Plant Physiology and Biochemistry 43 (2005) 147–154
www.elsevier.com/locate/plaphy
Effects of elevated pCO2 and/or pO3 on C-, N-, and S-metabolites in
the leaves of juvenile beech and spruce differ between trees grown in
monoculture and mixed culture
Xi-Ping Liu a, Thorsten E.E. Grams b, Rainer Matyssek b, Heinz Rennenberg a,*
a
Institute of Forest Botany and Tree Physiology, University of Freiburg, Georges-Köhler-Allee 53/54, D-79110 Freiburg, Germany
b
Ecophysiology of Plants, TU München, Am Hochanger 13, D-85354 Freising, Germany
Received 27 December 2004; accepted 17 January 2005
Available online 02 March 2005
Abstract
Three and four-year-old saplings of beech (Fagus sylvatica L.) and spruce (Picea abies (L.) Karst.) grown in monoculture and mixed
culture were exposed in phytotrons to (1) ambient air, (2) elevated pO3, (3) elevated pCO2, or (4) elevated pCO2 plus elevated pO3. After
5 months, the contents of soluble sugars, starch, soluble amino compounds, non-structural proteins (NSP), as well as reduced (GSH) and
oxidized (GSSG) glutathione were determined in the leaves of both species in order to assess the effects of the gaseous regimes on primary
metabolism. Elevated pO3 did not affect sugar and starch levels in beech leaves in monoculture, but significantly increased sugar levels in
beech leaves grown in mixed culture. In spruce needles, sugar levels tended to be enhanced in both culture types. Individual and combined
exposure of elevated pCO2 led to an increase in non-structural carbohydrate (soluble sugars plus starch) levels in beech and spruce leaves of
both culture types. Differences in the responses of non-structural carbohydrate levels to elevated pCO2 between beech and spruce were
apparent from different contributions of sugars and starch to the increase in carbohydrate levels. Exposure to elevated pCO2 and/or elevated
pO3 did not affect the levels of soluble amino compounds and NSP in beech leaves, but reduced amino compound levels in spruce needles of
both culture types. Elevated pO3 increased GSH levels in the leaves of both tree species in both culture types, while GSSG levels in monoculture were reduced in beech leaves, but significantly enhanced in spruce needles. Elevated pCO2 reduced GSSG levels in beech and spruce
leaves in monoculture, and GSH levels in spruce needles of both culture types. The combination of elevated pCO2 and pO3 increased GSSG
levels in beech leaves of both culture types and in spruce needles in monoculture, but reduced GSH levels in spruce needles of both culture
types. Apparently, under each gaseous regime, the culture type significantly altered primary metabolism of the leaves of beech and spruce.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Culture type; Elevated carbon dioxide; Elevated ozone; Fagus sylvatica; Glutathione; Non-structural carbohydrates; Non-structural proteins; Picea
abies; Soluble amino compounds; Soluble sugars; Starch; Thiols
1. Introduction
Since pre-industrial time the atmospheric carbon dioxide
concentration (pCO2) increased by ca. 30% from ca. 280 µl l–1
to the current level of 365 µl l–1 [21] and is predicted to double
during the 21st century as a result of anthropogenic combustion of fossil fuels, deforestation and biomass burning [60].
During the past century, also tropospheric ozone concentra* Corresponding author. Tel.: +49-761-203-8301; fax: +49 761 203
8302.
E-mail address: [email protected]
(H. Rennenberg).
0981-9428/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.plaphy.2005.01.010
tion (pO3) increased by a factor of about two to four [55,14].
These changes in the atmospheric environment are supposed
to strongly affect plant metabolism and, hence, plant growth
and development [6,8,29,39,32]. Composition and levels of
C, N and S metabolites are often considered important parameters of primary metabolism of plant leaves that indicate
responses to climate changes, especially to elevated pCO2
and pO3.
CO2 in the atmosphere is the dominant carbon source for
plants that is fixed by the enzyme ribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco) in the foliage of C3 plants
and is translated into carbohydrates as primary products of
photosynthesis. Elevated pCO2 can lead to enhanced carboxy-
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X.-P. Liu et al. / Plant Physiology and Biochemistry 43 (2005) 147–154
lation efficiency and a reduction in oxygenation rate, thereby
increasing the rate of photosynthesis and, hence, carbohydrate production, carbon export from the leaves, and carbon
allocation inside the plants [52,54,6,24,45]. After an initial
increase in the rate of photosynthesis, a “down-regulation”
or an acclimation of photosynthesis is often observed in
response to elevated pCO2 ([43,37]. Still an increase in nonstructural carbohydrate levels is one of the most consistent
reactions of plants exposed to elevated pCO2 [40,45], although
such an effect may be small [25]. Another common response
of plants to elevated pCO2 levels is a reduced nitrogen level
in the leaves [7,9,53], which was often considered to be the
results of (a) a “dilution effect” caused by carbohydrate accumulation and/or (b) the acceleration of plant growth [7,45,53].
The reduced thiol contents of leaves as a consequence of
elevated pCO2 [49,50] may be attributed to the same causes,
but also to enhanced translocation out of the leaves [49]
In contrast, elevated pO3 is considered to be one of the
most detrimental environmental factors for plant growth and
development [42,51,32]. Elevated pO3 can not only affect CO2
uptake by altered stomatal conductance [15,32], but also dark
and light reactions of photosynthesis, by reducing the activity and concentration of Rubisco as well as the light reaction
efficiency. Thereby, elevated pO3 contributes to a decrease in
photosynthesis and assimilate production [27,32]. On the
other hand, O3-induced membrane injury and a collapse of
mesophyll as well as phloem cells may restrict phloemloading of sugar and assimilate translocation from leaves,
resulting in a reduction of carbohydrate allocation to belowground organs [23,30,31,42]; Andersen et al., 1997;
[2,17,1,26]. As a consequence, an accumulation of nonstructural carbohydrates in the leaves was observed in studies with deciduous and evergreen trees [31,13,27]. Elevated
pO3 may lead to an increase [28,59] or a decrease in foliar N
levels [44] or have no effects on foliar N levels at all
[47,12,34,46]. The differences in response between deciduous and coniferous species are likely related to species and
growth conditions (e.g. potted vs. field) as well as to the availability of nitrogen and water. Exposure to elevated pO3 can
result in a decrease or an increase in the levels of reduced
glutathione (c-glutamyl-cysteinyl-glycine, GSH) and/or oxidized glutathione (glutathionedisulfid, GSSG), depending on
species and experimental conditions [10,11,57].
The adverse effects of elevated pO3 on carbohydrate production and assimilate allocation may be counteracted by
elevated pCO2, when the two gaseous regimes are combined
(Volin et al., 1998; [20,26]). Such compensatory effects on
photosynthesis and carbohydrate production have been found
in the leaves of both deciduous and evergreen trees [22,15,38].
However, little is known about the physiological and biochemical basis for such compensation [33], especially the
responses of nitrogen and sulfur contents in plants to the combination of simultaneously elevated pCO2 and pO3.
The responses of different tree species to individual and
combined exposure to elevated pCO2 and pO3 are also dependent on monoculture or mixed culture. This difference in cul-
ture type can change resource gains and space sequestration
of plants [36,16]. It can affect the allocation of resources
(assimilates and nutrients) among organs, especially partitioning of assimilates to the roots and, as a consequence, both
biotic and abiotic conditions in the soil [45,1,26]. Thereby,
culture type can also alter the pCO2 and pO3 sensitivity of
herbaceous and woody plants (Andersen et al., 2001; [35];
Bender et al., 2003; Fuhrer et al., 2003). When plants are
grown in conditions of monoculture or mixed culture, elevated
pCO2 and pO3 may not only directly affect photosynthesis
and carbohydrate production, but also may alter these processes indirectly through its effects on tree competition for
resources [3].
Since temperate forests are considered to be nitrogenlimited ecosystems due to the often low availability on mineral nitrogen in soils [58], a parallel investigation of C, N,
and S levels appears necessary to estimate the consequences
of elevated atmospheric pCO2 and pO3 on primary metabolism. In the present study, we examined whether the responses
of C, N and S levels in the leaves of beech and spruce exposed
to elevated pCO2 and/or pO3 are dependent on culture type.
Juvenile beech (Fagus sylvatica L.) and spruce (Picea abies
(L.) Karst.), the most important tree species in Central Europe
with different sensitivity to elevated pCO2 and pO3
[51,15,32,26], were chosen for the experiments.
2. Material and methods
Plants and treatments: 2- and 3-year-old saplings of beech
and spruce were planted in May 1998 in 32 containers (0.7 ×
0.4 × 0.3 m3, volume = 84 l) filled with forest soil, in monoculture (beech and spruce, each) and mixed culture (beech:
spruce = 1:1), arranged in rows of 4 × 5 individuals [26]. In
mid-April 1999, the containers were transferred to the phytotrons (2.8 × 3.4 m) of the National Research Center for
Environment and Health (GSF) in Neuherberg, Germany, and
plants were exposed to (1) ambient air; (2) elevated pO3 (i.e.
twice-ambient pO3 levels, maximum restricted to < 150 ppb);
(3) elevated pCO2 (ambient + 300 ppm); or (4) elevated pCO2
and elevated pO3. In the phytotrons, the climate conditions
were adjusted hourly to match those of an air quality study at
the Kranzberg Forest near Freising (Germany, 490 m a.s.l.;
see Pretzsch et al., 1998). Monthly mean day values of photosynthetic photon flux were about 430–480 µmol m–2 s–1.
Monthly mean air temperature and relative humidity
amounted to about 18–20 °C and 57–63% at day and,
13–15 °C and 77–84% at night. During cultivation, liquid
fertilizer (Hoagland solution) was added five times to ensure
non-limiting nutrient supply in the containers. Soil water content of each container was monitored continuously at a depth
of 7 cm with tensiometers set to trigger irrigation with deionized water, whenever soil water tension reached 350 hPa. At
the beginning of September 1999, beech and spruce leaves
were collected. Aliquot samples were frozen immediately in
liquid nitrogen, powdered with mortar and pestle and stored
at –80 °C until biochemical analysis.
X.-P. Liu et al. / Plant Physiology and Biochemistry 43 (2005) 147–154
Analysis of soluble sugars and starch: To determine the
contents of soluble sugars in leaves of beech and spruce, aliquot samples of 30 mg powdered tissue were extracted with
1 ml of double-distilled H2O (Milli-Q filter, Millipore,
Schwalbach, Germany) at 100 °C for 5 min. The extracted
soluble sugars were measured colorimetrically after derivatization with anthrone-reagent at 578 nm [5]. Glucose was used
as a standard. Starch contents of the samples were determined colorimetrically after enzymatic hydrolysis using a
commercial test combination (Boehringer, Mannheim, Germany).
Analysis of soluble amino compounds: For extraction of
soluble amino compounds, 50 mg samples of powdered leaf
tissue were homogenized in a mixture of 200 µl Hepes puffer
(containing 20 mM hepes, 5 mM EGTA and 10 mM NaF, pH
7.0) plus 1 ml of methanol/chloroform (3.5:1.5, v/v). The
homogenate was incubated at 4 °C for 30 min. Water-soluble
amino compounds were extracted twice with 700 µl doubledemineralized water after centrifugation at 4 °C and 14,000 g
for 5 min. The contents of extracted amino compounds were
measured colorimetrically at 570 nm after derivatization with
ninhydrin reagent. Glutamate (Sigma, Munich, Germany) was
used as a standard.
Determination of non-structural proteins (NSP): To determine NSP contents, aliquots of ca. 50 mg powdered leaf tissue were homogenized with 1.5 ml 0.1 M Tris–HCl puffer
(pH 7.8) and shaken at 4 °C in the dark for 10 min. The contents of NSP were measured colorimetrically after derivatization with Bradford reagent at 595 nm [4]. Bovine serum
albumin (BSA, initial fraction V, Serva Feinbiochemica,
Heidelberg, Germany) was used as a standard.
Analysis of thiols: Low molecular weight thiols were analyzed by a modification of the method described by [48]. For
extraction, 80–100 mg samples of powdered leaves were
homogenized with 1500 ml of a mixture containing 0.1 M
HCl, 1 mM EDTA and 100 mg insoluble polyvinylpolypyrrolidon (PVPP). After centrifugation at 14,000 × g for 30 min,
aliquots of 180 µl of supernatants were adjusted to pH
8.3 ± 0.2 by adding 240 µl 0.2 M CHES buffer (pH 9.3).
Reduction of thiols was carried out by addition of 60 µl 6 mM
1,4-Dithiolthreitol (DTT) for 60 min at room temperature.
Thiols were derivatized by addition of 20 µl 30 mM monobromobimane for 15 min under dim light at room temperature. Derivatization was terminated by addition of 260 µl 10%
(v/v) acetic acid. For analysis of the mBBr-derivatives, 200 µl
of each sample were injected into a Beckman HPLC system
(Munich, Germany) and separated on an ODS-hypersil column (Octa-Decyl-Silicium, C-18; 5-µm particle size, 4.6 ×
250 mm; Beckman, Munich, Germany). The elution buffers
contained 0.25% (v/v) acetic acid, 10% (v/v) methanol, pH
4.3, for eluent A and 0.25% (v/v) acetic acid, 90% (v/v) methanol, pH 3.9, for eluent B. An attached Shimadzu RF 535 fluorescence detector mediated the determination of the thiol
derivatives at 480 nm, after excitation at 380 nm. Peaks were
identified and quantified using a standard solution containing
0.2 mM cysteine, 0.1 mM c-glutamylcysteine and 1.0 mM
149
glutathione. Recovery of cysteine, c-glutamylcysteine and
GSH amounted 75–90%.
To determine GSSG contents in the leaves, the free thiol
group of GSH was blocked by addition of N-ethylmaleimide
before reduction with DTT. The remaining GSSG was treated
as described above [56].
Data analysis: Two-way analysis of variance (ANOVA)
was used to test effects of elevated pCO2 and/or pO3 on
soluble sugars, starch, soluble amino compounds, NSP, and
thiols in the leaves of beech and spruce. Interactions of atmospheric treatments (pCO2, pO3) with culture types (monoculture and mixed culture) were tested by three-way ANOVA.
Significant differences were analyzed by means of pairedsample t-test. When significant differences were found,
Tukey’s multiple comparisons were used to determine the difference between treatments. The differences were considered significant at P < 0.05. Statistical analysis was performed with the SPSS 10.0 software (SPSS Science, Chicago,
IL).
3. Results and discussion
3.1. Soluble sugars and starch
As reported in previous investigations, effects of elevated
pO3 on leaf non-structural carbohydrate levels, i.e. soluble
sugars plus starch, of beech (Fig. 1A,B) and spruce
(Fig. 1C,D) were small in this study, when the trees were
grown in monoculture. This may be due to the combined effect
of two antagonistic processes. Elevated pO3 reduces photosynthetic efficiency [15] and, on other hand, limits phloemloading of sugars [31,42]. The limitation on carbohydrate
export from the leaves is likely to be more pronounced than
O3-induced reduction of photosynthesis [17,1], thereby,
enhancing leaf non-structural carbohydrate levels [13,27].
Adverse effects of elevated pO3 may be exhibited more distinctly by reduced carbon allocation to belowground organs
[2,1,26].
When grown in mixed culture, leaf sugar levels of beech
were significantly enhanced by elevated pO3; however, starch
levels were about 50% lower than in beech grown in mixed
culture at ambient air (Fig. 1A,B). As a consequence, nonstructural carbohydrate levels largely remained unchanged as
also observed with trees grown in monoculture. Apparently,
in beech transformation between sugars and starch is affected
differently by culture type, but non-structural carbohydrate
levels were still maintained at a similar level. Spruce grown
in mixed culture showed responses of sugar and starch levels
in the needles to elevated pO3 similar to those observed in
monoculture (Fig. 1C,D).
When grown in monoculture, elevated pCO2 significantly
enhanced leaf sugar levels of beech and also starch levels
tended to be increased (Fig. 1A,B). This effect was also
observed in the leaves of beech and spruce grown in mixed
culture. In needles of spruce grown in monoculture, starch
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X.-P. Liu et al. / Plant Physiology and Biochemistry 43 (2005) 147–154
Fig. 1. Effects of elevated pCO2 and/or pO3 on the levels of soluble sugars and starch in the leaves of beech (A, B) and spruce (C, D) grown in monoculture and
mixed culture (mean ± S.E. for n = 8–10). Open bars = gaseous control regime (ambient pCO2/ambient pO3); gray bars = elevated pO3 and ambient pCO2
(+O3); hatched bars = ambient pO3 and elevated pCO2 (+CO2); black bars = elevated pCO2 and elevated pO3 (+O3/CO2). Significant differences between
treatments are indicated by different letters above the bars (ANOVA, P < 0.05).
levels were significantly enhanced, whereas sugar levels
remained at the level of control (Fig. 1C,D). Hence, beech
and spruce grown in monoculture or mixed culture showed a
common increase in non-structural carbohydrates in the
leaves. Although the increase in leaf carbohydrate levels is a
general response of woody species to elevated pCO2 [40,45],
the present data show a considerable variability in the magnitude of the response and in the contribution of sugars and
starch to this increase (cf. [25]), depending on species and
cultivation type.
When beech and spruce were grown in monoculture, combined exposure to elevated pO3 and elevated pCO2 resulted
in a significant increase in leaf sugar levels of both tree species. While leaf starch levels of beech tended to be increased,
the stimulatory response of starch levels in spruce needles
was significant (Fig. 1A,B). When grown in mixed culture,
leaf sugar levels of both tree species were significantly
enhanced. Starch levels in beech leaves tended to be reduced,
but to be increased in spruce needles. Hence, combined exposure to elevated pCO2 and elevated pO3 also led to a common
increase in leaf non-structural carbohydrates of beech and
spruce, resembling exposure to elevated pCO2. Since the
responses to the combined exposure were more similar to
elevated pCO2 than to elevated pO3, apparently elevated pCO2
overruled the effects of elevated pO3 on non-structural carbohydrates.
3.2. Soluble amino compounds and NSP
When seedlings of beech and spruce were grown in monoculture, elevated pO3 did not affect the levels of soluble amino
compounds [34,46] and NSP in beech leaves compared to
the controls (Fig. 2A,B) as also observed by [28]. In spruce
needles, reduced levels of amino compounds were associated with enhanced protein levels (Fig. 2C,D). A similar
response was observed in the leaves of beech and spruce
grown in mixed culture (Fig. 2) and may be attributed to the
synthesis of defence proteins.
Elevated pCO2 tended to reduce the levels of amino compounds in the leaves of both tree species grown in monoculture and in mixed culture (Fig. 2). Hence, the reduction in the
levels of amino compounds was independent from tree species and culture type and appears to be a general response to
elevated pCO2 [7,9,53]. This response has previously been
observed; it may partially be explained by an increase in nonstructural carbohydrates, but may also be due to a decrease in
N availability of the leaves [52,8]. Protein levels were not
affected by elevated pCO2 irrespective of tree species and
culture type (Fig. 2B,D).
Combined exposure to elevated pCO2 and elevated pO3
did not affect the levels of amino compounds in leaves of
beech grown in monoculture as also observed for elevated
pO3 (Fig. 2A); it significantly reduced amino compounds in
spruce needles (Fig. 2C), whereas leaf protein levels of both
tree species showed a minor increase (Fig. 2B,D). When
grown in mixed culture, protein levels in beech leaves tended
to be increased, whereas the levels of amino compounds and
proteins in spruce needles were reduced significantly. As the
effects of combined exposure on the levels of amino compounds in spruce needles resembled those of elevated pCO2,
apparently, elevated pO3 did not modify the effects of elevated
X.-P. Liu et al. / Plant Physiology and Biochemistry 43 (2005) 147–154
151
Fig. 2. Individual and combined effects of elevated pO3 and/or elevated pCO2 on the levels of soluble amino compounds and NSP in the leaves of beech (A, B)
and spruce (C, D) grown in monoculture and mixed culture (mean ± S.E. for n = 8–10) (signature of bars, treatments and statistics as in Fig. 1).
pCO2 on amino compound levels in spruce needles of both
culture types [18,15]. Remarkably, combined exposure to
elevated pCO2 and pO3 showed effects on the levels of amino
compounds and proteins in beech leaves similar to elevated
pO3. Apparently, elevated pO3 was able to counteract the
effect of elevated pCO2 on N metabolism in beech but not in
spruce leaves.
3.3. Thiols
Irrespective of culture type, elevated pO3 tended to increase
GSH levels in the leaves of beech and spruce grown in monoculture (Fig. 3A,C). Whereas GSSG levels in beech leaves
tended to be reduced in monoculture, they were significantly
enhanced in spruce needles under these conditions
Fig. 3. Levels of glutathione (GSH and GSSG) in the leaves of beech (A, B) and spruce (C, D) grown in monoculture and mixed culture and exposed to elevated
pCO2 and/or elevated pO3 (mean ± S.E. for n = 8–10) (signature of bars, treatments and statistics as in Fig. 1).
152
X.-P. Liu et al. / Plant Physiology and Biochemistry 43 (2005) 147–154
(Fig. 3B,D). The positive effect of elevated pO3 on leaf GSH
levels in this study was similar to previous observations with
poplar grown as individual plants [57]. Significantly enhanced
levels of leaf GSSG of spruce grown in monoculture indicate
a detoxification of O3 that includes GSH oxidation at an extent
that exceeds GSSG reduction capacity [41,19]. Apparently,
mixed cultivation allowed O3 detoxification without a change
in the GSH level.
In monoculture, elevated pCO2 significantly reduced GSSG
levels in beech leaves, but did not affect the GSH level
(Fig. 3A,B). In spruce needles, the levels of both GSH and
GSSG tended to be reduced under these conditions
(Fig. 3B,D). When beech and spruce were grown in mixed
culture, elevated pCO2 tended to increase leaf GSSG levels
of beech, however, significantly reduced GSH levels in spruce
needles, while GSSG levels in the needles remained at the
level of control. These results are consistent with observation
of [50] for oak trees showing higher sugar, reduced foliar N
and lower thiol levels at short-term exposure to elevated pCO2.
The combination of elevated pCO2 and elevated pO3
slightly reduced GSH, but increased GSSG levels in leaves
of beech grown in monoculture and mixed culture (Fig. 3). In
spruce grown in monoculture, the levels of leaf GSH were
significantly reduced, whereas GSSG levels in the needles
tended to be enhanced. In mixed culture, the levels of both
GSH and GSSG tended to be decreased in spruce needles.
beech and spruce strongly depended on culture type. The
effects observed may be interpreted as higher flexibility of
primary metabolism at mixed cultivation or enhanced deviation from homeostasis under these conditions.
Acknowledgements
The authors are indebted to Dr. H.-D. Payer and Dr. H.
Seidlitz (GSF—National Research Center for Environment
and Health) and their technical staff for excellent support during the experiments. We gratefully acknowledge the skilful
technical assistance by Ing. T. Feuerbach, A. Jungermann, P.
Kuba and I. Süß. The investigation was funded through SFB
607 “Growth and Parasite Defense – Competition for
Resources in Economic Plants from Agronomy and Forestry,” by the “Deutsche Forschungsgemeinschaft” (DFG).
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Elevated pO3 had minor effects on the levels of leaf nonstructural carbohydrates, regardless of tree species and culture type. It did not affect the levels of leaf amino compounds
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