Tree Physiology 24, 323–330
© 2004 Heron Publishing—Victoria, Canada
Effects of elevated carbon dioxide concentration on growth and N2
fixation of young Robinia pseudoacacia
Z. FENG,1 J. DYCKMANS2–4 and H. FLESSA1
1
Institute of Soil Science and Forest Nutrition, University of Göttingen, Büsgenweg 2, 37077 Göttingen, Germany
2
Environmental Resource Management, Faculty of Agriculture, University College Dublin, Belfield, Dublin 4, Ireland
3
Present address: Institute of Soil Science and Forest Nutritition, University of Göttingen, Büsgenweg 2, 37077 Göttingen, Germany
4
Corresponding author ([email protected])
Received April 8, 2003; accepted July 20, 2003; published online January 2, 2004
Summary Effects of elevated CO2 concentration ([CO2]) on
carbon (C) and nitrogen (N) uptake and N source partitioning
(N2 fixation versus mineral soil N uptake) of 1-year-old
Robinia pseudoacacia were determined in a dual 13C and 15N
continuous labeling experiment. Seedlings were grown for
16 weeks in ambient (350 ppm) or elevated [CO2] (700 ppm)
with 15NH415NO3 as the only mineral nitrogen source. Elevated
[CO2] increased the fraction of new C in total C, but it did not
alter C partitioning among plant compartments. Elevated
[CO2] also increased the fraction of new N in total N and this
was coupled with a shift in N source partitioning toward N2 fixation. Soil N uptake was unaffected by elevated [CO2], whereas
N2 fixation was markedly increased by the elevated [CO2]
treatment, mainly because of increased specific fixation (mg N
mg –1 nodule). As a result of increased N2 fixation, the C/N ratio of tree biomass tended to decrease in the elevated [CO2]
treatment. Partitioning of N uptake among plant compartments
was unaffected by elevated [CO2]. Total dry mass of root nodules doubled in response to elevated [CO2], but this effect was
not significant because of the great variability of root nodule
formation. Our results show that, in the N2-fixing R. pseudoacacia, increased C uptake in response to increased [CO2] is
matched by increased N2 fixation, indicating that enhanced
growth in elevated [CO2] might not be restricted by N limitations.
Keywords: carbon uptake, nitrogen uptake, N source partitioning, stable isotope.
Introduction
The atmospheric CO2 concentration ([CO2]) has risen dramatically in the past 120 years, primarily as a result of the combustion of fossil fuels and deforestation (Houghton et al. 2001). A
doubling of the preindustrial atmospheric [CO2] is expected
within this century (Houghton et al. 1995). Increased atmospheric [CO2] increases CO2 assimilation and growth of many
plant species, especially when other environmental resources
do not limit productivity (Bazzaz 1990, Saxe et al. 1998).
Many terrestrial ecosystems, especially forests, are nitrogen
(N)-limited (Johnson 1992, Vitousek et al. 1997) and several
studies indicate that the response of woody plants to elevated
[CO2] decreases as N availability decreases (Field et al. 1992,
Murray et al. 2000, Poorter and Pérez-Soba 2001). The negative acclimation of photosynthesis that is often observed in
trees grown in elevated [CO2] (Gunderson and Wullschleger
1994) may be a result of the decline in leaf N content that often
occurs under conditions of low soil N availability (Centritto
and Jarvis 1999, Norby et al. 1999, Curtis et al. 2000). Furthermore, it has been suggested that a persistent increase in plant
biomass production in elevated [CO2] can only be maintained
by a concomitant increase in N uptake, either from the soil
mineral N pool or from the atmosphere (Norby et al. 1986,
Gifford 1994, Soussana and Hartwig 1996, Saxe et al. 1998).
Symbiotic N2 fixation is an important source of N in many
terrestrial ecosystems (Boring et al. 1988, Ibewiro et al. 2000),
and the close coupling between C assimilation and symbiotic
N2 fixation suggests that N2 fixation should increase as atmospheric [CO2] increases (Polley et al. 1997, Schortemeyer et al.
2002). Because N2-fixing plants are largely independent of
soil N availability, they may become more competitive compared with non-fixing plants in a future CO2-enriched atmosphere, as has been shown for a grassland ecosystem (Lüscher
et al. 1998). Increases in growth and N2 fixation in elevated
[CO2] are well documented in annual legume species (Hebeisen et al. 1997, Arnone et al. 1999). There is evidence that
N2-fixing trees also have the capacity to increase both C assimilation and total N2 fixation with increasing atmospheric [CO2]
(e.g., Thomas et al. 2000, for Gliricidia sepium (Jacq.) Kunth
ex Walp., Schortemeyer et al. 2002, for several Acacia species). Increased N2 fixation of trees grown in elevated [CO2]
has been attributed to a greater mass or a greater activity of
root nodules, or both, leading to greater nitrogenase activity
per plant (Norby 1987, Tissue et al. 1997, Vogel et al. 1997,
Olesniewicz and Thomas 1999, Thomas et al. 2000). Increased N2 fixation by legume trees may also affect the CO2 response of associated non-fixing trees, because it leads to
increased soil N availability and amelioration of N limitations.
324
FENG, DYCKMANS AND FLESSA
In turn, it may also ameliorate nitrogen limitations to ecosystem C storage under elevated [CO2] conditions (Schimel
1995).
Black locust (Robinia pseudoacacia L.) is an important
component of many temperate forest ecosystems (Seeling
1997, DeGomez and Wagner 2001) and has a high N2-fixing
capacity (Danso et al. 1995). However, little is known about
how elevated [CO2] affects its growth, N uptake and nutrient
partitioning among plant organs. The objectives of our study
were to analyze the responses of 1-year-old R. pseudoacacia
seedlings to elevated [CO2] and to determine the effects of increased [CO2] on (1) C assimilation, (2) N accretion and plant
N source partitioning (N2 fixation versus mineral soil N uptake), (3) nodule formation, and (4) the partitioning of C and
fixed N among tree organs. A continuous 13C and 15N labeling
experiment was performed in which black locust seedlings
were grown for 16 weeks in ambient or elevated [CO2] with
15
NH415NO3 as the only mineral nitrogen source.
growth conditions are comparable with those found in forest
undergrowth in central Europe. Details of the growth chamber
system are given in Dyckmans et al. (2000a, 2000b).
Sampling and analysis
At the beginning of the CO2 experiment, 10 pretreated plants
were analyzed for total fresh mass and total N content. From
these data, the initial N content of the trees used in the CO2 experiment was estimated (see below). After 16 weeks of growth
in the CO2 treatments, six plants were harvested from each
CO2 treatment. The plants were divided into leaves + branches,
stem, fine roots (< 2 mm), coarse roots (> 2 mm) and nodules.
All samples were dried at 65 °C and finely ground.
Total C and N contents, and 13C/12C and 15N/14N isotope ratios were measured with an elemental analyzer (EA 1108,
Fison Instruments, Milan, Italy) coupled to an isotope ratio
mass spectrometer (IRMS Delta plus, Finnigan Mat, Bremen,
Germany) operating in the online mode.
Assimilation and partitioning of C
Materials and methods
Plant material
Black locust seeds (R. pseudoacacia) were germinated and the
seedlings were grown in vermiculite under roofed-over freeair conditions for 1 year before the CO2 treatments were applied. The seedlings were fertilized with 15NH415NO3
(9.3 atom% 15N) as the only nitrogen source along with a
Hoagland-based nutrient solution (Hoagland and Arnon
1950). No root nodule formation was observed during this pretreatment. The mean 15N abundance of the seedlings was
9.16 atom% at the end of the first year. The seedlings used for
the CO2 experiment had a mean dry mass (estimated from
fresh mass) of 2.6 ± 0.5 g.
Microcosms and growth chamber
At the beginning of the CO2 experiment, the 1-year-old trees
were inoculated with Rhizobium spp. (Liphatec, Milwaukee,
WI) and Co2+ (0.02 mM) was added to the nutrient solution to
enhance infection and nodule formation. Two weeks before
leafing, the trees were planted in PVC cylinders (height 0.3 m,
diameter 0.14 m) containing sand. The planted trees were installed in growth chambers and watered weekly with 130 cm3
of a Hoagland-based nutrient solution (4 mM 15NH415NO3,
9.3 atom% 15N). This mineral N supply has been shown to increase seedling growth but affect nodule formation and activity only moderately compared with seedlings growing without
mineral N supply (Johnsen and Bongarten 1991). We supplied
CO2-free air continuously to the growth chambers at a rate of
125 cm3 s –1. A CO2 analyzer (UNOR 610, Maihak, Hamburg,
Germany) connected to the growth chambers controlled the injection of CO2 containing δ13C = –48.0‰ (Messer-Griesheim,
Duisburg, Germany) and maintained the [CO2] at 350 and
700 ppm for the ambient and elevated [CO2] treatments, respectively. The trees were grown in a 12-h photoperiod at an
irradiance of 130 µmol m –2 s –1 and day/night temperatures of
18/13 °C. Relative humidity was maintained at 75%. These
The labeling of the chamber atmosphere allowed us to distinguish between C taken up during the CO2 experiment and C
present in the pre-existing plant material. Hereafter, the C
taken up by the trees during the experiment is referred to as
new C.
The isotopic signal for C was expressed as δ13C versus the
international standard (Belemnite (δ13C = 0.00‰) from Pee
Dee formation, SC):
 R

δ13C =  sample – 1 1000
 Rstandard

(1)
where R is 13C/12C ratio.
Relative specific allocation (RSA), which describes the
fraction of newly incorporated C or labeled nitrogen in the tissue ( flabeled) relative to total C or N in a given sample (Deléens
et al. 1994) was calculated from:
E p = f labeled E l + (1 − f labeled )E c
f labeled =
Ep − Ec
El − Ec
= RSA labeled E
(2)
(3)
where Ep is the isotopic signal of the plant sample, Ec is the isotopic signal of the unlabeled control plant, and El is the isotopic signal of the labeled C in the plant.
The isotopic signal of labeled C in the plant was influenced
by both the isotopic signal of the air supply and discrimination
during plant uptake. We assumed that new root tips consisted
exclusively of new assimilates, and used the isotopic signals of
root tips harvested at the end of the experiment as a standard
for El (Dyckmans et al. 2000a). This approach accounts for
both 13C labeling of the chamber atmosphere and 13C discrimination during C assimilation in the ambient and elevated [CO2]
treatment, respectively.
Partitioning describes the proportion of the newly acquired
TREE PHYSIOLOGY VOLUME 24, 2004
EFFECTS OF ELEVATED [CO 2] ON N2 FIXATION OF BLACK LOCUST
(labeled) element in a given plant organ relative to the total labeled element in the whole plant (Deléens et al. 1994). The
partitioning of new C and labeled N was calculated as:
PE % =
E organ f labeled organ
E plant f labeled plant
(4)
100
where Eorgan is the amount of C or N in the specific plant organ
and Eplant is the amount of C in the whole plant.
N2 fixation, soil N uptake and partitioning of N
Because soil N was labeled, we could distinguish between N
uptake from the soil and N derived from seed or N2 fixation by
the 15N dilution method. The isotopic signal for N was expressed as atom% 15N (A%) and was derived as:
15
A% =
14
N
100
N + 15N
(5)
where 14N and 15N are the amounts of 14N and 15N isotopes in
the sample, respectively.
Relative specific allocation was calculated as the fraction of
labeled (i.e., soil derived) and unlabeled N (i.e., from seed or
N2 fixation) relative to total N in a given sample according to
Equation 3. The isotopic signal of the nutrient solution was
used as El.
To distinguish between mineral N taken up from soil during
pretreatment and during the CO2 experiment (both labeled) on
the one hand, and between N2 fixation and N derived from the
seed (both unlabeled) on the other hand, we estimated the N
content of the plants at the beginning of the CO2 experiment.
One-year-old trees were sampled before the beginning of the
CO2 experiment and fresh mass was correlated with N content
of these trees. The trees had a mean N concentration of 0.78%
(related to fresh biomass) and the initial N content of the individual trees after the pretreatment (Ni; g) could be estimated
reliably from fresh mass according to:
N i = 0.0062 FM + 0.0107
(6)
where FM is fresh mass of individual trees. The r 2 of the correlation was 0.955.
Seed N content (Nseed) was determined from mean seed dry
mass (20.3 mg) and mean seed N concentration (6.15%).
Unlabeled N refers to the N derived either from the seed or
from atmospheric N2 fixation, and Nfix refers to the amount of
N2 fixed, which was calculated as:
N fix = Nt RSA Nu − N seed
(7)
N soil / old = N i − N seed
(8)
The abbreviation Nsoil/new refers to N that was taken up from
the soil during the CO2 experiment, and was calculated as:
N soil / new = Nt RSA Nl − N soil / old
(9)
where RSANl is the fraction of labeled (i.e., soil-derived) N in
the plant.
Because the contribution of seed N to the unlabeled N in
trees at the end of the CO2 experiment was < 5%, partitioning
of the nitrogen from N2 fixation (Nfix) was estimated from the
partitioning of total unlabeled N according to Equation 4.
Statistics
Results were expressed as arithmetic means with standard deviation. The significance of treatment effects was tested by the
t-test, assuming a normal distribution of the data. Probabilities
less than 0.05 were considered to be significant, whereas probabilities > 0.1 were considered to indicate a trend.
Results
Assimilation and partitioning of C
Elevated atmospheric [CO2] had a strong positive effect on C
assimilation of black locust trees. At the whole-plant level,
RSA of new C increased by 31%, from 51.2% in ambient
[CO2] to 67.0% in elevated [CO2] (Table 1). Increased C assimilation was found in all plant compartments (Figure 1a);
however, the contribution of stored C to the formation of
leaves, branches and nodules was less than 20% in ambient
[CO2] and less than 10% in elevated [CO2] (Figure 1a).
At the whole-plant level, the amount of C assimilated almost doubled in response to elevated [CO2], for both above-
Table 1. Dry mass, relative specific allocation (RSA) of new carbon
(C), nitrogen (N) concentration, C/N ratio, nodule dry mass and N2
fixation per nodule dry mass (Nfix) of trees grown for 16 weeks in ambient or elevated [CO2] and relative changes between the treatments.
Values are means with standard deviation in parenthesis. An asterisk
denotes significant differences between means (P < 0.05) and the degree symbol (°) indicates a trend (P > 0.1).
Property
Ambient
Elevated
Elevated/ambient
Dry mass (g)
4.73
(1.30)
51.2
(6.5)
2.25
(0.30)
19.5
(2.5)
91.7
(60.1)
0.21
(0.07)
6.68
(2.61)
67.0
(11.4)
2.41
(0.20)
17.5
(1.4)
186.3
(123.4)
0.37
(0.23)
1.41
RSA Cnew (%)
N concentration (%)
C/N ratio
where Nt is total N in the plant at the end of the experiment,
RSANu is the fraction of unlabeled N in the plant and Nseed is
the N derived from the seed.
The abbreviation Nsoil/old refers to N uptake from the soil
during the pretreatment and was calculated as:
325
Nodule dry mass (mg)
−1
)
Nfix (mg N mg DM
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1.31*
1.07
0.90°
2.03
1.76*
326
FENG, DYCKMANS AND FLESSA
Figure 2. Effect of a 16-week elevated [CO2] treatment on (a) total
carbon (C) uptake per plant calculated as the sum of aboveground
(solid and open bars) and belowground (hatched bars) allocated carbon for the 350 and 700 ppm CO2 treatments, and (b) total nitrogen
(N) uptake per plant calculated as the sum of N2 fixation (solid and
open bars) and N uptake from soil (hatched bars) for the 350 and
700 ppm CO2 treatments. Means and standard deviations are presented. An asterisk indicates significant differences between treatments (P < 0.05) and the degree symbol (°) indicates a trend (P < 0.1).
Figure 1. Effects of a 16-week elevated [CO2] treatment on (a) the
fraction of new carbon in total carbon among compartments, and (b)
the fraction of unlabeled (i.e., from seed and N2 fixation) nitrogen in
total nitrogen among compartments. Means and standard deviations
are presented. An asterisk indicates significant differences between
treatments (P < 0.05) and the degree symbol (°) indicates a trend (P <
0.1).
ground and belowground allocation (Figure 2a). Despite
increased C assimilation in response to elevated [CO2], there
was no significant increase in total dry mass of the trees at the
end of the CO2 experiment (Table 1).
We analyzed the partitioning of new C to obtain information
about C assimilation into different compartments. In both CO2
treatments, the strongest sink was the leaves + branches, where
nearly half of the new C was allocated. Stem and roots (fine
roots + coarse roots) each accounted for about 25% of the new
C. Nodule biomass accounted for 5 and 6% of the new C in
ambient and elevated [CO2], respectively. Partitioning of new
C expressed as a root/shoot ratio was 0.32 in the ambient
[CO2] treatment and 0.33 in the elevated [CO2] treatment.
There was no significant effect of elevated [CO2] on the partitioning of new C among tree compartments or on the
root/shoot ratio of new C (Figure 3a); we note, however, that
we did not account for belowground respiration, which can be
a major C sink especially in N2-fixing plants.
N2 fixation, soil N uptake and partitioning of nitrogen
Total nitrogen uptake increased threefold in response to elevated [CO2] (P = 0.066; Figure 2b). The fraction of new N in
total N significantly increased from 29% in ambient [CO2] to
51% in elevated [CO2] (Figure 4). The N concentration of the
trees was not altered significantly by the elevated [CO2] treatment and the C/N ratio of the whole plant tended to decrease in
the elevated [CO2] treatment compared with the ambient
[CO2] treatment (Table 1).
The main source of the increased N uptake in elevated [CO2]
was N2 fixation, which increased 3.5-fold compared with N2
fixation in ambient [CO2] (Figure 2b, P = 0.069), yielding
75.8 mg N from N2 fixation per tree in elevated [CO2]. The
amount of new N uptake from soil increased by 92%
(P = 0.250) to 18.9 mg. However, the variability in total
amount of new N uptake was extremely high as shown by the
standard deviations presented in Figures 2 and 4. The fraction
of N derived from N2 fixation in total N (RSAN fix ) was more
than doubled in the elevated [CO2] treatment compared with
the ambient [CO2] treatment (P = 0.036; Figure 4). In contrast,
the fraction of new N from soil uptake in total N (RSANsoil(new) )
was unaffected by atmospheric [CO2] (Figure 4).
The fraction of unlabeled N (which mainly represents N
from N2 fixation but also includes the seed N) increased in all
plant compartments in elevated [CO2] compared with ambient
[CO2], although the increase was only a trend in the fine roots
and in the nodules (Figure 1b).
Mean nodule dry mass doubled in elevated [CO2], but this
effect was not significant because of the high variability in root
nodule formation (Table 1). Specific nodule activity expressed
as mg N fixed mg –1 nodule dry mass was significantly greater
in elevated [CO2] than in ambient [CO2] (Table 1).
Partitioning of unlabeled N and labeled N, and consequently
of total N, was similar between CO2 treatments (Figure 3b).
The main N sinks were leaves + branches, which contained
TREE PHYSIOLOGY VOLUME 24, 2004
EFFECTS OF ELEVATED [CO 2] ON N2 FIXATION OF BLACK LOCUST
327
more than 40% of the N from N2 fixation (Figure 3b). The
aboveground plant compartments accounted for about 60% of
total N. Among organs, root nodules contained the greatest
fraction of unlabeled N in total N (70 to 80%) (Figure 1b);
however, only about 5% of total N in trees was found in root
nodules in both CO2 treatments (data not shown).
Discussion
Figure 3. Effects of a 16-week elevated [CO2] treatment on the partitioning of (a) new carbon and (b) unlabeled nitrogen (i.e., from seed
and N2 fixation) among compartments. Means and standard deviations are presented. An asterisk indicates significant differences between treatments (P < 0.05) and the degree symbol (°) indicates a
trend (P < 0.1).
Figure 4. Effect of a 16-week elevated [CO2] treatment on the fraction
of nitrogen (N) uptake during the CO2 experiment from the soil
( RSA N soil(new) ), N2 fixation (RSA N fix ), and total new N (from soil and
N2 fixation, RSA N new ) on total plant N. Means and standard deviations
are presented. An asterisk indicates significant differences between
treatments (P < 0.05).
Elevated [CO2] increased C allocation in all plant compartments (Figure 1a) and nearly doubled (85% increase) total C
uptake (Figure 2a). In both treatments, newly developed tree
organs comprised mainly new assimilates (more than 80% of
the C in leaves + branches and nodules was derived from new
C uptake), with greater relative importance of new C uptake in
trees grown in elevated [CO2] than in trees grown in ambient
[CO2] (Figure 1a). This indicates that tree internal C stores
played only a minor role in the formation of leaves and root
nodules. Internal C stocks were more important for the formation of leaves in beech trees (which exhibit a largely determinate growth pattern), where about 45 and 30% of the leaf C
originated from previously formed C stocks of trees grown in
ambient and elevated [CO2], respectively (Dyckmans et al.
2000a).
Elevated [CO2] led to a 194% increase in N uptake (Figure 2b) that resulted in a 78% increase in RSAN new along with
strongly altered N source partitioning in elevated [CO2] compared with ambient [CO2] (Figure 4). The increased N uptake
in elevated [CO2] was mainly caused by increased N2 fixation,
because soil N uptake was not significantly affected (Figures 2b and 4). This resulted in a doubling of the RSAN fix in elevated [CO2] compared with ambient [CO2], whereas the
contribution of soil N uptake to total N was unaltered. Several
studies on N2-fixing trees have also shown a higher responsiveness to elevated [CO2] of N2 fixation compared with soil N
uptake. Olesniewicz and Thomas (1999) reported that the fraction of N from N2 fixation was almost doubled in elevated
[CO2] compared with ambient [CO2] in 56-day-old nonmycorrhizal R. pseudoacacia, whereas this effect was not observed in mycorrhizal seedlings. Thomas et al. (2000) analyzed the effect of elevated [CO2] on N2 fixation of seedlings
of the tropical tree G. sepium and found a fourfold increase in
N2 fixation, whereas N uptake from soil was unaffected by elevated [CO2], despite high N fertilization rates. These findings,
together with our results, show that N2-fixing trees acclimate
to the increased N demand induced by accelerated growth in
elevated [CO2] by enhancing N2 fixation. We note that this
conclusion is based on studies with tree seedlings, because
there is no information available on the effects of elevated
[CO2] on N uptake and N source partitioning in older N2-fixing
trees.
The ability to acquire N from the atmosphere by symbiotic
N2 fixation may offset the growth limitation resulting from insufficient soil N availability that is often observed under elevated [CO2] conditions (Field et al. 1992, Johnson et al. 1998,
Maillard et al. 2001). However, the costs (in terms of respira-
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328
FENG, DYCKMANS AND FLESSA
tory losses of C) are higher for N2 fixation than for soil N uptake (Warembourg and Roumet 1989). Consequently, N2 fixation is decreased by high N or low C supply and promoted by
low N or high C supply (Houwaard 1980, Hartwig et al. 1987,
Walsh 1990). In both treatments, N2 fixation was the main
source of N during the experiment. Similarly, Danso et al.
(1995) reported that, on average, 80% of total N is derived
from N2 fixation in 4-year-old field-grown black locust.
Although the RSA data showed a strong CO2 effect on uptake and source partitioning of N, there was only a trend of increased N2 fixation in response to elevated [CO2] (P = 0.069;
Figure 2b) because of the high variability in tree N uptake,
which originated mainly from two sources. First, growth of individual trees differed, although 1-year-old trees with a similar
biomass and height were selected at the beginning of the experiment. Second, because we used 1-year-old trees, the
amount of N in the trees at the beginning of the CO2 experiment had to be estimated from control plants.
Because elevated [CO2] increased N2 fixation, the C/N ratio
of total tree biomass tended to decrease in elevated [CO2] (Table 1), indicating that N assimilation of R. pseudoacacia kept
pace with C assimilation and that the N status even improved
under elevated [CO2] conditions compared with ambient
[CO2] conditions. Similar findings have been reported in several other N2-fixing trees. Thus, tissue N concentrations were
unaffected by elevated [CO2] in mycorrhizal R. pseudoacacia
(Olesniewicz and Thomas 1999) and in Alnus glutinosa (L.)
Gaertn. (Vogel et al. 1997, Temperton et al. 2003) and the tropical tree species G. sepium (Tissue et al. 1997, Thomas et al.
2000). Cotrufo et al. (1998) reviewed the effects of elevated
[CO2] on plant N concentrations and found that the mean reduction in N concentration was significantly smaller in N2-fixing species (7%, n = 68) than in non-fixing C3 species (16%,
n = 278).
Partitioning of assimilated C was unaltered by elevated
[CO2]. This finding contrasts with model predictions (Lacointe 2000) and with the results of studies on non-fixing trees
where belowground allocation of C increased, especially under conditions of low-N availability (Zak et al. 1993, Pregitzer
et al. 2000, Dyckmans and Flessa 2002). The partitioning of N
was also unaffected by elevated [CO2], probably as a result of
the altered N source partitioning in black locust in response to
elevated [CO2]. Under elevated [CO2] conditions, the trees did
not increase their root system to increase soil N uptake, but
rather increased root nodule mass, nodule activity and N2 fixation (Table 1). This finding is in agreement with other studies
on the response of N2-fixing trees to elevated [CO2], where either increased nodule activity, increased nodule mass, or both
has been found (Norby 1987, Tissue et al. 1997, Vogel et al.
1997, Olesniewicz and Thomas 1999, Thomas et al. 2000).
We did not include belowground CO2-C losses, which are
expected to increase with increasing [CO2] as a result of increased N2 fixation. Energy expenditure for N2 fixation is estimated to be 3 mg C mg –1 N fixed (Warembourg and Roumet
1989, Schulze et al. 1999). Based on this value, we estimated
that belowground CO2-C losses due to N2 fixation (21.8 and
75.8 mg N in the ambient and elevated [CO2] treatments,
respectively) in our experiment were 65 and 227 mg C in the
ambient and elevated [CO2] treatments, respectively. These
losses correspond to 21 and 42% of the new C allocated to the
root system for the ambient and elevated [CO2] treatments, respectively.
In a model simulating the role of N2-fixing trees in forest
succession, Rastetter et al. (2001) identified elevated [CO2] as
a factor favoring N2 fixation and canopy closure, with canopy
closure being one of the main reasons for the disappearance of
N2 fixing trees in later successional states. Our finding that elevated [CO2] increases C uptake and N2 fixation of black locust
even at relatively low irradiances (130 µmol m –2 s –1) indicates
that the dominance of N2-fixing species might be prolonged
during ecosystem development under elevated [CO2] conditions.
We showed that growth of R. pseudoacacia in elevated
[CO2] will not primarily be limited by N availability. The species will therefore be at a competitive advantage with nonN2-fixing tree species. The observed increase in N2 fixation in
response to elevated [CO2] may also improve N availability for
non-fixing trees and thus play a key role in the growth response of forest ecosystems to elevated [CO2].
Acknowledgments
Z.F. was financially supported by DAAD (Deutscher Akademischer
Austauschdienst). J.D. was financially supported by COFORD (National Council for Forest Research and Development), Dublin, Ireland. The authors thank Lars Szwec for excellent technical support.
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