.
O3 and its reaction products (reactive oxygen species,
ROS; Kanofsky and Sima 1995) are oxidative stressors,
and can cause severe damage to plants leading to reduced
growth and photosynthesis as well as to premature senes-
cence (Calatayud et al. 2003, 2004, Langebartels and
Kangasjärvi 2004). Chronic and short-term O3 fumigation
can also modify gs (McAinsh et al. 2002) as observed in
beech (Löw et al. 2006, Kitao et al. 2009) and birch (Frey
et al. 1996) by changing the sensitivity of stomatal regula-
tion (Matyssek and Sandermann 2003). O3 is assumed to
activate Ca2+ channels, leading to an increased intracellu-
lar Ca2+ concentration (Clayton et al. 1999, Kwak et al.
2003, Dietrich et al. 2004, Suhita et al. 2004). As a conse-
quence, chronic ozone exposure might lead to a reduction
in both assimilation rate and gs over the long term.
In recent decades, measurements of leaf carbon isotope
composition (d13C) and discrimination (D13C) have been
increasingly used to estimate the ratio between A and gs
[intrinsic water-use efficiency of plants (WUEint; Farquhar
et al. 1989a)]. The advantages of stable isotopes are: (1) that
they can integrate physiological parameters during the
whole lifetime of the tissues examined. This is in contrast
to the gas exchange measurements that provide information
about instantaneous A and gs at a single time point; (2) they
are easy to apply to a large number of samples across the
canopy, allowing spatial integration as well as the assess-
ment of intra-canopy gradients.
Rubisco discriminates against 13C, and this discrimina-
tion is positively linked to the ratio of intercellular to atmo-
spheric CO2 concentration (ci/ca; Farquhar et al. 1982,
Brugnoli et al. 1988). Therefore, d13C reflects variations in
A and as well in gs, as both are influencing ci and, depend-
ing on the kind of tissue examined, integrates this informa-
tion over days (e.g., phloem organic matter; Brandes et al.
2006) to years (tree rings). To determine to what extent the
variations in ci are caused by the changes in A or gs, d13C
TREE PHYSIOLOGY VOLUME 29, 2009
measurements are often applied in combination with the
analysis of oxygen isotope composition (d18O) or 18O
enrichment above source water (D18O) (Yakir and Israeli
1995, Farquhar et al. 1998, Grams et al. 2007). As an exam-
ple, the conceptual model derived by Scheidegger et al.
(2000) semi-quantitatively links d18O and d13C values in
organic matter with gs and photosynthetic capacity.
The d18O in leaf water at the sites of evaporation is char-
acterized by the composition of the source water and the
evaporative enrichment during phase transition and diffu-
sion (Dongmann et al. 1974, Roden and Ehleringer 1999).
The extent of the evaporative enrichment is influenced by
the gradients of water vapour pressure between the leaf
intercellular space and the ambient air (Craig and Gordon
1965, Dongmann et al. 1974, Farquhar and Lloyd 1993). A
decrease in the relative humidity (RH) of the ambient air, as
well as an increase of the leaf temperature, can decrease the
ratio of ambient to intercellular water vapour pressure and
thus influence D18O. The changes in leaf temperature are
governed by transpiration and gs and, hence, by the
changes in water availability, light exposure, atmospheric
CO2 concentration and/or by air pollutants.
The average lamina mesophyll water, the isotopic com-
position of which is imprinted on the newly assimilated
organic matter, is, however, often observed to be less 18O
enriched than the water at the evaporative sites. This differ-
ence has been theoretically explained by the convection of
unenriched xylem water (i.e., source water) via the transpi-
ration stream opposing the diffusion of 18O-enriched water
from the sites of evaporation to the mesophyll (Péclet
effect). Thus, variations in the RH of ambient air as well
as in E and/or the leaf temperature can both be the origin
for variations in D18O. They either lead to (RH) or rather
are the consequence (E) of changes in gs. As differences in
d18O of the source water additionally influence d18O of leaf
water and leaf organic matter, but are not related to leaf
level physiology, D18O rather than d18O is suited for sepa-
rating effects of A and gs on d13C.
Correlation analyses between d13C and d18O or D18O
indicating a dependency of d13C on gs or A have been suc-
cessfully applied under experimentally modified environ-
mental conditions, in diel, diurnal and annual courses,
with plant species growing in habitats differing in their
climatic and/or meteorological conditions and along
geographical transects (Barbour and Farquhar 2000,
Scheidegger et al. 2000, Xu et al. 2000, Adams and
Grierson 2001, Cernusak et al. 2005). Conversely, studies
on d13C and d18O have indicated that the approach of
Scheidegger et al. (2000) is not applicable when gs is pre-
dominantly governed by factors other than vapour pressure
deficit (Brandes et al. 2007). In such cases, the Péclet effect
can be the prevailing factor influencing d18O/D18O in leaf
material when plants exhibit a similar leaf-to-air vapour
pressure difference (Sullivan and Welker 2007).
Despite these constraints, the combined analysis of d13C
and D18O has been successfully applied to assess the effects
 WITHIN-CANOPY AND OZONE EFFECTS ON STABLE ISOTOPES
of O3 on the time-integrated leaf conductance of different
grassland species (Jäggi and Fuhrer 2007). The authors con-
cluded that mainly stomatal conductance and not the
assimilation rate was affected by elevated O3 concentra-
tions. Recently, Grams et al. (2007) applied a combined
analysis of the two relevant isotope signatures to differenti-
ate between the effects of A and gs on d13C in the leaves of
adult beech and spruce trees. To cover the extrema in A and
gs, the leaves of the sun and shade crown of experimentally
O3-exposed and control trees were considered, although
assessment was restricted to one time point during the
whole growing season. In this case, no O3 effect on the leaf
isotope composition was detected. In contrast, Löw et al.
(2007) observed that the ozone treatment reduced the max-
imum photosynthesis rate of beech at the same field site.
However, this effect was variable during the growing season
and among years.
Based on the exploratory study of Grams et al. (2007), we
aimed to assess the potential of the dual isotope approach
to differentiate O3 effects on gs and/or A over various time
integrals during two growing seasons with contrasting
meteorological conditions. As a short-term integrating mea-
sure of gs and A, we determined D18O and d13C in the
phloem organic matter. In addition, we assessed bulk leaf
material and leaf cellulose, which are assumed to integrate
the information over the growing season.
We hypothesize that, besides the effect on d13C, modifica-
tions in gs caused by elevated ozone might also influence
D18O in the adult beech trees upon changes in leaf tempera-
ture and/or E. The clarification of light exposure effects (sun
versus shade crown) on d13C and D18O of leaves and phloem
sap under the ambient and twice-ambient ozone regimes was
another aim of this study. In addition, instantaneous gas
exchange performance and meteorological parameters were
related to the isotopic analysis. To obtain a mechanistic
understanding of the processes controlling evaporative
enrichment, we modelled D18O of leaf cellulose taking into
account the oxygen isotope fractionation during phase tran-
sition, the water vapour diffusion and the Péclet effect.
Materials and methods
Experimental set-up and plant material
This study was carried out in a mixed ca. 60-year-old beech
(Fagus sylvatica L.) and 52-year-old spruce [Picea abies (L.)
Karst.] stand near Kranzberg, Germany (4825 0 0800 N and
1139 0 4100 E, elevation 490 m; Matyssek et al. 2007).
Throughout the growing seasons in 2003 and 2004, the trees
were exposed either to the ambient (1 · O3) or to the twice-
ambient (2 · O3) O3 regimes. The experimentally enhanced
O3 regime was generated using a free-air O3 exposure sys-
tem employed within the forest canopy (for a detailed
description, see Nunn et al. 2002, Werner and Fabian
2002, Karnosky et al. 2007). To prevent the risk of acute
TREE PHYSIOLOGY ONLINE at http://www.treephys.oxfordjournals.org
1351
O3 injury under 2 · O3, maximum O3 concentrations were
restricted to 150 nl O3 l1. Ambient atmospheric ozone
concentrations ranged from 22.5 to 48.6 ppb in 2003 and
from 11.8 to 37 ppb in 2004. Ozone concentrations in the
2 · O3 regime ranged from 42.6 to 95.4 ppb in 2003 and
from 16.3 to 64.4 ppb in 2004. Ozone concentrations of
the ambient regime in 2003 were 1.2- to 1.9-fold higher than
during the respective months of 2004 (Haberer et al. 2007).
Five beech trees were investigated at ambient and
enhanced ozone conditions each. Plant material from the
sun (24 m) and shade (16 m) crown was harvested once per
month (May 19, June 16, July 29, September 15, October
17 2003 and May 6, June 15, July 28, September 13, October
5 2004) between 11.00 and 13.00 after exposure to sunlight
for at least 3 h. Buds were harvested only in May 2004
(sun and shade crown from five trees; n = 5). Sun leaves
were collected in May, June, July, September and October
2003 and 2004 (n = 5). Shade leaves were sampled in June,
July, September and October 2003 and June, July and
September 2004 (n = 5). Phloem material and xylem sap
were harvested in June, July and September 2003 and 2004
in the sun and shade crown (n = 5). Cellulose was extracted
from sun leaf material in May, June, July (n = 5) and
September and October (n = 3) and from shade leaf mate-
rial in June, July, September and October 2003 (n = 5). In
2004, cellulose samples were extracted from sun and shade
leaves in June, July, September and October (n = 5).
Each of the 10 trees (five in the 1 · O3 treatment and five
in the 2 · O3 treatment) was statistically considered as an
individual case (Matyssek et al. 2007). Leaves and buds
were shock frozen in liquid nitrogen immediately after
harvesting.
Environmental parameters and gas exchange
Tair in combination with RH was measured at 24 and
16 m aboveground within the canopy using an aspiration
psychrometer (model Assman, Theiss, Göttingen,
Germany). Data were averaged over 10 min. Mean Tair of
the growing season (May to October) in 2003 (2004) was
16.9 C (14.5 C) in the sun and 16.3 C (14.1 C) in the
shade crown. Mean RH in 2003 (2004) was 67.6%
(77.1%) in the sun and 68.2% (80.3%) in the shade crown.
Global radiation and wind velocity above the canopy were
used for modelling purposes and were measured with a
pyranometer (type CM 11, Kipp and Zonen, Delft, The
Netherlands) and a cup anemometer, respectively, and
calculated as 10-min averages.
Leaf gas exchange was measured at five dates during the
growing season at the same day when the plant material
was harvested or one day earlier (September 2003) or later
(September 2004) than the sampling dates (with the excep-
tion of October 2003 when gas exchange was measured
7 days after the harvest) as described in Löw et al. (2006,
2007). Gas exchange was determined using a Li-Cor 6400
CO2/H2O diffusion porometer with a ‘Leaf Chamber
1352 GESSLER ET AL.
Fluorometer 6400-40’ (Li-Cor, Lincoln, NE) under both
ozone regimes at 24 m (June to October 2003 and May to
October 2004) and 16 m (September 2003 and June to
October 2004) above the ground level. Measurements were
done at 40–60% relative air humidity, about 25 C air tem-
perature, 360 lmol mol1 CO2 of the ambient air and satu-
rating light conditions (1500 lmol m2 s1 photosynthetic
photon flux density (PPFD) in the sun crown and 900 lmol
m2 s1 in the shade crown) within the chamber. The PPFD
of light-saturated photosynthesis (Amax) had been previ-
ously determined by separate assessments (data not shown).
In each of the five trees of each treatment (n = 5), one leaf
was measured in the sun and one in the shade crown. For
further details, see Löw et al. (2006). Mean values of gas
exchange parameters are given in Table 1.
Stomatal conductance and transpiration during the whole
growing season were also modelled, applying the ANAFORE
model (Analysis of Forest Ecosystems; Deckmyn et al.
2008). This process-based forest model simulates tree and
forest growth starting from leaf-scale gas exchange pro-
cesses. With measured input values of spatial dimension
and organ biomass (stem, branches and roots) for each sam-
ple tree, the model was calibrated to branch cuvette gas
exchange measurements in the top canopy (data not
shown). After calibration, ANAFORE was run for 2003
and 2004 to measure the site meteorological and soil mois-
ture data, simulating vertical gas exchange crown profiles on
a 4-min time resolution. Single modelled transpiration and
stomatal conductance values for the crown layers between
15 and 16 m (shade crown) and between 23 and 24 m
(sun crown) were averaged for the light period of each day.
Bulk leaf material and cellulose extraction
Sugars, which are the main constituents of phloem sap, mir-
ror short-term variations in the oxygen isotope ratio/enrich-
ment of the leaf lamina water with some time lag, temporal
integration and damping of the amplitudes (e.g., Barnard
et al. 2007, Gessler et al. 2007). Structural organic matter
in deciduous trees is likely to carry an isotopic signature
influenced by both the present and previous growing sea-
sons, and by the effect of carbon storage and remobilization
(Kozlowski and Pallardy 2002, Helle and Schleser 2004).
However, because the newly fixed carbon is continuously
incorporated into structural organic matter, the isotopic
Table 1. Mean values of gas exchange parameters during the growing seasons in 2003 and 2004.
gs [mol m2 s1]
2003 Sun 1 · O3 0.09
crown 2 · O3 0.08
2004 Sun 1 · O3 0.16
crown 2 · O3 0.12
Shade 1 · O3 0.06
crown 2 · O3 0.05
TREE PHYSIOLOGY VOLUME 29, 2009
signatures in this leaf pool might – beyond all limitations
– still provide a useful integrative measure of the environ-
mental influence on leaf physiology at the seasonal scale
(Fotelli et al. 2003, Keitel et al. 2006). Bulk leaf organic
matter is, however, a mixture of various compounds with
different turnover times and metabolic histories. To avoid
the influence of these various components and the poten-
tially varying contribution of single compounds to the bulk
leaf material, cellulose was extracted from leaf bulk mate-
rial, and the isotopic composition of both cellulose and
total bulk organic matter was analysed.
Bulk leaf material was ground with liquid nitrogen into a
fine homogeneous powder and dried at 60 C for 4 days.
Cellulose was extracted according to the method reported
by Updegraf (1969). Cell walls were prepared by adding
MES buffer containing ascorbic acid and Triton X to the
homogenized leaf material. The cell wall fraction was cen-
trifuged (20,124g; 10 min; RT) and washed several times
with a solution of NaCl, MeOH and bidistilled water to
remove the cytoplasmic components. For the determination
of crystalline cellulose, an acetic/nitric reagent (Updegraf
1969) was added, and the suspension was placed in a heat-
ing block (100 C; 60 min). Cellulose pellets were obtained
by centrifugation (20,124g; 5 min; RT), washed again five
times with bidistilled water and dried at 60 C in an oven,
followed by gravimetric analysis. The very aggressive treat-
ment with the acetic acid/nitric acid reagent efficiently
removed lignin, hemicellulose and xylosans, leaving behind
crystalline cellulose. We are aware of the fact that different
extraction methods might give slightly different results for
the isotope composition of cellulose (e.g., Gaudinski et al.
2005). As a consequence, comparison among different
methods should be made with care.
Collection of phloem exudates
Phloem exudates were collected according to the method
described by Rennenberg et al. (1996) and were adapted
for stable isotope analysis according to Brandes et al.
(2007). Bark pieces were removed from twigs and incubated
in distilled water for 5 h on ice. After incubation, the liquid
phase of the samples was transferred into a reaction tube,
shock frozen in liquid nitrogen and stored at 80 C until
analysis. With this method, contamination of the exudates
by cellular constituents is negligible (Schneider et al. 1996).
E [mmol m2 s1] Amax [lmol m2 s1]
1.94 7.43
1.73 5.98
2.85 11.14
2.38 8.98
1.08 5.23
0.91 3.0
 WITHIN-CANOPY AND OZONE EFFECTS ON STABLE ISOTOPES
Sampling of xylem sap
Xylem sap of twigs was collected by applying the method of
Scholander et al. (1965) as modified by Schneider et al.
(1996). In summary, twigs were harvested, cut to a length
of 30–50 cm and fixed in a pressure chamber (Soilmoisture,
Santa Barbara, CA). By raising the chamber pressure up to
0.6 MPa, the xylem sap was protruded and transferred into
a reaction tube, frozen immediately in liquid N2 and stored
at 80 C until analysis. For a detailed description, see
Hofer et al. (2008).
Analysis of stable C and O isotopes
Dried bulk leaf material (0.5–1.0 mg) and extracted cellu-
lose were transferred into tin capsules for d13C and into sil-
ver capsules for d18O analyses (IVA Analysentechnik,
Meerbusch, Germany). From phloem exudates, 200-ml ali-
quots were transferred into silver and tin capsules and
oven-dried at 70 C.
For the determination of d13C, samples were combusted
in an elemental analyser (NA 2500, CE Instruments, Milan,
Italy or EA 3000, Euro Vector, Milan, Italy). For d18O
analyses, samples were pyrolysed in a high temperature
conversion/elemental analyser (TC/EA Finnigan MAT
GmbH, Bremen, Germany or EA 3000, Euro Vector,
Milan, Italy). Both were coupled to an isotope ratio mass
spectrometer (Delta Plus, Finnigan MAT GmbH, Bremen,
Germany or an Isoprime, Micromass, Manchester, UK).
Standard deviations (SDs) for the repeated standard mea-
surements (n = 10) were ±0.1 and ±0.4& for measure-
ments of d13C and d18O, respectively.
To estimate the effect of possible adsorption of atmo-
spheric water vapour to dried sample material not enclosed
in gastight cups, as previously observed by Cernusak et al.
(2003), we compared the d18O values of the dried sample
material that was always stored under ambient air conditions
of the laboratory without gastight sealing, with the material
sealed under argon immediately after removal from the dry-
ing oven. d18O of ground bulk leaf material was not signifi-
cantly different between the treatments (n = 10). By
contrast, phloem organic matter from beech twigs showed
significantly higher d18O values in samples sealed under
argon, with the linear correlation being significant between
argon-treated and untreated samples (n = 10; R = 0.79,
P < 0.0001). These results confirm the previous studies of
Cernusak et al. (2003) and Brandes et al. (2007) on phloem
organic matter. Additional assessments exhibited a similar
effect for cellulose. We therefore applied the following cor-
rections to unsealed and not argon-treated phloem and cellu-
lose samples: d18Ocorrected = 1.228 · d18Ounsealed  3.64.
For the d18O measurement of xylem water samples,
0.3 ml of sap was injected by a GCPal autosampler
(CTC, Zwingen, Switzerland) into a TC/EA coupled online
to an isotope ratio mass spectrometer (Delta plus XP) via a
ConFlo III interface (both Finnigan MAT GmbH). The
SDs for repeated measurements of standard water samples
TREE PHYSIOLOGY ONLINE at http://www.treephys.oxfordjournals.org
1353
(n = 10) was ±0.2&. Isotopic values are expressed in d
units denoting parts per thousand deviations (&) relative
to VPDB (Vienna Pee Dee Bee Belemnite) in the case of
d13C and to Vienna standard mean ocean water (VSMOW)
for d18O. For oxygen, we calculated the enrichment of 18O
in organic matter above the source water (D18O) to account
for the potential variations in the oxygen isotope composi-
tion of the source water:
d18 OOM  d18 Osource

18 O ¼ ; ð1Þ
1 þ d18 Osource
where d18OOM is the oxygen isotope ratio of organic mat-
ter as deviating from VSMOW [&] and d18Osource is the
oxygen isotope ratio [&] of xylem water.
Oxygen isotopic theory and modelling
Steady-state enrichment of water at the site of evaporation
in the leaf (D18Oe) can be calculated according to
Dongmann et al. (1974), with e+ denoting the equilibrium
fractionation (&) between liquid water and water vapour,
ek the kinetic fractionation (&) during water vapour diffu-
sion (Farquhar et al. 1989b), D18Ov the isotopic difference
(according to Eq. (1)) of water vapour in the atmosphere
compared to source water and ea/ei the ratio of ambient
to intercellular water vapour pressure.

ea

18 Oe ¼ eþ þ ek þ
18 Ov  ek ; ð2Þ
ei
where e+ can be calculated according to Bottinga and
Craig (1969) from a regression equation relating the equi-
librium fractionation to leaf temperature and ek can be
calculated according to Farquhar et al. (1989b) taking
into account the stomatal and boundary layer resistance
to water vapour and the associated fractionation factors
(Farquhar and Cernusak 2005) based on the determina-
tion of isotopic effect for diffusion of H218O in air (Cappa
et al. 2003).
The steady-state enrichment of mean leaf lamina water
(D18OL) depends on the steady-state enrichment at the
evaporative site of the leaf (D18Oe) and on the lamina radial
Péclet number } (Farquhar and Lloyd 1993).

18 Oe ð1  e} Þ

18 OL ¼ ; ð3Þ
}
where } is defined as EL/CD, where E is the transpiration
rate (mol m2 s1), L is a scaled effective path length (m),
C is the molar concentration of water (mol m3) and D is
the diffusivity of H218O in water (m2 s1).
Oxygen isotope signatures in organic plant material
record the 18O signature of the water in which they were
formed (Epstein et al. 1977, Yakir and Deniro 1990).
Enrichment of 18O above source water in the newly pro-
duced organic matter depends on D18O of average lamina
mesophyll water (D18OL, Eq. (3)) and on the equilibrium
1354 GESSLER ET AL.
fractionation between organic carbonyl oxygen and water
(ewc; +27&; Yakir and Deniro 1990). We applied Eq. (3),
taking into account ewc for predicting D18O of leaf cellulose
for the sampling dates during the growing seasons in 2003
and 2004. In our calculation, we used mean daytime (i.e.,
light period) values of environmental and physiological
parameters either determined or modelled for each day from
the beginning of the growing season (24th April) until leaf
harvest. These mean daytime values were weighted for aver-
age global radiation of the respective days as a proxy of
CO2-assimilation rate, as we assumed continuous assimila-
tion-dependent cellulose accumulation in the leaves during
the growing season. Stomatal conductance and transpira-
tion rates used for the prediction of leaf cellulose D18O were
calculated using the ANAFORE model (see above). Input
data of air temperature, RH, wind velocity and global radi-
ation originated from the datasets recorded at the study site.
Modelling was based on the following assumptions of
Keitel et al. (2006) and Brandes et al. (2007): Water vapour
was in isotopic equilibrium with source water, and the dif-
ference between air temperature and leaf temperature can
be calculated according to a leaf energy balance model
described in detail by Barbour et al. (2000a) and Cernusak
et al. (2003) assuming an average surface area of a single
leaf of 7.9 cm2. Isothermal net radiation as an input
parameter for the energy balance model was estimated as
described by Barbour et al. (2000a), with the global radia-
tion values measured above the canopy for leaves from
the sun crown; for the shade crown, we assumed the above
canopy global radiation to be attenuated by 40%. Internal
water vapour ei was assumed to equal the leaf-temperature-
dependent saturation pressure. As an effective path length
for beech we chose L = 0.015 m according to Keitel
et al. (2006). Boundary layer resistance for sun crown leaves
was calculated from the wind velocity above the canopy
and from the leaf length, according to Jones (1992). In
the shade crown, we assumed the wind velocity to be
reduced by 10%. During cellulose synthesis, a proportion
of the organic oxygen atoms exchange with the reaction
water. As the reaction water for leaf cellulose synthesis is
the evaporatively enriched mean leaf lamina water, we
assume this exchange to not affect D18O of cellulose incor-
porated into the leaf tissue. We acknowledge, however, that
any diel rhythm in the synthesis of cellulose (e.g., main cel-
lulose production during night when leaf water is less
enriched in 18O) might have some effect as discussed by
Gessler et al. (2008).
Statistics
d13C and d18O were determined in leaves and twig phloem
exudates of five individual trees for each ozone treatment
and light exposure. Cellulose was extracted from leaves of
the same trees as the bulk and phloem material (n = 5
for all measurement months, except September and Octo-
ber 2003 with n = 3). For statistical analyses, the general
TREE PHYSIOLOGY VOLUME 29, 2009
linear model (GLM) with two fixed factors (sun/shade
exposure and ambient/enhanced ozone concentration) was
applied. Significant differences between the crown positions
and the ozone regimes were determined using the univariate
GLM. Significant changes of the measured parameters dur-
ing the annual course (combining the measurements of the
1 · O3 and 2 · O3 regimes) were assessed by the analyses
of ‘repeated measures’ of the GLM. Homogeneity of vari-
ance was ascertained by Levene’s test. Selectively, data were
log-transformed to homogenize variances.
For the examination of relationships between argon-
treated and untreated d18O values, bivariate correlation anal-
yses were performed. Comparisons between meteorological
conditions (RH, Tair) in the sun and shade crown and in
the 1 · O3 and 2 · O3 regimes and between isotope ratios
of leaf bulk and phloem material (combining the measure-
ments of the 1 · O3 and 2 · O3 regimes) were made using
Student’s t test with a confidence level of 95%. The t test
was also used to compare the isotope ratios (combining all
samples with the exception of buds, treatments and months),
RH (as means of day) and gas exchange parameters (all
measurements per vegetation period) measured in 2003 and
2004 and to compare the twig water potential of the different
light exposure sites. To determine the significant differences
between d18O in xylem source water of the different light
and ozone regimes (1 · O3 sun crown, 2 · O3 sun crown,
1 · O3 shade crown and 2 · O3 shade crown), one-way
ANOVA with the Tukey-HSD post hoc test was applied.
All statistical analyses were performed using SPSS
(Chicago, IL) for Windows.
Results
Meteorological and gas exchange parameters
During the growing seasons of 2003 and 2004, air temper-
ature was in general slightly lower in the shade than in the
sun crown independent of the time periods used for calcu-
lating the ratio between shade and sun crown (Table 2). The
difference between the light exposure sites (i.e., sun and
shade crowns) was significant at four dates when the ratios
were calculated for the day of harvest of plant material and
for the day before. Ratios of shade to sun crown ranged
from 0.94 to 1.0 in all months of measurements in 2003
and 2004.
The RH tended to be comparable between the shade and
the sun crown (Table 2) in 2003 and 2004. Only in October
2004, during the 29 h before leaf sampling, RH was signif-
icantly higher in the shade than in the sun crown. Especially
for the time periods of 5 and 29 h before leaf sampling,
measurement of RH failed from July 2003 until July 2004
due to technical problems, and a calculation of the shade-
to-sun ratio was not possible. The RH ratio between shade
and sun crown ranged from 1.0 to 1.18 in all months when
a calculation was possible.
 WITHIN-CANOPY AND OZONE EFFECTS ON STABLE ISOTOPES 1355

Table 2. Air temperature and RH measured in the shade crown in relation to the sun crown (2003 and 2004).
Ratio shade/sun

Whole month Cumulative months 8.00–14.00 8.00 (day before) to 14.00

(measurement day) (measurement day)
Air temperature
2003 May 0.97 0.97 0.97 0.96
June 0.99 0.98 0.94 0.96
July 0.98 0.98 1.00 0.98
September 0.98 0.98 X 0.99
October 0.96 0.98 0.91** 0.94*
2004 May 0.96 0.97 0.98 0.97
June 0.97 0.96 0.98 0.96
July 0.97 0.97 0.98 0.95*
September 0.97 0.97 0.97 0.95*
October 0.97 0.97 0.96 0.94*
Relative humidity
2003 May 1.03 1.04 1.05 1.07
June 1.01 1.02 1.12 1.06
July 1.06 1.03 X X
September 1.18 1.00 X X
October X 1.01 X X
2004 May X X X
June X X X X
July 1.03 1.00 X X
September 1.04 1.02 1.02 1.03
October 1.06 1.02 1.04 1.05*

The column ‘whole month’ indicates monthly mean values of the Julian calendar used for the calculation of the ratio shade/sun
(n ranged between 14 and 31, mean values of the day, within the treatment groups depending on sampling month and parameter). The
column ‘cumulative months’ gives mean values calculated from the beginning of May (start of the vegetation period) to the respective
sampling day (n = 11–158; mean values of the day). For the calculation of the ratio shade/sun during 6 and 30 h before sampling,
values with the time resolution of 10 min were used (n = 38 and 132–180, respectively), *, **significant at P < 0.05 and P < 0.01. X,
not enough data for statistical analyses.

Measurement-derived gs of leaves grown under 2 · O3 Carbon isotope composition

conditions were generally decreased, with a tendency show-
Leaf bulk material Significant differences between d13C
ing 48–96% of the 1 · O3 values (with the exception of
of the ambient and the twice-ambient O3 regimes and
September 2003, Table 3). E was decreased under elevated
between 2003 and 2004 were not observed. d13C in leaf bulk
ozone especially in June 2003, but was slightly increased in
material was significantly lower in the shade than in the sun
July to October 2003 relative to 1 · O3. In 2004, E was
crown in most months in 2003 and 2004 (Figure 1A and B).
slightly decreased in 2 · O3 than in 1 · O3 (ratio 2 · O3 ver-
In sun leaves, there was a tendency for d13C to decrease in
sus 1 · O3: 0.72 to 0.93). No significant effects of ozone on E
unfolded leaves from May to October. A significant sea-
were observed in 2003 and 2004. Amax was lower under
sonal pattern was, however, not found in 2003 (Figure
2 · O3 than under 1 · O3 in June and July 2003 and during
1A), whilst d13C of bulk material in both sun and shade
all months of measurement in 2004 (Table 3). With the
leaves in 2004 showed significant differences during the
exception of September 2004, this decrease in Amax due to
growing season (Figure 1B; P = 0.002 and 0.034 for sun
the ozone fumigation was insignificant, and the ratio of
and shade crowns, respectively).
2 · O3 versus 1 · O3 ranged from 0.46 to 1.1 in both years.
Instantaneous gs and E were significantly lower in the
shade than in the sun crown in both years (Table 3). A sig- Leaf cellulose The d13C in cellulose samples was similar
nificant reduction in the shade crown also occurred in Amax to the patterns in the leaf bulk material (Figure 1C and
in 2004. The shade-to-sun crown ratios ranged from 0.27 to D). d13C was significantly lower in shade than in sun leaves
0.54 for gs, from 0.26 to 0.42 for E and from 0.32 to 0.41 for in both years, and no influence due to the enhanced ozone
Amax. The ANAFORE model predicted an even stronger regime was observed. In contrast to the leaf bulk material,
reduction of gs and E in the shade than in the sun crown cellulose d13C was significantly higher in 2003 than in 2004
(Table 3). Note that the ANAFORE calculations comprise (P < 0.001; n = 62 and 80 for 2003 and 2004, respec-
monthly daytime values. tively). There was a continuous, but an insignificant decrease

TREE PHYSIOLOGY ONLINE at http://www.treephys.oxfordjournals.org

1356 GESSLER ET AL.

Table 3. Measured and modelled gas exchange parameters in beech leaves. The table shows measured stomatal conductance,
transpiration rate and maximum photosynthetic activity (Amax) as well as ANAFORE-modelled stomatal conductance and
transpiration rates of beech leaves from the shade crown in relation to the sun crown and in the 2 · O3 regime in relation to the 1 · O3
regime (2003 and 2004).
Measured Modelled

Ratio shade/sun Ratio 2 · O3/1 · O3 Ratio shade/sun

Stomatal conductance (gs)

2003 June nm 0.50 0.16
July nm 0.96 0.16
September 0.27** 1.01 0.16
October nm 0.96 0.17
2004 June 0.54** 0.48 0.20
July 0.34*** 0.66 0.19
September 0.35*** 0.68 0.19
October 0.33** 0.73 0.18
Transpiration rate (E)
2003 June nm 0.56 0.14
July nm 1.04 0.14
September 0.26** 1.09 0.13
October nm 1.13 0.13
2004 May nm X 0.23
June 0.42*** 0.86 0.17
July 0.37*** 0.72 0.16
September 0.30*** 0.78 0.19
October 0.34*** 0.93 0.17
Photosynthetic rate (Amax)
2003 June nm 0.55
July nm 0.78
September nm 1.07
October nm 1.10
2004 May nm 0.46
June 0.41*** 0.78
July 0.38*** 0.74
September 0.41*** 0.71*
October 0.32*** 0.61

Depending on the sampling month and parameter, n ranged between 3 and 10 within the treatment groups. For the calculation of the
ratios, shade/sun (2 · O3/1 · O3) data of both ozone (light exposure) regimes were combined to one mean value. Asterisks indicate
significant differences between the compared groups as achieved by the Student t test analyses. *, **, ***significant at P < 0.05,
P < 0.01, P < 0.001. X, not enough data for statistical analyses. nm, not measured in the shade crown. The ratio shade/sun from the
modelled transpiration and stomatal conductance values is calculated for the monthly daytime averages.

of d13C from May to October of 2003 (Figure 1C) and a sig-
nificant (P = 0.039) seasonal pattern with increasing d13C
towards September and a following decline at the end of
the vegetation period in the sun crown in 2004 (Figure 1D).
Twig phloem The d13C in phloem exudates showed simi-
lar levels and similar patterns between sun and shade twigs
and between ozone treatments to those observed for the
bulk and cellulose material (Figure 2A and B). Significant
differences between the ozone regimes and between the years
2003 and 2004 were not observed for phloem d13C. The d13C
in phloem exudates was significantly lower in the shade than
in the sun crown in June, July and September 2003 (Figure
2A). In 2004, this relation was observed only in September,
whilst d13C was higher in the shade than in the sun crown in
July and remained unchanged in June (Figure 2B). The d13C
of phloem exudates did not vary significantly in the sun and
the shade crown during the 2003 growing season. By con-
trast, significant differences were found within the growing
season of 2004, which were caused by negative peak values
in July (sun crown; P = 0.007) and September 2004 (shade
crown; P = 0.028).
When comparing d13C in bulk leaf organic matter with
that in phloem organic matter, significant differences were
not found at most time points. Neither in the sun nor in
the shade crown in June of both years, in July 2003 and

TREE PHYSIOLOGY VOLUME 29, 2009

 WITHIN-CANOPY AND OZONE EFFECTS ON STABLE ISOTOPES 1357

ne
ne

pt

pt
ay

ay

ly
ly
ud

ct

t
nm c
Se

Se
Ju

Ju

Ju

Ju
nm M

nm M
nm O

O
-b
ne

ne
pt

pt
ay

ay

ay
ly

ly
ct

nm ct
Se

Se
Ju

Ju

Ju

Ju
M

M
O

O
-20

nm
nm
-22

δ C [‰] in phloem exudates

-22
δ C [‰] in leaf bulk material

-24
-24
-26
-26
-28
-28
-30
-30

13
-32 ***
*** * ***
13

-32 ** *** *** ** ***

***
A ** *** B
A B -34
2003 2004
nm

Figure 2. Carbon isotope composition (d13C) of phloem exudates

-22 organic matter under ambient and enhanced ozone regimes in
δ C [‰] in leaf cellulose

the sun and shade crowns of adult beech trees in 2003 (A) and
2004 (B). Data shown are mean values of five individual
-24
trees + SD. Asterisks indicate significant differences between
the sun and the shade crown. *significant at 95% and ***sig-
-26 nificant at 99.9% probability level. nm, not measured. White
bars, 1 · O3 sun crown; hatched white bars, 2 · O3 sun crown;
grey bars, 1 · O3 shade crown; and hatched grey bars, 2 · O3
-28 shade crown.
13

*** ***
-30
C **
* D
2003
Figure 1. Carbon isotope composition (d13C) of leaf bulk
material (A and B) and leaf cellulose (C and D) under ambient
and enhanced ozone regimes in the sun and shade crowns of
adult beech trees in 2003 and 2004. Data shown are mean values
of five individual trees + SD. Asterisks indicate significant
differences between the sun and the shade crown. *significant at
95% and ***significant at 99.9% probability level. nm, not
measured. White bars, 1 · O3 sun crown; hatched white bars,
2 · O3 sun crown; grey bars, 1 · O3 shade crown; and hatched
grey bars, 2 · O3 shade crown.
in September 2004, were the differences between leaf and
phloem d13C significant. Only in September 2003 (sun
crown) and in July 2004 (shade crown), significant by
higher d13C levels were found in phloem exudates than in
leaf bulk material.
Oxygen isotope composition of xylem sap and oxygen
isotope enrichment of organic matter
Xylem water When comparing d18O in xylem sap, i.e.,
source water, no significant differences between the sun
and the shade crown or between the ambient and the
twice-ambient ozone regimes were observed during the
growing seasons of 2003 and 2004 (data not shown). Simi-
lar observations have been reported in the study of
Cernusak et al. (2005), which showed no differences
between xylem sap d18O in the sun and shade crowns of
Eucalyptus globulus Labill. Despite a lack of significant
*** *** *** ***
seasonal patterns, xylem sap d18O was significantly higher
2004 in 2004 than in 2003 (P < 0.001). Mean d18O of 2003
(10.83 ± 1.6 &; n = 56) and 2004 (6.85 ± 1.9 &;
n = 51) were determined for the calculation of D18O in
bulk organic matter, cellulose and phloem organic matter
(cf. Eq. (1)).
Leaf bulk material Leaves grown under enhanced ozone
concentrations exhibited significantly increased D18O in
May 2003, June and July 2004 and decreased levels in Octo-
ber 2004 compared to those grown under ambient ozone
conditions. The D18O in leaf bulk material was significantly
higher in the shade than in the sun crown in June, July and
September 2003 and in June 2004 (Figure 3A and B). In
general D18O was significantly lower in 2004 than in 2003
(P < 0.001; n = 80). In both years, significant variation
in D18O during the growing season was observed in the
sun (P < 0.001 in 2003 and P = 0.023 in 2004) and in
the shade crown (P < 0.001 in 2003 and P = 0.012 in
2004). In 2003, the highest D18O was found in May and
the lowest in October. In 2004, the annual pattern was more
pronounced: D18O was lowest in buds, increased from May
and June (when leaves were fully expanded) to July and
decreased towards the end of the growing season.
Leaf cellulose A significant decrease of D18O in leaf cellu-
lose as a consequence of ozone fumigation was observed in
May 2003 in the sun crown, whilst D18O increased under
2 · O3 compared to 1 · O3 in September 2003 in the shade
crown (Figure 3C). Consistent with the findings from leaf

TREE PHYSIOLOGY ONLINE at http://www.treephys.oxfordjournals.org

1358 GESSLER ET AL.
Figure 3. Oxygen isotope enrichment (D18O) of leaf bulk
material (A and B) and leaf cellulose (C and D) under ambient
and enhanced ozone regimes in the sun and shade crowns of
adult beech trees in 2003 and 2004. Data shown are mean values
of five individual trees + SD. Asterisks indicate significant
differences between the sun and the shade crown. *significant at
95%, **significant at 99% and ***significant at 99.9% proba-
bility level. Letter ‘a’ indicates significant differences between the
1 · O3 and 2 · O3 regime. ‘a’, significant at 95% probability
level. nm, not measured. White bars, 1 · O3 sun crown; hatched
white bars, 2 · O3 sun crown; grey bars, 1 · O3 shade crown;
and hatched grey bars, 2 · O3 shade crown. The symbols down
triangle (1 · O3 sun crown), diamond (2 · O3 sun crown), up
triangle (1 · O3 shade crown) and hexagon (2 · O3 shade
crown) denote the modelled D18O values calculated according
to Eq. (3) taking into account ewc of 27& and an effective length
L = 0.015 m.
bulk material, D18O in cellulose samples was higher in
shade than in sun leaves from June through September
2003, whilst no significant differences between different light
exposures were observed in 2004 (Figure 3C and D). As
shown for leaf bulk material, D18O was significantly lower
in 2004 than in 2003 (P < 0.001; n = 74 and 80 for 2003
and 2004, respectively). 18O enrichment declined during
the seasonal course of 2003 (significant in the shade crown,
P = 0.009) from May to October. In 2004, enrichment lev-
els peaked in June followed by a rather stable plateau from
July to October. These variations in D18O during the 2004
TREE PHYSIOLOGY VOLUME 29, 2009
growing season were significant at both light exposure sites
(sun crown: P = 0.033 and shade crown: P = 0.002).
D18O modelled for leaf cellulose in the shade and sun
crown under the two ozone treatments explains the
observed seasonal pattern between June and October rea-
sonably well (Figure 3C and D). This is also indicated by
the linear regression analysis between observed and pre-
dicted values shown in Figure 4, with the coefficient of
determination (R2) amounting to 0.71. However, the model
does not account for the high range of variations between
the sun and the shade leaves in 2003, partially overestimat-
ing the evaporative enrichment in sun leaves (low measured
evaporative enrichment) and underestimating it in shade
leaves (September 2003). As a consequence, the slope of
the regression line shown in Figure 4 is clearly lower than
unity. In agreement with the measured leaf cellulose
D18O, the model predicts a higher evaporative enrichment
in shade than in sun leaves in 2003. However, this pattern
is also predicted in 2004, when the observed values did
not differ between sun and shade leaves. In both years,
D18Oe, as calculated according to Eq. (2) (not taking into
account the Péclet effect), did not strongly differ between
sun and shade crown (data not shown). The Péclet effect
thus reduced the evaporative enrichment of lamina leaf
water and newly produced organic matter more intensively
in the sun crown due to higher transpiration rates.
Twig phloem A significant decrease in D18O of twig
phloem exudates caused by the elevated ozone was
observed only in July 2004, whereas no differences were
Figure 4. Modelled D18O of leaf cellulose (D18O predicted)
plotted against measured D18O values (D18O observed) in leaves
from the sun and shade crowns during the growing seasons in
2003 and 2004. We assumed L to be 0.015 m according to the
previous observations with beech (Keitel et al. 2006). Down
triangle (1 · O3 sun crown), diamond (2 · O3 sun crown), up
triangle (1 · O3 shade crown) and hexagon (2 · O3 shade
crown). The regression line for all data points displayed is
characterized by the following linear equation: D18O predicted
(&) = 0.34 · D18O observed (&) + 27.7 (&); R2 = 0.71. The
bold solid line denotes the 1:1 line.
 WITHIN-CANOPY AND OZONE EFFECTS ON STABLE ISOTOPES 1359

2003 2004 site in 2001. However, juvenile trees in phytotron experiments

42 A B were affected by enhanced ozone concentrations as a conse-
Δ O [‰] in phloem organic matter

* ** quence of lowered gs (Grams et al. 2007). Even though in this

40
38 study the reduced measured instantaneous stomatal conduc-
36
tance of beech trees under 2 · O3 compared to 1 · O3 could
34
have reduced ci (and thus increased d13C), especially in 2004,
a this effect seems to be compensated for by the concomitant
32
reduction in photosynthetic activity in leaves exposed to
30
the double ozone regime, which in turn increases ci.
28
D18O was enhanced under the double ozone regime com-
26 pared to the ambient ozone regime, in the bulk material
24 sampled in June and July 2004. In 2004, E was reduced
18

22 in leaves grown under the double ozone regime in all mea-

nm

nm

nm

nm
nm
20 surements as a consequence of stomatal closure under ele-
y
ne

pt
vated ozone. In contrast, leaf samples in 2003 showed an
ne
ct

ct
ay

ay

ly
s
ep
l

ud
Ju

Ju
O

O
Se
M

M
Ju

Ju
S

-b

enhanced D18O under 2 · O3 compared to 1 · O3 only in

ay
M

June. Accordingly, E was decreased under 2 · O3 only in

Figure 5. Oxygen isotope enrichment (D18O) in phloem organic June 2003, whilst the ratio 2 · O3/1 · O3 remained almost
matter in the sun and shade crowns of adult beech trees growing unchanged throughout the remainder of the year. Since the
at ambient and double ozone conditions in 2003 (A) and 2004 RH between 1· and 2 · O3 was not different in the shade
(B). Data shown are mean values of five individual trees + SD.
Asterisks indicate significant differences between the sun and the
crown (data not shown), the changes in D18O as a conse-
shade crown. *significant at 95% and **significant at 99% quence of ozone fumigation are not assumed to be driven
probability level. Letter ‘a’ indicates significant differences by a different evaporative demand of the atmosphere, but
between the 1 · O3 and 2 · O3 regimes, significant at the 95% seem to be caused exclusively by the changes in stomatal
probability level. nm, not measured. White bars, 1 · O3 sun aperture, E and/or leaf temperature.
crown; hatched white bars, 2 · O3 sun crown; grey bars, 1 · O3
shade crown; and hatched grey bars, 2 · O3 shade crown.
Phloem sap showed less pronounced or even inverted pat-
terns for D18O in ambient and twice-ambient ozone-exposed
materials compared to bulk material with a significant
observed on the other dates. Twig phloem exudates (Figure decrease as a consequence of the ozone treatment in July
5A and B) differed from bulk material as they exhibited 2004. It might be assumed that several hours before sampling
lower D18O in the shade than in the sun crown; these data the differences in gs and E between the two ozone regimes
being significant in June and July 2003. In contrast to 2003 might not have been sufficiently strong to change the oxygen
(Figure 5A), D18O did not vary significantly between differ- isotope signature significantly. In addition, it had been pro-
ent light exposure sites in 2004 (Figure 5B). D18O in phloem posed previously that the phloem D18O might be influenced
exudates was significantly lower in 2004 than in 2003 by the assimilates originating from bark photosynthesis
(P = 0.001, n = 59 for 2003 and 2004) and also varied sig- (Brandes et al. 2007). Organic matter (re-)fixed in the bark,
nificantly during the growing season at both contrasting where the reaction water is not or only slightly enriched,
light exposures in both years (P = 0.01 for both, sun and should have a D18O well below the enrichment for sugars
shade crowns 2003; P = 0.003 and P < 0.001 for sun fixed in leaves. The phloem sugars were 18O depleted com-
and shade crowns 2004, respectively). In 2003, the D18O pared to the leaf bulk material (Figures 3 and 5), and thus this
in phloem organic matter from sun and shade twigs was finding supports the hypothesis of bark assimilation. Varying
highest in July and reduced in June and September, whilst proportions of phloem sugars derived from bark synthesis
the opposite was found for 2004. could therefore explain the difference between the oxygen
D18O was significantly lower in phloem exudates than in isotope patterns in leaves and phloem. In this study, D18O
leaf bulk material in September (sun crown) and from June of leaf bulk and cellulose material correlated (Pearson’s cor-
to September (shade crown) 2003 and in June and July (sun relation coefficient: 0.379, P < 0.001; n = 143) and the
and shade crown) 2004. average offset amounted to 5.97 ± 3.14& in all measure-
ments of 2003 and 2004. This value is similar to that given
in previous studies (Cernusak et al. 2004, Sullivan and
Discussion
Welker 2007). The offset varied between the measurement
months exhibiting maximum values in June and minimum
Differences in isotope composition between the ambient
values in July. The differences in D18O between the ozone
and the enhanced ozone regime
regimes are less pronounced than the differences caused by
In this study, d13C in leaf bulk, phloem and cellulose material varying light intensities. In addition, the effect of doubling
was not affected by doubling the ozone levels. This is consis- ozone concentrations on stomatal conductance and E is less
tent with the findings from adult beech trees at the same field pronounced than the influence caused by light exposure.

TREE PHYSIOLOGY ONLINE at http://www.treephys.oxfordjournals.org

1360 GESSLER ET AL.

Differences in the isotope composition between the sun -26

and the shade crown
A
-27
The significantly lower d13C in leaf bulk organic matter and
-28
cellulose as well as in phloem organic matter in the shade
compared to the sun crown in both study years indicate a -29

δ C (‰)
generally higher leaf internal CO2 concentration (ci) in the
-30

13
shaded crown foliage, irrespective of whether leaf physiol-
ogy was integrated over a few hours before sampling (of -31
phloem) or the entire life span of the leaf (leaf bulk organic
-32 13 18
δ CJuly = -0.90 * Δ OJuly + 5.93
2
R = 0.39 P = 0.003
matter and cellulose). Similar reports exist on the leaf bulk 13 18
δ CSept = -1.06 * Δ OSept + 11.54
2
R = 0.41 P = 0.002
material of E. globulus and F. sylvatica (Cernusak et al. -33
2005, Grams et al. 2007). Possible causes for an increase 35 36 37 38 39 40 41 42

in ci can be an increase in gs as well as a decrease in Amax, -23

which is mainly due to the reduced light availability. The -24
13 18
δ C = -0.68 * Δ O -2.64
2
R = 0.37 P < 0.001

oxygen isotopic enrichment above source water (D18O) of -25

plant organic matter provides additional information to
-26
separate the effects of gs from the influence of changes in

the photosynthetic capacity on d13C (Farquhar et al.
1998, Scheidegger et al. 2000, Grams et al. 2007).
The conceptual model proposed by Scheidegger et al.
(2000) used for this purpose is based on the assumption that
any change in D18O is predominantly caused by the changes
in RH. In addition, the model assumes that gs is coupled
closely to RH. Applying the theoretical consideration this
model is based on, the higher enrichment of 18O in the shade
crown especially observed in the leaf bulk organic matter and
cellulose in 2003 should indicate a lower RH and, as a conse-
quence, a lower gs in the shade than in the sun crown.
Moreover, from the negative relationship between d13C
and D18O as illustrated for the sun and shade leaf bulk
organic matter for July and September 2003 (Figure 6A),
as well as for the combined data of the 2 years (Figure 6B),
the Scheidegger model denotes Amax to be the factor decisive
for the generally reduced d13C values in the shade crown
observed in our study. From the two potential scenarios that
might lead to an increase in ci and thus a decrease in d13C,
which are: (1) an increase in gs and (2) a decrease in Amax,
the model selects the second based on the assumed influence
of RH on D18O and gs. The meteorological data within this
study, however, clearly show that RH did not differ between
the sun and the shade crown and, thus, can be excluded as a
decisive factor for changes in D18O and gs. As a consequence,
other factors, such as the Péclet effect and/or an increase in
leaf temperature not explicitly accounted for by Scheidegger
et al. (2000), might be the main reasons for changes in D18O
(cf. Grams et al. 2007).
Measurements of leaf gas exchange on the sampling days
showed a clear reduction of gs, E and Amax in shade com-
pared to sun leaves. We might assume that a decrease in
gs results, at constant RH, in (i) a decline in E and conse-
quently in (ii) an increase in leaf temperature. A decline
in E (i) would result in less dilution of enriched apoplastic
water from the site of evaporation with unenriched source
water (Péclet effect) and (ii) in a decrease in the ratio of
ambient to intercellular water vapour pressure both
δ C (‰)
-27
-28
13
-29
-30
-31
-32
B
-33
34 36 38 40 42 44
18
Δ O (‰)
Figure 6. Carbon isotope composition (d13C) of bulk leaf
material from the sun and shade crowns plotted against oxygen
isotope enrichment (D18O) of bulk leaf material for July and
September 2003 (A) and combined for June, July and September
2003 and 2004 (B). In (A) circles indicate July and squares
September values and filled symbols indicate shade leaves,
whereas half-filled indicate sun leaves. Each symbol in (A) and
(B) represents shade or sun leaves from a single tree under the
1 · O3 or 2 · O3 treatment.
increasing D18O. We applied the energy balance model of
Barbour et al. (2000b) to estimate the leaf temperature,
and only slight differences between shade and sun crown
were observed as shade leaves also obtained less net radia-
tion. Thus (ii) cannot be regarded as the main factor
explaining the D18O difference between sun and shade
crown. The isotope modelling approach showed, on the
other hand, that D18Oe (cf. Eq. (2)) was mostly comparable
between sun and shade leaves and that the Péclet effect
(included in Eq. (3)) reduced lamina leaf water enrichment
and, thus, D18O of organic matter in the sun leaves signifi-
cantly stronger than in the shade leaves. The calculated
mean 2003 growing season Péclet number (}), as depending
on E amounted to 0.1 in sun leaves, thus being comparable
to previous results with beech sun leaves (Keitel et al. 2006),
and to 0.02 in shade leaves. The calculated difference
between D18Oe (evaporative enrichment at the sites of
evaporation; calculated according to Eq. (2)) and D18OL
(mean lamina leaf water enrichment; calculated according

TREE PHYSIOLOGY VOLUME 29, 2009

 WITHIN-CANOPY AND OZONE EFFECTS ON STABLE ISOTOPES
to Eq. (3)) ranged between 0.8& and 1.1& in sun and
between 0.1& and 0.2& in shade leaves during the 2003
growing season. Thus, the reduction of the Péclet effect
with lower transpiration rates in the shade leaves is most
likely to explain the observed light versus shade leaf differ-
ences in organic matter in 2003. This assumption is consis-
tent with the results of Sullivan and Welker (2007) who
investigated Salix arctica L. at different sites with similar
air temperatures and relative humidities, but with divergent
soil temperatures and soil water content. The dual isotope
conceptual model tested by Sullivan and Welker (2007)
and Grams et al. (2007) is also based on the changes in E
as the cause for varying D18O. These authors found that
the dual isotope approach under the above-mentioned
conditions of constant air humidity is capable of revealing
the qualitative contributions of gs and Amax to d13C.
We demonstrated in this study that, at constant RH,
light-dependent reduction of stomatal conductance (and
thus transpiration) and of Amax can drive a D18O increase
with a concomitant decrease of d13C in the shade crown.
It is not relevant if any change in D18O is driven by RH
and only indirectly connected to a change in gs (assumption
of Scheidegger et al. 2000) or at constant RH directly gov-
erned by gs and E (our observation) and so results of the
Scheidegger model and our conclusions are similar, but
on the basis of different physiological mechanisms. We
could also show that despite the reduction of gs in the shade
crown (which by itself should lead to more positive d13C
values), the concomitant reduction of A causes an overall
decrease in d13C in shade as compared to sun leaves.
Figure 3 shows that the observed differences between leaf
cellulose D18O in shade and sun leaves were higher than
predicted. For the model calculation, we applied a value
for L, which is the scaled effective path length over which
mixing of evaporatively enriched water with unenriched
source water occurs within the leaf lamina, as previously
calculated for the sun leaves of beech along an European
gradient (Keitel et al. 2006). There is, however, some indi-
cation that the effective path length L and, thus, the Péclet
effect vary with leaf anatomy and physiology (Wang et al.
1998, Barnard et al. 2007, Kahmen et al. 2008). We cannot
exclude the possibility that the pronounced differences in
D18O between sun and shade leaves in 2003 are partially
due to the variations in L. In 2004, the predicted difference
in leaf cellulose D18O between sun and shade crown is
somewhat lower than in the year before, since in 2004 the
difference in ANAFORE-modelled transpiration between
shade and sun crown was less pronounced (Table 3). We
have, however, to acknowledge that the model – though
explaining the D18O patterns over the growing season as
well as the sun-to-shade crown differences in 2003 – does
not fully predict the absolute D18O values of cellulose.
Figures 3 and 4 show that the observed D18O values
< 42& are overestimated, whereas higher values are under-
estimated. As a consequence, the high D18O values of cellu-
lose in May 2003 are underestimated by up to ca. 4&,
TREE PHYSIOLOGY ONLINE at http://www.treephys.oxfordjournals.org
1361
whereas the observed values in October 2003 and in the sec-
ond half of the growing season in 2004 are overestimated.
These findings cannot be explained by an erroneous estima-
tion of the effective path length L and thus } alone. In
May, June and July 2003, the observed values even
exceeded the calculations of evaporative enrichment with-
out considering the Péclet effect (as calculated according
to Eq. (2) taking into account ewc of +27&), and thus
any variation in L would not lead to correct estimates.
One possible reason for this observation could be related
to the diel timing of cellulose synthesis. For lignin, diurnal
variations in the expression of key enzymes for the synthesis
pathways are known (Rogers et al. 2005), and for cellulose
comparable circadian patterns have been assumed (Gessler
et al. 2008). Hymus et al. (2005) have suggested that –
depending on the extent of photosynthetic carbon accumu-
lation – sugars are distributed differently to various meta-
bolic pathways over the day. We therefore might assume
that cellulose is not laid down continuously over the day
as assumed in our model but during particular periods of
the day, which might even change over the growing season.
Preferential cellulose synthesis during midday, when the
maximum of leaf water evaporative enrichment is observed
(e.g., Cernusak et al. 2005), would thus lead to an underes-
timation of the actual value by our model, whereas prefer-
ential deposition during the night would result in an
overestimation. In addition, there is no information
whether a different timing of cellulose deposition causes
the evaporative enrichment of leaf water to be variably
transferred to leaf organic matter in shade and sun leaves.
We also have to acknowledge that part of the input param-
eters for the isotope model (stomatal conductance and tran-
spiration) came from the ANAFORE model calculations.
Any potential inaccuracy of the ANAFORE model esti-
mates will propagate to the isotope model and might also
partly explain the differences between the observed and pre-
dicted values.
In contrast to leaf bulk and cellulose material, D18O of
phloem organic matter showed an opposite pattern with a
decrease in 18O enrichment in shade compared to the sun
crown. D18O in twig phloem organic material integrates
over a period of some hours before the sampling of exu-
dates (Barnard et al. 2007), and it may be assumed that
the values of D18O in phloem exudates of the sun and shade
crown mirror the short-term gradients in RH in the lower
part of the crown overruling other factors such as the Péclet
effect. In addition, bark photosynthesis, which produces
organic matter with lower D18O (see above), might variably
contribute to phloem-transported organic matter. A higher
relative proportion of bark photosynthates in the shade
phloem organic matter might thus explain the D18O pat-
terns observed.
Cellulose material of adult beech trees at the same field
site analysed in 2001 showed increasing d18O from shade
to half-shade crown position, but decreasing values
from half-shade to sun-lit positions (Grams et al. 2007).
1362 GESSLER ET AL.
In contrast to the present results, no significant differences
in D18O were observed between the upper and lower crown
of E. globulus, neither in the leaves nor in the phloem
organic matter (Cernusak et al. 2005).
Inter-annual and seasonal changes of stable isotope
composition
d13C of bulk leaf material and phloem organic matter did
not differ significantly between 2003 and 2004. As observed
for 2 · O3 relative to 1 · O3, both measured gs and Amax
were significantly reduced in 2003 compared to 2004
(P < 0.001 for both parameters; n = 32 and 35 in 2003;
n = 42 and 44 in 2004, respectively). The changes in both
gas exchange parameters might outweigh each other, result-
ing in an unchanged discrimination of 13C. As these results
were observed in bulk leaf organic matter integrating over
the whole lifespan of the leaf as well as in phloem material
integrating over several hours before sampling, it can be
assumed that this effect occurs over a major part of the
growing season. In contrast, cellulose material was more
enriched in 13C in 2003 compared to that in 2004. This dif-
ference is mainly due to the relatively high d13C in June
2003, whereas in July, September and October the levels
were comparable between years. A pattern of decreasing
d13C from spring to autumn in leaf material, as observed
here for bulk material in 2003 and 2004 and for cellulose
in 2003, was previously observed in beech (Helle and
Schleser 2004). These authors attributed this phenomenon
to the spring remobilization of starch. Carbon remobilized
from reserve starch is 13C-enriched, and thus leads to the
observed enrichment in leaf structural organic matter.
During the growing season, these starch-derived isotope
signals become diluted by a gradual incorporation of new
assimilates into the newly formed cell wall material, leading
to a decline of d13C in leaf organic matter. It remains
unclear why this seasonal pattern was not observed in the
cellulose during 2004.
The isotope enrichment model explains the seasonal pat-
terns of cellulose D18O during both years reasonably well.
For our approach, we assumed continuous incorporation
of new assimilates in the cellulose pool during the growing
season. It was assumed previously that the leaf structural
organic matter was mainly produced in young leaves and
that its D18O mainly reflected the evaporative conditions
of the year before (when the starch used for the production
of new leaf material was laid down) and the beginning of
the growing season (Keitel et al. 2006). The observed suc-
cessive changes in d13C and D18O of whole leaf organic mat-
ter and leaf cellulose, however, indicate that even after the
leaves are fully expanded, they change their structure dur-
ing the growing season and/or continue to thicken, as pre-
viously postulated by Helle and Schleser (2004). The
continuous addition of new assimilates to the existing struc-
tural organic matter explains why d13C and d18O vary and
are not constant in cellulose and bulk leaf material over the
TREE PHYSIOLOGY VOLUME 29, 2009
growing season. The significantly lower 18O enrichment
found in phloem organic matter as well as in leaf cellulose
and bulk material in 2004 can be readily explained by the
higher RH in 2004 compared to the exceptionally dry and
hot summer of 2003.
We conclude that even though instantaneous gas
exchange measurements indicated a slight reduction in A,
gs and E as a consequence of the ozone treatment, the dif-
ferences were not clearly mirrored in the isotope signals in
the various organic matter pools examined. We could, how-
ever, show that when applying dual isotope approaches, the
consideration of varying influence of the Péclet effect can
explain the differences in D18O without changes in RH. This
is of particular importance for the interpretation of isotope
data when comparing experimental treatments, which do
not differ in climatic conditions (as the ozone treatment)
or shade and sun crowns of forest stands. There is, how-
ever, also the need to critically evaluate the Scheidegger
model. Even though the model prediction derived from
the relation between D18O and d13C is obviously correct
for our study, the underlying mechanisms are not consid-
ered by the model. It remains to be tested if the different
scenarios given by Scheidegger et al. (2000) are reconcilable
with the Péclet effect.
Acknowledgments
This study is part of the Project ‘CASIROZ – the carbon sink
strength of beech in a changing environment: Experimental risk
assessment by mitigation of chronic ozone impact’, which is sup-
ported by European Commission – Research Directorate-General,
Environment Programme, ‘Natural Resources Management and
Services’ (EVK2-2002-00165, Ecosystem Vulnerability). We thank
Maria Alexou for providing phloem exudates and xylem saps of
the sample trees. We gratefully acknowledge Dan Yakir for an
intensive discussion about the results obtained in this study. We
also thank Gaby Deckmyn for developing and providing the
ANAFORE model to simulate detailed gas exchange of the sample
trees and William Teale for language editing.
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