Tree Physiology 17, 231--240
© 1997 Heron Publishing----Victoria, Canada
Photosynthetic responses of Scots pine to elevated CO2 and nitrogen
supply: results of a branch-in-bag experiment
SEPPO KELLOMÄKI and KAI-YUN WANG
University of Joensuu, Faculty of Forestry, P.O.Box 111, FIN-80101 Joensuu, Finland
Received February 28, 1996
Summary Naturally seeded Scots pine (Pinus sylvestris L.)
trees, age 25--30 years, were subjected to two soil-nitrogensupply regimes and to elevated atmospheric CO2 concentrations by the branch-in-bag method from April 15 to September
15 for two or three years. Gas exchange in detached shoots was
measured in a diffuse radiation field. Seven parameters associated with photosynthetic performance and two describing
stomatal conductance were determined to assess the effects of
treatments on photosynthetic components. An elevated concentration of CO2 did not lead to a significant downward regulation
in maximum carboxylation rate (Vcmax ) or maximum electron
transport rate (Jmax ), but it significantly decreased light-saturated stomatal conductance (g sat ) and increased minimum
stomatal conductance (gmin ). Light-saturated rates of CO2 assimilation were higher (24--31%) in shoots grown and measured
at elevated CO2 concentration than in shoots grown and measured at ambient CO2 concentration, regardless of treatment
time or nitrogen-supply regime. High soil-nitrogen supply
significantly increased photosynthetic capacity, corresponding
to significant increases in Vcmax and Jmax . However, the combined elevated CO2 + high nitrogen-supply treatment did not
enhance the photosynthetic response above that observed in the
elevated CO2 treatment alone.
Keywords: carboxylation rate, electron transport rate, Pinus
sylvestris, stomatal conductance.
Introduction
Photosynthetic responses of plants to elevated atmospheric
concentrations of CO2 are complex and depend on many physiological and environmental variables. Despite numerous studies on photosynthetic responses to elevated atmospheric CO2
concentrations, many uncertainties remain (Cure and Acock
1986, Ziska et al. 1990, 1991, Idso et al. 1991, Pettersson et al.
1992, Dufrêne et al. 1993, Gunderson et al. 1993).
Depending on growth conditions and experimental methods, responses of leaf-level photosynthesis to elevated CO2
concentrations may reflect combined changes in biochemical
capacity and leaf morphology. Results from gas exchange and
metabolic studies (Farquhar and von Caemmerer 1982,
Sharkey 1985, von Caemmerer and Edmondson 1986) suggest
that changes in the biochemical capacity of photosynthesis can
be induced by Rubisco activity, the rate of RuP2 regeneration
or triosephosphate consumption (Sage et al. 1989, Sage 1990,
Stitt 1991). In addition, plants grown under inadequate nutritional supplies are often unable to respond to elevated atmospheric CO2 concentrations (Eamus and Jarvis 1989, Evans
1989, Arp 1991). Morphological changes in response to elevated CO2 may include accumulation of carbohydrates as a
result of inadequate sink demands for photosynthates (Clough
et al. 1981, Sage et al. 1989), changes in leaf thickness and
changes in mesophyll cell number (Vu et al. 1989). In principle, well-established models can be used to quantify modifications in these key photosynthetic components (Harley et al.
1992, McKee and Woodward 1994, Mitchell et al. 1995).
The response of trees to elevated CO2 concentrations may be
expected to mirror that of other C3 species; however, forest tree
longevity raises questions about long-term effects about which
little is known. For example, it is not known whether shortterm enhancement of photosynthesis by elevated CO2 concentration can be sustained over several growing seasons. For
several reasons, most CO2 enrichment experiments have been
confined to short-term studies on juvenile trees (Eamus and
Jarvis 1989), but it is not known whether these observations
can be extrapolated to mature trees (Cregg et al. 1989). The
branch-in-bag method makes it possible to examine the effects
of elevated CO2 concentrations on mature trees without the
technical difficulties and expense of subjecting entire trees to
the CO2 treatment (Barton et al. 1993, Dufrêne et al. 1993).
The validity of branch-in-bag experiments is based on the
finding that branches are relatively autonomous with respect to
their carbon budget during the growing season. The assumption that branches do not import carbon but only export it to
woody tissues and roots is considered valid for large branches
after initial shoot elongation has ceased (Sprugel et al. 1991).
We measured gas exchange in shoots of Scots pine (Pinus
sylvestris L.) trees grown in two atmospheric CO2 concentrations and two soil-nitrogen regimes for two to three years.
Specifically, we determined: (1) how long-term elevation of
CO2 concentration affects photosynthetic performance; (2)
whether a high soil-nitrogen supply enhances the response of
photosynthesis to elevated atmospheric CO2 concentrations;
(3) whether different lengths of exposure (two or three years)
to elevated CO2 concentration lead to differences in photosyn-
232
KELLOMÄKI AND WANG
thetic capacity; and (4) whether there is a coupled relationship
between modifications in photosynthetic components and leaf
nitrogen concentration. The main physiological parameters
concerning photosynthetic processes were determined by fitting the data to a set of photosynthetic models and to an
empirical model of stomatal conductance.
Materials and methods
Experimental design
The experiment was established in a naturally seeded stand of
Scots pine near the Mekrijärvi Research Station (62°47′ N,
30°58′ E, 145 m elevation), University of Joensuu, Finland.
The soil on the site is sandy with a water retention of 7 mm at
field capacity and 3 mm at the wilting point for the top 30-cm
layer of soil. The mean density of the pure Scots pine stand was
about 2500 stems per hectare. The trees were 25--30 years old
and had a mean height of 4--5 m.
Three branches of the third whorl from the stem apex on
each tree were chosen for the treatments: one branch was
placed in a bag and subjected to an elevated CO2 concentration
(Bagged + CO2); one branch was placed in a bag and subjected
to ambient CO2 concentration (Bagged); and one branch was
left as an unbagged control (Unbagged). Starting in early
spring of 1993, six sample trees representing six replicates of
each treatment were exposed to the CO2 treatments. In early
spring of 1994, a similar experiment was set up with 12 trees
per CO2 treatment. Within each CO2 treatment, half of the trees
were grown under normal soil conditions of the site (NormalN), whereas the remaining trees received an improved nitrogen
supply (High-N); i.e., 0.7 kg of pure nitrogen in the form
NH4NO3 was added to a 4 × 4 m area around each tree.
The design of the branch bags was similar to that described
by Barton et al. (1993). Briefly, the bags consisted of a cylindrical wire frame covered with a polyethylene sleeve which
was made of PVC plastic (thickness 0.4 mm, protected from
deterioration by UV radiation) and had a volume of 0.379 m3
(length 1.65 m, diameter 0.6 m). The sleeve material prevented
only a small fraction of radiation (approximately 10%), within
the spectral range 400--800 nm, from entering the bags, and
had only a small impact on the spectral composition of visible
radiation.
Air was supplied to each bag through a 10-cm-diameter
duct. Air in the duct was driven by a blower fan (maximum air
flow = 190 dm3 s −1) that was mounted in a weather-proof
housing. A butterfly valve was positioned in the duct, close to
the housing, to enable manual setting of the air flow rate which
was usually 0.02--0.025 m −3 s −1.
The CO2 concentration inside the bags providing the elevated CO2 treatment was increased to 670--820 µmol mol −1
from April 15 to September 15, by injecting pure CO2 into the
duct (Figure 1). The CO2 concentration inside the bags was
monitored with a CO2 analyzer (Model 7MB1300-0BA00,
Siemens, Germany).
Relative humidity and temperature inside and outside the
bags were recorded separately with a probe combining a tem-
Figure 1. Frequency distribution of atmospheric CO2 concentration in
bags supplied with CO2-enriched air. The plot is based on hourly
means of 30-s readings. Data are for the period April 15 to September
15, 1995.
perature sensor and a Humicap relative humidity sensor (Model
HMP131Y, Vaisala, Vantaa, Finland). During most of the day,
temperature and relative humidity inside the branch bags were
only slightly higher than ambient values (Figures 2a and 2b)
except during short periods of intense sunshine, when temperatures inside the bags exceeded ambient by 4--6 °C (about
10% of the time) and relative humidity inside the bags exceeded ambient by 10--15% (about 20% of the time).
Figure 2. Daily courses of temperature (a) and relative humidity (b)
inside and outside the bags. The plot is based on hourly means of 30-s
readings. Data are for the period from August 1 to August 31, 1995.
PHOTOSYNTHETIC RESPONSES IN SCOTS PINE
233
Measurement and analysis of gas exchange
Measurements of CO2 exchange in a diffuse radiation field
were conducted in the laboratory with an automatic open-gasexchange system as described by Wang (1996).
A one-year-old shoot was excised under water and transported to the laboratory. After the shoot had been recut under
water, a 10-cm-long section of shoot with only 30 needle
fascicles (surplus needles had been thinned evenly one week
before conducting the measurements to minimize shoot structure effects) was sealed in the assimilation cuvette. The base of
the shoot was inserted in a tube providing a constant water
supply throughout the measurements.
Gas-exchange measurements were conducted on two shoots
from each branch bag through July and August 1995. Photosynthetic parameters were first estimated on the basis of a
single branch bag. Then the mean of the estimated parameters
from the six replicated branch bags representing the same
treatment was used to analyze the results. During all measurements, air temperature in the assimilation cuvette was maintained at 20 ± 0.5 °C and water vapor pressure deficit remained
below 0.6 kPa, except during measurements to estimate stomatal conductance parameters. Gas-exchange parameters
were calculated by the method of von Caemmerer and Farquhar (1981). Rate of net assimilation (An) was based on
projected needle area measured with a leaf-area meter (LI3100, Li-Cor, Inc., Lincoln, NE).
Immediately after the gas-exchange measurements, all needles were removed from the shoot, after which their projected
area, fresh weight, and weight after drying at 75 °C were
determined. In addition, their nitrogen concentration was determined by the micro-Kjeldahl method.
Estimation of photosynthetic parameters
Three sets of response curves were obtained for each shoot.
First, slopes of An versus the calculated intercellular concentration of CO2 (An/Ci curve) at photon flux densities (Qp) of 150
and 700 µmol m −2 s−1 were determined by least-squares linear
regression. Respiration rate in light (Rd) and CO2 compensation point in the absence of nonphotorespiratory respiration
(Γ∗) were estimated from the intercept of the two slopes, as
described by Brooks and Farquhar (1985). Representative examples of An/Ci curves, measured at the two Qp values used to
estimate Rd and Γ*, are shown in Figure 3.
Then An/Ci curves were measured at nearly saturating Qp
(1500 µmol m −2 s −1). Based on the estimated Rd and Γ*, Equation 1 was fitted to each An/Ci curve. The maximum RuP2-saturated rate of carboxylation (Vcmax ) was estimated for each
branch bag:
Anc =
Vcmax (C i − Γ∗)
− Rd,
Ci + K c(1 + O i /K o)
(1)
where Anc is RuP2-saturated rate of CO2 assimilation, Oi is
intercellular concentration of O2 (198 µmol mol −1), and Kc and
Ko are the Michaelis constants for carboxylation and oxygena-
Figure 3. Representative partial An/Ci curves measured at two irradiances, 150 µmol m −2 s −1 (s) and 700 µmol m −2 s −1 (d) air temperature at 20 ± 0.5 °C and vapor pressure deficit less than 0.6 kPa, for
shoots grown under different environmental conditions (Bagged +
CO2, Bagged, and Unbagged) for three growing seasons. Slopes were
fitted by least-squares linear regression. The CO2 compensation point,
Γ*, in the absence of nonphotorespiratory respiration and respiration
rate in light, Rd, were estimated by solving for the intercept of the two
slopes (Brooks and Farquhar 1985).
tion, respectively. Values of Kc (358.6) and Ko (255.9) at 20 °C
were derived from the measurements as described by Badger
and Collatz (1977).
Finally, An/Qp curves were measured at a CO2 concentration
of 2000 ± 10 µmol mol −1. Three parameters, maximum rate of
electron transport (Jmax ), effectivity factor for use of light (q)
and convexity factor of light-response curve (θ) were estimated in Equation 2 by the nonlinear least-squares regression
techniques described by Ziegler-Jöns and Selinger (1987):
234
Anj =
KELLOMÄKI AND WANG
Jmax + qQ p − [(Jmax + qQ p)2 − 4θqQ pJmax ]0.5
− Rd, (2)
9θ
where Anj is the RuP2-regeneration limited rate of CO2 assimilation.
Estimate of stomatal parameters
The model of Ball et al. (1987), modified by Leuning (1990,
1995) and Collatz et al. (1991), was applied to describe the
response of stomatal conductance, gs, to variations in net
assimilation rate (An), leaf-to-air vapor pressure difference
(Ds), and CO2 mole fraction (Cs) at the leaf surface:
g s = gmin + g 1
1000 An
,
D s(C s − Γ∗)
(3)
where g min is minimum stomatal conductance to water vapor
and g1 is an empirical coefficient. We assumed: (1) Cs equals
the CO2 mole fraction inside the cuvette (Ca); (2) units are
corrected by the factor 1000 when all units in Equation 3
coincide with those presented in the Appendix; (3) Ds is the
difference between saturated vapor pressure at air temperature
and actual vapor pressure inside the cuvette, because leaf
temperature maintained a relatively constant boundary conductance that was fairly large (750--800 mmol m −2 s −1). Two
stomatal parameters, gmin and g1, were estimated by fitting
Equation 3 to observations within the ranges: 0 < Qp < 1500
µmol m −2 s −1, 50 < Cs < 1400 µmol mol −1, and 0.3 < Ds <
2.0 kPa (Figure 4).
Statistical analysis
Responses of photosynthesis to environmental factors were
analyzed in terms of the parameters of the fitted function.
Analysis of variance was applied to test the effects of growth
conditions. First, effects of CO2 concentration and soil-nitrogen supply on each parameter were analyzed (multifactor
ANOVA) using the SPSS/PC software package (SPSS, Chicago, IL) based on shoots grown in bags, assuming that significant responses were attributable to the aforementioned
treatments, and not to some unknown bagging effects. Treatment time was analyzed as repeated measures during the two
growth seasons. Finally, one-way ANOVA was applied to test
for the effect of bagging.
Results
Maximum RuP2-saturated rate of carboxylation, Vcmax , and
maximum rate of electron transport, Jmax
Both Vcmax and Jmax were significantly affected by the High-N
treatment (Tables 1 and 2). Compared to the Normal-N treatment, the High-N treatment increased Vcmax and Jmax on average by 33 and 11%, respectively. Regardless of duration of
exposure, elevated CO2 alone did not significantly affect
Vcmax or Jmax , although Vcmax was depressed 11% after the third
Figure 4. Stomatal conductance, gs, plotted against the stomatal index
(An/[(C s − Γ∗)D s]) of Equation 3 for shoots of Scots pine grown in
different environmental conditions (Bagged + CO2, Bagged, and Unbagged). Observations are within the ranges: photon flux densities of
50--1500 µmol m −2 s −1, vapor pressure deficits of 0.3--2.0 kPa, air
temperature of 20 °C, and CO2 concentrations of 20--2000 µmol mol−1.
Symbols represent measured values. The lines are fitted curves obtained by Equation 3. (a) With Normal-N after three growth seasons,
(b) with High-N after two growth seasons.
growing season. Effects of elevated CO2 on Vcmax and Jmax
were not enhanced by High-N (Table 2). Bagging resulted in
only a slight decrease in Jmax (Table 1).
Carbon dioxide compensation point in the absence of dark
respiration, Γ*, and respiration rates
The CO2 compensation point (Γ*), and respiration rates in the
light (Rd) and the dark (Rn) were significantly affected by
elevated CO2 (Tables 1 and 2). Compared to the bagged treatment alone, elevated CO2 concentration decreased Γ* on average by 9%, and increased Rd and Rn by 22 and 18%,
respectively. Furthermore, only in the case of Rn was this
increase significantly enhanced by both High-N and treatment
time. The High-N treatment resulted in a slight increase in the
CO2-induced increase in Rd. There was no effect of bagging on
any of the three parameters; however, treatment time led to a
significant increase (17%) in Rn (Table 1).
PHOTOSYNTHETIC RESPONSES IN SCOTS PINE
235
Table 1. Parameter estimates as functions of treatments. Data are means ± SE of estimated values from six replicated branch bags per treatment.
‘‘Bagged + CO2’’ are bagged shoots with CO2 concentration of 700 µmol mol--1; ‘‘Bagged’’ are bagged shoots with CO2 concentration of 350 µmol
mol --1 and ‘‘Unbagged’’ are unbagged shoots with CO2 concentration of 350 µmol mol --1.
θ
gmin
After two growing seasons with High-N
60.3 ± 2.7 172.6 ± 6.1 32.11 ± 2.4 1.090 ± 0.06 1.678 ± 0.04 0.388 ± 0.03
Bagged + CO2
Bagged
59.8 ± 1.4 169.7 ± 4.5 35.07 ± 1.8 0.918 ± 0.04 1.311 ± 0.07 0.396 ± 0.04
Unbagged
61.6 ± 2.2 177.1 ± 4.2 35.22 ± 2.1 0.929 ± 0.07 1.347 ± 0.06 0.401 ± 0.06
0.87 ± 0.05
0.74 ± 0.03
0.72 ± 0.07
53.7 ± 1.3 3.55 ± 0.20
47.9 ± 2.1 3.83 ± 0.30
44.6 ± 1.4 3.91 ± 0.34
After two growing seasons with Normal-N
43.7 ± 2.1 156.3 ± 6.3 31.86 ± 2.2 0.874 ± 0.07 1.203 ± 0.04 0.349 ± 0.02
Bagged + CO2
Bagged
47.8 ± 2.7 152.8 ± 7.4 34.79 ± 1.9 0.788 ± 0.05 1.112 ± 0.05 0.347 ± 0.03
Unbagged
45.5 ± 3.2 158.4 ± 4.1 35.00 ± 1.7 0.780 ± 0.08 1.147 ± 0.04 0.350 ± 0.05
0.70 ± 0.04
0.60 ± 0.03
0.59 ± 0.08
52.8 ± 2.0 3.08 ± 0.26
47.2 ± 1.7 3.57 ± 0.21
42.3 ± 1.8 3.56 ± 0.27
After three growing seasons with Normal-N
39.7 ± 3.7 145.2 ± 9.3 30.84 ± 2.4 0.963 ± 0.04 1.474 ± 0.06 0.320 ± 0.07
Bagged + CO2
Bagged
44.8 ± 3.1 147.6 ± 8.0 34.01 ± 2.0 0.714 ± 0.05 1.239 ± 0.04 0.346 ± 0.04
Unbagged
47.3 ± 3.4 156.1 ± 6.1 32.99 ± 2.0 0.744 ± 0.06 1.045 ± 0.05 0.354 ± 0.04
0.76 ± 0.06
0.72 ± 0.02
0.54 ± 0.07
59.6 ± 2.6 2.90 ± 0.23
53.2 ± 2.8 3.22 ± 0.31
37.4 ± 4.0 3.44 ± 0.35
Treatment
Vcmax
Γ*
Jmax
Rd
Rn
q
g1
Table 2. Summary of F-values from multifactor ANOVA for the effects of growth CO2 regime and soil-nitrogen supply on photosynthetic
parameters, assuming that significant responses were due to CO2 and nitrogen, and not to some unknown bagging effect. The environmental
treatments are CO2 concentration (ambient [CO2 ] (Bagged) and elevated [CO2] (Bagged + CO2)), and soil-nitrogen supply (normal nitrogen supply
(Normal-N) and high soil-nitrogen supply (High-N)). Treatment times were treated as repeated measures during two growth seasons. Bagging
effects were analyzed by one-way ANOVA. Least significant differences (LSD) was used for the range test. Legend: * P < 0.05; **P < 0.001.
Source of variation
Vcmax
Jmax
Γ*
Rd
Rn
q
θ
gmin
g1
Asat
gs
CO2 (Bagged and Bagged + CO2)
1.02
0.97
4.84*
4.99*
5.06*
0.42
1.99
5.08*
6.40*
7.51*
6.25*
N (Normal-N and High-N)
8.22**
4.30*
1.01
6.34*
4.76*
4.90*
4.23*
1.14
4.28*
5.04*
3.66
CO2 × High-N
1.16
1.67
0.69
0.75
1.10
0.88
4.40*
0.87
0.76
4.73*
2.18
Season (2 and 3)
1.82
2.03
1.44
0.86
5.00*
1.04
3.78*
4.11*
1.03
2.01
0.62
Bagging
0.87
0.55
0.08
1.10
1.23
0.04
4.01*
5.22*
0.03
1.44
0.87
‘‘Effectivity factor’’ for the use of light, q, and convexity
factor of light-response curve, θ
Light-saturated rate of assimilation, Asat , and stomatal
conductance, g sat
Values of θ were significantly increased 23% by High-N, 13%
by elevated CO2 concentration, 14% by time of treatment, and
13% by the bagged treatment (Tables 1 and 2). In contrast,
values of q were significantly increased (by 13%) only by the
High-N treatment. The bagged treatment caused a 2% reduction in values of q.
Figure 5 presents Asat and g sat as functions of the treatments.
Compared to bagged shoots alone, elevated CO2 concentration
increased Asat by 31, 29 and 24%, and decreased gsat by 17, 19
and 16% separately for shoots grown in High-N), Normal-N
after two growth seasons (Norm + 2) and Normal-N after three
growth seasons (Norm + 3). However, there were no significant differences in Asat or gsat between bagged shoots and
unbagged shoots.
Minimum stomatal conductance, gmin , and the empirical
coefficient, g1
Compared with the bagged treatment alone, elevated CO2
significantly increased gmin by 12% on average, but this increase was not enhanced by High-N (Tables 1 and 2). Both
time of treatment and the bagged treatment significantly increased gmin by 13 and 20%, respectively. Treatment effects on
g1 were the opposite of those observed on g min . Thus, elevated
CO2 significantly decreased g1 by 10%, whereas High-N significantly increased g1 by 11%, and the bagged treatment and
treatment time caused slight decreases in g1 (Table 2).
Nitrogen concentration of needles
Regardless of CO2 concentration and treatment time, High-N
resulted in a significant increase (P < 0.01) in area-based
needle nitrogen concentration, but not in mass-based needle
nitrogen concentration (Figure 6). Elevated CO2 treatment
decreased needle nitrogen concentration, but the decrease was
only significant (P < 0.05) for mass-based needle nitrogen
concentration. Neither the bagged treatment nor treatment
236
KELLOMÄKI AND WANG
Figure 5. Light-saturated rate of assimilation, Asat , and stomatal conductance, gsat , as a function of treatment. Value is the mean of measurements from six replicated bags per treatment. Measuring
conditions: photon flux density of 1500 µmol m −2 s −1, vapor pressure
deficit of 0.4 kPa and air temperature of 20 °C for all treatments; CO2
concentrations, Ca, of 350 µmol mol −1 for Bagged and Unbagged
shoots; 700 µmol mol −1 for Bagged + CO2 shoots. High-N = high
soil-nitrogen supply after two growth seasons; Norm + 2 = normal
soil-nitrogen content after two growth seasons; Norm + 3 = normal
soil-nitrogen content after three growth seasons.
Figure 6. Nitrogen concentrations based on projected area of foliage (a)
and on dry weight (b) as a function of treatment. Values are the mean
and standard deviation of measurements from six replicated bags per
treatment. Significant differences (P < 0.05) between means (by Duncan’s multiple range test) are indicated by different letters (a or b).
High-N = high soil-nitrogen supply after two growth seasons; Norm +
2 = normal soil-nitrogen content after two growth seasons; Norm + 3
= normal soil-nitrogen content after three growth seasons.
time led to a significant change in needle nitrogen concentration.
Smith 1985). We attempted to overcome these technical difficulties by using shoots with relative few needles (i.e., needles
were thinned evenly to give 30-needle fascicles per sample
shoot) so that no point on the needle surface was shaded in all
directions under a uniform diffuse radiation field. Therefore,
the photon flux density incident on the needle surface was
assumed to be equal to the irradiance incident on any surface
in the diffuse radiation field (Oker-Blom et al. 1992). Further,
the projected area of needles can be measured directly in the
laboratory.
The total radiation in the spectral range 400--800 nm entering the bag was about 90% of that outside the bag, implying
that bagged branches did not have to acclimate to a substantially altered radiation regime. Therefore, bag effects on some
photosynthetic parameters (Table 1) are probably associated
with increases in temperature or relative humidity (Figure 1);
however, bag effects did not lead to significant differences in
the acclimation of key parameters such as Vcmax and Jmax .
Although branches were exposed to the elevated-CO2 treatment from April 15 to September 15, a period longer than the
average growing season outside the bags, there was no significant downward regulation in the capacity for CO2 carboxylation (Vcmax ) or maximum electron transport (Jmax ) regardless of
Discussion
We assessed whether long-term treatment with elevated CO2
concentration and high soil-nitrogen supply modifies the
photosynthetic capacity of Scots pine. We applied the branchin-bag technique (Barton et al. 1993), where single branches in
the same whorl are subjected to CO2 treatments or are used to
control the effect of the bag itself. By this means it is possible
to reduce the effects of genetic and physiological status of the
tree on the results. This technique also facilitates study of the
effects of elevated CO2 concentration on physiological and
developmental processes in mature trees. However, despite
these advantages, the technique subjects only a small fraction
of the tree to elevated CO2 concentrations, and subsequent
source--sink relationships could differ greatly from those of
whole trees under natural conditions.
Because of the complex structure of a Scots pine shoot,
irradiation of needle surfaces is not uniform (Oker-Blom et al.
1992), and the needle area receiving direct radiation is often
difficult to determine (Leverenz and Jarvis 1978, Carter and
PHOTOSYNTHETIC RESPONSES IN SCOTS PINE
soil-nitrogen supply and treatment time (Besford and Hand
1989, Sage et al. 1989, Harley et al. 1992, Nie and Long 1992,
Van Oosten et al. 1992) (Tables 1 and 2). When measured at
the CO2 concentration in which the shoots were grown, the
light-saturated rate of CO2 assimilation was higher (24--31%)
in shoots grown at elevated CO2 concentration than in shoots
grown at current ambient CO2 concentration (Figure 5).
Reasons for this difference in response could be that: (i) only
a small part of each tree was subjected to elevated CO2 concentrations; (ii) the trees were in a stage of rapid growth (a big
‘‘sink’’ in other parts of the trees and a high demand for
carbohydrates would have accompanied (i) and (ii)); (iii) compared to trees grown in a pot, relatively unrestricted root
growth of field-grown trees may help prevent sink limitation
and end-product inhibition; and (iv) the presence of active
sinks for recently assimilated metabolites may also be important for maintenance of photosynthetic capacity (Stitt 1991,
Thomas and Strain 1991). The finding that Scots pine can
sustain increased capacity for carboxylation after long-term
exposure to elevated CO2 concentrations suggests that this
species will exhibit increased growth and carbon storage with
future increases in atmospheric CO2 concentration.
Values of Γ* were similar among treatments, except in the
elevated CO2 treatment (Table 1). This absence of treatment
effects was expected because the value of Γ* depends mainly
on the kinetic characteristics of Rubisco (Laisk 1977, Jordan
and Ogren 1981, von Caemmerer et al. 1994), which are
unlikely to change rapidly (Long and Drake 1991). The average Γ* value of 33.54 µmol mol −1 is close to the value of 35.5
µmol mol −1 found by Brooks and Farquhar (1985) for Spinacia oleracea L. leaves. The relatively low value of Γ* in the
elevated CO2 treatment may have resulted from using the
x coordinate (intercellular CO2 partial pressure) at the intercept
of the two slopes as the value of Γ* (see Figure 2). This
estimate ignores the effect of conductance for CO2 transfer
from substomatal cavities to the sites of carboxylation (gw), on
the value of Γ* (see Brooks and Farquhar 1985 and von
Caemmerer et al. 1994, for detailed discussion on measurements of Γ*). If valid, this explanation implies that the decrease in Γ* in response to the elevated CO2 treatment was
associated with changes in gw and Rd.
Our results showing a significant increase in Rd with elevated CO2 and high soil-nitrogen supply, are consistent with
the results of McKee and Woodward (1994), but differ from
Thomas et al. (1993) who found no effect. Studies on the
long-term effect of elevated CO2 on dark respiration (Rn) have
shown increases (Hrubec et al. 1985, Poorter et al. 1988),
decreases (Reuveni and Gale 1985, Bunce 1990, Wullschleger
et al. 1992) or no effect (Gifford et al. 1985, Hrubec et al. 1985)
depending on species, age, plant part, or reference units. Wang
et al. (1996) have shown that the direction of change in Rn of
Scots pine shoots grown in open-top chambers in elevated CO2
depends on both growing conditions and temperature during
measurements.
Many studies (e.g., Sharp et al. 1984, Brooks and Farquhar
1985) have shown that Rd is correlated with Rn. We observed
that different treatments induced similar changes in Rd and Rn
237
(Table 1) resulting in relatively small variations (0.58--0.72) in
the ratio of Rd to Rn. The mean ratio value of 0.68 was higher
than that of 0.34 estimated by Brooks and Farquhar (1985) for
Spinacia oleracea, but still within the range (0.25--1.0) measured by others (Peisker et al. 1981, Azcón-Bieto and Osmond
1983).
The elevated CO2 treatment decreased the slope of stomatal
functions (g1 in Equation 3), but increased minimum stomatal
conductance (g min ; Table 1, Figure 4) (cf. Harley et al. 1992).
This finding is consistent with the measured decrease in lightsaturated stomatal conductance in shoots grown in bags and
elevated CO2 (Figure 5), and implies that elevated CO2 could
limit stomata adaptability to changing environmental conditions.
Because leaf nitrogen is a major component of Rubisco
(Evans 1989), it is often closely correlated with photosynthetic
performance (Brix 1981, Matyssek and Schulze 1987). Although there is not a direct relationship between leaf nitrogen
concentration and soil nitrogen content, fertilization usually
results in increased foliar nitrogen concentrations in Scots
pine, perhaps because pine forests commonly occur on nutrient-poor forest soils (Miller et al. 1979, Sheriff et al. 1986,
Näsholm and Ericsson 1990, Mitchell and Hinckley 1993). In
response to nitrogen fertilization, the increase in area-based
needle nitrogen concentration was closely coupled with the
increase in light-saturated assimilation rate (Figures 5 and 6),
regardless of growing CO2 concentration. Similar responses
have been reported by Mitchell and Hinckley (1993) for
Pseudotsuga menziesii (Mirb.) Franco.
Long-term exposure to elevated CO2 often alters photosynthetic capacity as a result of nitrogen reallocation from Rubisco to other photosynthetic components, or to nonphotosynthetic processes (Evans 1989, Newton 1991, Stitt 1991,
Conroy 1992, Tissue et al. 1993). Such acclimation is variable
because it depends on several factors. Compared to the NormalN, High-N increased maximum carboxylation rate more than
maximum electron transport rate (33 versus 11%), indicating
that high soil-nitrogen supply had a greater effect on carboxylation rate than on electron transport capacity (cf. Evans and
Terashima 1987, Tissue et al. 1993). However, a comparison of
the Bagged shoots and Bagged + CO2 shoots indicated that
increasing soil-nitrogen supply did not significantly increase
the magnitude of photosynthetic response to elevated CO2
concentration, implying that photosynthetic response to elevated CO2 was not limited by soil-nitrogen content at the site.
An analytical model is only as good as the set of assumptions underlying it. Our parameter estimates were based on
simplified model assumptions: Rubisco was assumed to be
fully activated; and Ci was assumed to equal the C concentration at the site of fixation, which ignores possible liquid resistances between the intercellular space and carboxylation site.
The error resulting from this assumption has been estimated by
Farquhar and von Caemmerer (1982) (see also Harley et al.
1985). We believe, however, that this approach represents an
advance in our ability to understand CO2 exchange kinetics at
the whole-leaf level, and that it enhances our ability to make
238
KELLOMÄKI AND WANG
ecophysiological comparisons among plants acclimated to different environments.
We conclude that two or three years of exposure to elevated
CO2 concentrations did not significantly reduce the photosynthetic capacity of Scots pine. However, elevated CO2 concentrations decreased light-saturated stomatal conductance. A
high soil-nitrogen supply significantly increased the photosynthetic capacity in Scots pine as a result of significant increases
in maximum carboxylation rate, Vcmax and maximum electron
transport rate, Jmax . However, high soil-nitrogen supply did not
increase photosynthetic response to elevated CO2 concentration.
Acknowledgments
This study is part of a research project entitled ‘‘Likely Impact of
Elevating CO2 and Temperature on European Forests’’ under the coordination of Prof. Paul Jarvis, University of Edinburgh. The study was
funded through the Finnish Climate Change Study Programme
(SILMU), and the University of Joensuu. We express our appreciation
to Jorma Aho for providing the facilities at the Mekrijärvi Research
Station; thanks also to Matti Lemettinen and Alpo Hassinen for developing and maintaining the experimental infra-structure.
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Appendix
Symbols
Unit
Definition
An
µmol m −2 s −1
µmol m
−2 −1
Anj
µmol m
−2 −1
Asat
µmol m −2 s −1
Anc
Ca
Cs
Ci
Net assimilation rate of foliage per unit leaf area (projected area)
s
Rubisco-limited rate of net assimilation
s
µmol mol
−1
µmol mol
−1
µmol mol
−1
RuP2-limited rate of net assimilation
Light-saturated rate of assimilation
Molar fraction of CO2 in the air inside cuvette
Molar fraction of CO2 at surface of leaf
Intercellular concentration of CO2
−2 −1
gmin
mmol m
g1
dimensionless
An empirical coefficient
gs
mmol m −2 s −1
Stomatal conductance to water vapor
gsat
mmol m −2 s −1
Light-saturated stomatal conductance to water vapor
Qp
Jmax
s
µmol m
−2 −1
µmol m
−2 −1
s
Kc , Ko
µmol mol
Oi
µmol mol
--1
Rn
Incident photon flux density
s
−1
Rd
Minimum stomatal conductance to water vapor
µmol m
−2 --1
µmol m
−2 --1
Maximum rate of electron transport
Michaelis constants for CO2, O2
Intercellular concentration of O2
s
Respiration rate in light
s
Respiration rate in dark
Rubisco
Ribulose 1,5-bisphosphate carboxylase/oxygenase
RuP2
dimensionless
Vcmax
µmol m
Ds
kPa
Γ*
µmol mol
θ
dimensionless
q
--2
s
Ribulose 1,5-bisphosphate
−1
Maximum RuP2 saturated rate of carboxylation
Leaf-to-air vapor pressure difference at leaf surface
--1
CO2 compensation point in the absence of dark respiration
Convexity factor of light-response curve
--1
electron quanta
Effectivity factor for the use of light