Tree Physiology 23, 97–108
© 2003 Heron Publishing—Victoria, Canada
Contrasting effects of elevated carbon dioxide concentration and
temperature on Rubisco activity, chlorophyll fluorescence, needle
ultrastructure and secondary metabolites in conifer seedlings
LEENA SALLAS,1 EEVA-MARIA LUOMALA,2 JARKKO UTRIAINEN,1 PIRJO
KAINULAINEN1,3 and JARMO K. HOLOPAINEN1,3,4
1
Department of Ecology and Environmental Science, University of Kuopio, POB 1627, FIN-70211 Kuopio, Finland
2
Finnish Forest Research Institute, Suonenjoki Research Station, FIN-77600 Suonenjoki, Finland
3
Agricultural Research Center of Finland, Plant Protection, FIN-31600 Jokioinen, Finland
4
Author to whom correspondence should be addressed ( jarmo.holopainen@uku.fi)
Received March 25, 2002; accepted June 30, 2002; published online January 2, 2003
Summary Scots pine (Pinus sylvestris L.) and Norway
spruce (Picea abies (L.) Karst.) seedlings were grown for
50 days in growth chambers in an ambient or twice ambient
carbon dioxide concentration ([CO2]) at a day/night tempera-
ture of 19/12 °C or 23/16 °C. Although elevated [CO2] (EC)
had only slight effects on the growth parameters measured, ele-
vated temperature (ET) increased aboveground dry mass of
both species. Among treatments, biomass accumulation of
both species was greatest in the combined EC + ET treatment.
The EC treatment induced thylakoid swelling and increased
numbers of plastoglobuli observed in Scots pine needles.
Although EC had little effect on Rubisco protein or N concen-
tration of needles, ET had a large effect on N-containing com-
pounds and enhanced N allocation from 1-year-old needles.
Terpenoids were more responsive to EC and ET than total
phenolics. Generally, terpene concentrations were reduced by
EC and increased by ET. Increased terpenoid concentrations in
response to ET might be associated with thermotolerance of
photosynthesis. In Norway spruce, EC decreased total pheno-
lic concentrations in needles, probably as a result of increased
growth. We conclude that, in seedlings of these boreal species,
the effects of elevated [CO2] on the studied parameters were
small compared with the effects of elevated temperature.
Keywords: climate change, CO2 concentration, ecophysiol-
ogy, growth, monoterpenes, Norway spruce, Picea abies,
Pinus sylvestris, resin acids, Scots pine, soluble proteins, total
phenolics.
Introduction
Although there are many uncertainties in measuring and pre-
dicting global warming (Krupa 1997), a doubling of pre-
industrial atmospheric carbon dioxide concentration ([CO2])
together with increases in other greenhouse gases is predicted
to raise global mean surface temperature by about 2–4 °C in
northern latitudes by the end of the 21st century (IPCC 2001).
Trees respond to CO2 enrichment in several ways including
increased photosynthesis and growth, acceleration of ontog-
eny, and decreased foliar nitrogen (N) concentrations and res-
piration (Ward and Strain 1999). There is little evidence of
long-term loss of sensitivity to elevated [CO2], as was sug-
gested by earlier pot experiments (Norby et al. 1999). Photo-
synthetic acclimation to high [CO2] is mostly related to
reduced sink strength and low nutrient availability, especially
of N (Ward and Strain 1999).
Elevated temperature causes a reduction in photosynthesis,
and elevated [CO2] counteracts this effect (Wang et al. 1995,
Wang and Kellomäki 1997). Because of the properties of
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase,
EC 4.1.1.39), the temperature optimum for assimilation in-
creases with increasing [CO2], and the relative stimulation of
assimilation by elevated [CO2] should be greater at higher
temperatures (Long 1991). In general, high temperatures stim-
ulate growth of meristems and organs, accelerating plant de-
velopment; however, the duration of the growing phases is
shortened, resulting in fewer, smaller organs and less biomass
accumulation. In contrast to the effects of elevated [CO2], high
temperatures increase respiration rates and decrease carbohy-
drate concentrations (Morison and Lawlor 1999). A combina-
tion of elevated [CO2] and temperature increases the
mineralization rate of soil organic matter and thus increases N
availability, thereby helping to sustain increased plant growth.
The long-term fate of N, however, is a key uncertainty in pre-
dicting the future response of boreal ecosystems to climate
change (van Breemen et al. 1998).
The wide distribution of conifers is partly associated with
their highly evolved systems of defense against herbivore pre-
dation and pathogens (Phillips and Croteau 1999). Conifers
have developed two major defense mechanisms: constitutive
resin production in resin ducts and impregnation of necrotic
areas with resinous and phenolic compounds by the induced
98 SALLAS, LUOMALA, UTRIAINEN, KAINULAINEN AND HOLOPAINEN
reaction mechanism (Christiansen et al. 1998). Changes in
plant secondary metabolism are important because they are
likely to affect plant–herbivory and plant–pathogen interac-
tions and decomposition of litter (Lambers 1993). Carbon di-
oxide enrichment increases the C/N ratio of plant tissue and
leaf carbohydrate content, which may result in the accumula-
tion of carbon-based secondary compounds (Ward and Strain
1999). The protein competition model (PCM) of Jones and
Hartley (1998) predicts a decrease or increase in total phenolic
concentration in response to elevated [CO2], depending on
photosynthetic acclimation. If full acclimation takes place,
photosynthesis is unchanged and the concentration of total
phenolics increases. According to Peñuelas and Estiarte
(1998), elevated [CO2] increases foliar concentrations of solu-
ble phenolics and condensed tannins, but not of lignins or
terpenoids. Elevated [CO2] may down-regulate monoterpene
synthesis in plant leaves (Loreto et al. 2001). There are few
studies on responses of conifer secondary metabolites to ele-
vated temperature or to a combination of elevated [CO2] and
temperature. Constable et al. (1999) predicted an increase in
monoterpene emission rates for Douglas-fir in response to the
combined effects of elevated [CO2] and temperature, but re-
ported no significant effect on the concentration of total mono-
terpenes in needles.
We investigated the combined effects of elevated [CO2] and
temperature on compounds associated with defense and pho-
tosynthesis of two conifer species dominating the boreal for-
ests in northern Europe. Based on the studies of Jones and
Hartley (1998), Peñuelas and Estiarte (1998) and Singsaas et
al. (1997), we tested three hypotheses: (1) elevated tempera-
ture enhances the effects of elevated [CO2] on carbon assimila-
tion (decreased carboxylation capacity), reducing nitrogen
content and promoting growth; (2) elevated [CO2] increases
the foliar concentration of total phenolics, but does not affect
terpenoid concentrations; and (3) elevated temperature in-
creases terpenoid concentrations but has no effect on the con-
centration of total phenolics.
Materials and methods
Plant material
One-year-old Scots pine seedlings (Pinus sylvestris L.,
M29-91-0033) and 2-year-old Norway spruce seedlings
(Picea abies (L.) Karst., T3-89-0141), both originating from
southern central Finland, were provided by the Finnish Forest
Research Institute, Suonenjoki Nursery. At the nursery, the
potted seedlings were grown in fertilized Sphagnum peat in
plastic greenhouses. In late July, the containerized seedlings
were transferred outdoors and irrigated frequently. Seedlings
were overwintered in the field, covered with excised spruce
branches and snow. In February, dormant seedlings were
transferred to a cool (+8 °C) greenhouse and transplanted to
1.5-l pots containing a 1:2 (v/v) mixture of sand and fertilized
peat. After 10 days in the greenhouse, the seedlings were
placed in computer-controlled environmental chambers (for
details see Holopainen and Kärenlampi 1984). The seedlings
TREE PHYSIOLOGY VOLUME 23, 2003
were watered when needed and fertilized weekly with a 0.05%
fertilizer solution (19:5:20, N:P:K) starting on the third week
in the chambers. A bank of metal halide lamps (Osram
POWER Stars) provided a photosynthetic photon flux density
(PPFD) of 500 µmol m –2 s –1 at the top of the seedlings. The
22-h photoperiod simulated the late-June photoperiod in cen-
tral Finland.
Experimental design
Thirty-three randomly selected seedlings per species were
placed in each of the four growth chambers. The seedlings
were grown for 50 days at ambient (measured daily mean =
325 ± 5 ppm) or twice ambient (731 ± 15 ppm) [CO2], at a
day/night temperature of 19/12 °C (control) or 23/16 °C
(elevated temperature). The control temperature simulated
weather conditions in June in central Finland (mean weather
data from 1970 to 1975). The CO2 fumigation regime was ap-
plied day and night. Pots and treatments were rotated once a
week within and between growth chambers to minimize error
due to chamber effects. The experimental treatments were:
ambient [CO2] + control temperature (Control); elevated
[CO2] + control temperature (EC); elevated temperature + am-
bient [CO2] (ET); and elevated [CO2] + elevated temperature
(EC + ET).
Measurement of seedling growth
After 7 weeks of exposure, six seedlings per treatment were
harvested, separated into needles (and needle age classes) and
stems and weighed, and leaf area of current-year needles was
measured (LI-3000, Li-Cor, Lincoln, NE). All components
were dried to constant mass at 60 °C and reweighed. Stem base
diameter and height of all seedlings were measured before
starting the experiment and at harvest. The current-year shoot
was also measured at harvest.
Analysis of starch and nutrients
At harvest, current-year needles of the main shoot of six seed-
lings per treatment were collected in liquid nitrogen and stored
at –80 °C. Starch was analyzed by an enzymatic method
(Boehringer kit for food analysis) on samples of freeze-dried
and milled needles (Karkalas 1985). Nitrogen concentrations
were determined by the standard Kjeldahl method, phospho-
rus was assayed spectrophotometrically (Shimadzu UV-1201,
Shimadzu, Kyoto, Japan), and K, Ca and Mg concentrations
were determined by atomic absorption spectrometry (Perkin
Elmer 460, Perkin Elmer, Boston, MA) (Allen 1989).
Determination of amount and activity of Rubisco and
concentrations of chlorophyll and soluble proteins
Samples of current-year and 1-year-old needles of the same
six seedlings per treatment that were used for analysis of
starch and nutrients were collected, frozen in liquid nitrogen
and stored at –80 °C. For determination of Rubisco, soluble
protein and chlorophyll (Chl) contents, frozen needles were
homogenized in ice-cold extraction buffer containing 50 mM
2-(N-morpholino)-ethanesulfonic acid, pH 6.8, 20 mM
MgCl2, 50 mM 2-mercaptoethanol and 1% Tween 80.
 TEMPERATURE AND CO2 EFFECTS ON GROWTH AND DEFENSE
Aliquots of the crude extract were analyzed for Chl content by
the method of Arnon (1949). After centrifugation, the activity
of Rubisco was determined as incorporation of 14C into acid-
stable products (Lorimer et al. 1977). The assay was per-
formed at 25 °C in a reaction mixture containing 50 mM
Epps-NaOH, pH 8.2, 20 mM MgCl2, 0.26 mM EDTA, 10 mM
NaH14CO3 (3.7 × 10 9 Bq mol –1; Amersham) and 0.3 mM
ribulose bisphosphate (RuBP; Sigma). Initial activity was
measured immediately after extraction. To obtain total activity
of the enzyme, the supernatant was preincubated in the reac-
tion mixture without RuBP for 10 min at 25 °C before adding
RuBP to start the reaction. The incorporation of 14C into acid-
stable products was determined by liquid scintillation spec-
trometry. Rubisco activation state is expressed as the ratio of
initial activity to total activity. The amount of Rubisco protein
was determined by polyacrylamide gel electrophoresis
(Ruuska et al. 1994) and soluble protein content was deter-
mined by the method of Bradford (1976).
Chlorophyll fluorescence
Chlorophyll fluorescence was measured at room temperature
in samples of current-year and 1-year-old needles of the same
six seedlings per treatment that were used for the chemical and
biochemical analyses with a portable pulse-amplitude-modu-
lated fluorometer (MINI-PAM, Heinz Walz, Effeltrich, Ger-
many). Detached needles were mounted side by side on a
piece of removable adhesive tape and put in a dark-leaf-clip
(DLC-8, Heinz Walz). Two replicate samples of both needle
age classes were measured on six seedlings per treatment. Af-
ter a 20-min dark adaptation, minimal fluorescence intensity
(F0) with all PSII reaction centers open was determined using
a low intensity modulated measuring beam (< 0.1 µmol m –2
s –1). A 1.0-s white light pulse of about 9000 µmol m –2 s –1 was
used to produce a transient closure of PSII reaction centers,
and maximal fluorescence intensity (Fm) was recorded.
Measuring pulse frequency was set at 0.6 kHz during the mea-
surements. Maximal photochemical efficiency of PSII in the
dark-adapted state (Fv /Fm) was calculated as (Fm – F0)/Fm
(Genty et al. 1989).
Needle ultrastructure
Samples for ultrastructural studies were collected from seed-
lings just before harvest. One representative needle from the
current-year leading shoot of each of six replicate seedlings
was collected and placed in 2% glutaraldehyde. For electron
microscopy, one needle segment (1.5 mm long) one-third from
the tip of each needle was cut and prefixed in 2% glutaral-
dehyde and postfixed in 1% OsO4 solution in phosphate buffer
(0.1 M, pH 7.0) (Holopainen et al. 1996). Samples were
stained with uranyl acetate and lead citrate and examined with
an electron microscope (JEOL JEM 1200 EX, JEOL, Tokyo,
Japan). To assess the condition of mesophyll cells (chloroplast
shape, condition of chloroplast stroma and thylakoids and the
numbers of cytoplasmic ribosomes and lipid bodies), three
digital electron micrographs (10,000×) were taken from the
middle part of the mesophyll tissue of each needle sample.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
99
From these micrographs, chloroplast starch grain areas and
their proportion relative to total chloroplast area, as well as
number of plastoglobuli, were determined with the Adobe
Photoshop 4.0 PC program.
Analysis of secondary metabolites
At harvest, current-year needles and stems of 10 seedlings per
treatment were frozen immediately in liquid nitrogen and
stored at –80 °C. Mono- and sesquiterpenes were extracted
with n-hexane (Kainulainen et al. 1992) using 1-chloro-octane
as an internal standard. Resin acids were extracted from
freeze-dried and milled needles and stems with petroleum
ether:diethyl ether following the procedure of Gref and Erics-
son (1985). The internal standard was heptadecanoic acid.
Terpene and resin acid extracts were analyzed by gas chro-
matography–mass spectrometry (GC-type 6890, MSD-type
5973, Hewlett Packard, New York, NY) using a 30-m-long
capillary column (HP-5MS, 0.25 mm ID, 0.25 µm film thick-
ness, Hewlett Packard) and helium as the carrier gas. The tem-
perature was increased at a rate of 5 °C min –1 from 40 to
250 °C for terpenes and from 150 to 250 °C for resin acids. In
both analyses, the technique of selected ion monitoring (SIM)
was used. Individual substances were quantified by their peak
areas and identified by comparison of their mass spectra and
retention times with those of pure reference compounds.
Quantities of individual terpenes and resin acids were summed
to obtain total terpenes and total resin acids.
For analysis of total phenolics, current-year needles and
stems of 10 seedlings per treatment were freeze-dried, ground
to a fine powder and stored in a desiccator at 4 °C. The fine-
powdered current-year needle and stem samples were ex-
tracted separately with 80% acetone, and the residue washed
three times with acetone. Total phenolics were analyzed with
Folin-Ciocalteu reagent as described by Julkunen-Tiitto
(1985). Absorbances of the samples were measured with a
spectrophotometer (Shimadzu UV-2100) at 735 nm.
Statistical analysis
When necessary, data were normalized by log(x + 1) transfor-
mations. Significance of treatment effects was determined by
separate analyses of variance for each species using the Gen-
eral Linear Models procedure in SPSS version 8.0. For ultra-
structural studies, the relative values were analyzed by the
Kruskall-Wallis test.
Results
Growth
In both Scots pine and Norway spruce seedlings, ET increased
final dry mass of aboveground plant parts (Table 1). When
compared with seedling height and stem base diameter at the
beginning of the experiment, the final height of Scots pine
seedlings increased in response to EC, whereas EC decreased
the final height of Norway spruce seedlings and ET increased
it. The ET treatment also increased stem base diameter of Nor-
way spruce seedlings. The EC treatment decreased specific
100 SALLAS, LUOMALA, UTRIAINEN, KAINULAINEN AND HOLOPAINEN

Table 1. Effects of elevated [CO2] (EC) and temperature (ET) on mean dry mass (± SEM) of aboveground parts (g), height (cm), stem diameter
(mm) and specific leaf area (SLA; cm2 g –1) of Scots pine and Norway spruce seedlings, and summary of analysis of variance of the treatment ef-
fects on the growth parameters (***, P = 0.001; **, P ≤ 0.01; *, P ≤ 0.05; and ns = not significant); n = 6.

Parameter Control EC ET EC + ET CO2 T CO2 × T

Scots pine
Dry mass 3.2 ± 0.2 3.4 ± 0.4 4.8 ± 0.4 5.5 ± 0.4 ns *** ns
Height 23.8 ± 1.2 24.2 ± 1.4 26.6 ± 1.0 28.0 ± 0.7 ** ns ns
Stem diameter 4.8 ± 0.1 4.7 ± 1.6 4.9 ± 1.3 5.2 ± 1.1 ns ns ns
SLA 15.6 ± 0.4 16.1 ± 1.1 14.3 ± 0.3 14.7 ± 0.7 ns ns ns
Norway spruce
Dry mass 4.2 5.9 7.0 7.5 ns * ns
Height 29.3 ± 1.4 25.1 ± 1.2 33.3 ± 0.8 31.8 ± 0.8 ** *** *
Stem diameter 4.4 ± 0.2 4.4 ± 0.1 5.2 ± 0.1 5.0 ± 0.1 ns *** ns
SLA 17.6 ± 0.9 12.1 ± 3.5 20.2 ± 1.2 16.8 ± 0.6 * ns ns

leaf area (SLA; P = 0.019) in current-year needles of Norway
spruce (Table 1), but had no effect on SLA of Scots pine.
Nutrients and starch
The EC treatment decreased concentrations of Ca and Mg in
current-year needles of Scots pine and increased the K concen-
tration in current-year needles of Norway spruce (Table 2).
The ET treatment decreased concentrations of Mg, N and P in
Scots pine and N in Norway spruce, and increased concentra-
tions of Ca, K and starch in Norway spruce.
Amount and activity of Rubisco
In 1-year-old needles of Scots pine and Norway spruce, ET de-
creased the amount of Rubisco protein (P < 0.001, P = 0.029,
respectively) (Table 3), whereas EC had no effect on the
amount of Rubisco. Total activity of Rubisco reflected the
changes observed in the amount of Rubisco (Table 3). Total
activity decreased in response to ET in 1-year-old needles of
Scots pine (P < 0.001) and Norway spruce (P = 0.027). The
activation state of Rubisco was decreased by ET only in
1-year-old needles of Norway spruce (P = 0.030). Specific ac-
tivity of Rubisco was increased by EC and decreased by ET in
current-year (P = 0.002, P = 0.079) and 1-year-old needles
(P = 0.104, P = 0.001, respectively) of Scots pine. In contrast,
ET increased specific activity in current-year needles of Nor-
way spruce (P = 0.024).
The Rubisco protein to Chl ratio (Rbc/Chl), an estimate of
relative N allocation between Rubisco and thylakoid compo-
nents (Evans 1989), decreased in 1-year-old Scots pine need-
les (P = 0.001) and in current-year (P = 0.063) and 1-year-old
Norway spruce needles (P = 0.003) in response to ET. There
was a CO2 × T interaction effect on Rbc/Chl in current-year
needles of Scots pine (P = 0.092), leading to lower Rbc/Chl
compared with the additive effects of EC and ET (Table 4).
The Rubisco to total soluble protein ratio (Rbc/Prot) was
low in seedlings in all treatments, especially in current-year
needles. The treatments did not affect Rbc/Prot in current-year
needles in either species. In 1-year-old needles of Scots pine,
Rbc/Prot was decreased by ET (P < 0.001) and EC (P =
0.031), whereas in 1-year-old needles of Norway spruce,
Rbc/Prot was increased by EC (P = 0.072) (Table 4).
Concentrations of chlorophyll and soluble proteins
In current-year needles of Scots pine and Norway spruce, EC
decreased Chl concentration (P = 0.021, P = 0.004, respec-
tively). Chlorophyll concentration of 1-year-old needles was

Table 2. Effects of elevated [CO2] (EC) and temperature (ET) on mean concentrations of nutrients and starch (mg g –1 dry mass) in current-year
needles of Scots pine and Norway spruce, and summary of analysis of variance of the treatment effects (***, P = 0.001; **, P ≤ 0.01; *, P ≤ 0.05;
and ns = not significant).

Treatment Scots pine Norway spruce

N P K Mg Ca Starch N P K Mg Ca Starch
Control 13.33 2.25 7.23 1.01 1.14 105 9.35 1.96 5.89 0.59 1.43 211
EC 13.28 2.11 6.49 0.83 1.07 125 11.40 2.07 6.05 0.64 1.35 170
ET 8.23 1.49 6.00 0.90 1.43 177 7.00 2.10 6.10 0.66 2.14 260
EC + ET 6.20 1.45 5.84 0.73 1.05 223 5.37 1.66 3.72 0.45 1.49 341
Main effects
CO2 ns ns ns ** * ns ns ns * ns ns ns
T ** ** ns * ns ns ** ns * ns * *
CO2 × T ns ns ns ns ns ns * ns * * ns ns

TREE PHYSIOLOGY VOLUME 23, 2003

 TEMPERATURE AND CO2 EFFECTS ON GROWTH AND DEFENSE 101

Table 3. Effects of elevated [CO2] (EC) and temperature (ET) on amount (mg g –1 fresh mass), total activity (µmol CO2 min –1 g –1 fresh mass), acti-
vation state (%) and specific activity of Rubisco (µmol CO2 s –1 g –1 Rubisco protein) in current-year and 1-year-old needles of Scots pine and Nor-
way spruce, and summary of analysis of variance of the treatment effects (***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; and ns = not significant).
Values are means of 3–6 replicate samples (± SEM) (where no SEM is indicated, only one sample was analyzed).

Control EC ET EC + ET CO2 T CO2 × T

Amount of Rubisco (mg g –1 fm)

Scots pine
Current-year 1.38 ± 0.29 1.17 ± 0.31 1.21 ± 0.13 0.92 ± 0.07 ns ns ns
One-year-old 5.24 ± 0.47 5.04 ± 0.61 2.18 ± 0.34 1.40 ± 0.06 ns *** ns
Norway spruce
Current-year 0.50 ± 0.09 0.44 0.62 ± 0.30 0.44 ± 0.18 ns ns ns
One-year-old 2.60 ± 0.69 2.74 ± 0.61 1.14 ± 0.30 1.57 ± 0.32 ns * ns
Total activity of Rubisco (µmol CO2 min –1 g –1 fm)
Scots pine
Current-year 1.54 ± 0.33 1.68 ± 0.37 1.04 ± 0.17 1.21 ± 0.13 ns ns ns
One-year-old 7.46 ± 1.12 8.49 ± 1.66 2.03 ± 0.38 1.60 ± 0.17 ns *** ns
Norway spruce
Current-year 0.54 ± 0.08 0.17 ± 0.03 0.65 ± 0.25 0.47 ± 0.19 ns ns ns
One-year-old 3.21 ± 0.97 3.04 ± 0.82 1.14 ± 0.41 1.82 ± 0.35 ns * ns
Activation state of Rubisco (%)
Scots pine
Current-year 32.7 ± 1.7 26.2 ± 1.4 32.0 ± 1.1 31.9 ± 2.6 ns ns ns
One-year-old 35.0 ± 1.8 30.2 ± 2.0 28.2 ± 1.4 31.5 ± 3.9 ns ns ns
Norway spruce
Current-year 48.0 ± 4.1 49.8 ± 11.3 50.5 ± 6.6 42.9 ± 5.5 ns ns ns
One-year-old 61.8 ± 10.4 60.8 ± 3.5 39.5 ± 3.9 50.3 ± 6.7 ns * ns
Specific activity of Rubisco (µmol CO2 s –1 g –1 Rubisco protein)
Scots pine
Current-year 18.9 ± 1.7 25.4 ± 2.4 15.5 ± 1.3 22.0 ± 1.5 ** ns ns
One-year-old 23.7 ± 2.8 27.4 ± 3.2 14.6 ± 1.3 18.9 ± 1.5 ns *** ns
Norway spruce
Current-year 18.5 ± 0.7 11.0 22.5 ± 1.9 23.1 ± 3.9 ns * ns
One-year-old 19.8 ± 2.5 21.3 ± 7.4 18.3 ± 3.8 17.9 ± 1.4 ns ns ns

decreased by ET in Scots pine (P = 0.015), whereas it was in-
creased by ET in Norway spruce (P = 0.049) (Table 4). In cur-
rent-year needles of Scots pine, EC increased (P = 0.001) and
ET decreased the chlorophyll a to b ratio (Chl a/b; P = 0.022).
The ET treatment also decreased Chl a/b in 1-year-old needles
of Scots pine (P = 0.001). In Norway spruce, Chl a/b of cur-
rent-year needles was unaffected by the treatments, but in
1-year-old needles EC increased (P = 0.002) and ET decreased
Chl a/b (P < 0.001). In Scots pine, ET decreased soluble pro-
tein concentration in current-year (P = 0.002) and 1-year-old
needles (P < 0.001). In Norway spruce, EC decreased soluble
protein concentration in current-year needles (P = 0.007), but
the soluble protein concentration did not decrease signifi-
cantly in 1-year-old needles. The ET treatment decreased solu-
ble proteins (P = 0.059) in 1-year-old needles of Norway
spruce (Table 4).
Chlorophyll fluorescence
Maximal photochemical efficiency of PSII (Fv /Fm) was high
in Scots pine needles (Figure 1a), indicating normal potential
for PSII photochemistry in seedlings grown in ambient condi-
tions. The low values of Fv /Fm in Norway spruce may be a
result of incipient nitrogen deficiency (Figure 1b). The ET
treatment increased Fv /Fm in current-year needles of Scots
pine (P < 0.001) and Norway spruce (P = 0.008) and in
1-year-old needles of Norway spruce (P = 0.008). In contrast,
in 1-year-old needles of Scots pine, Fv /Fm was significantly
decreased by ET (P < 0.001), and the EC + ET treatment had a
negative effect on Fv /Fm (P < 0.001).
Needle ultrastructure
In both species, EC increased starch accumulation in chloro-
plasts of current-year needle mesophyll cells (P = 0.003 in
Scots pine and P = 0.045 in Norway spruce), increasing the
starch grain area to total chloroplast area ratio (Table 5). How-
ever, starch accumulation was more evident in Norway spruce
current-year needles in all treatments. Although mean starch
grain area was unaffected by ET, ET depressed both EC-in-
duced starch accumulation and the EC-induced increase in
number of chloroplast plastoglobuli in Scots pine (Table 5). A
nonsignificant decrease in the number of plastoglobuli was ob-
served in response to EC and ET in Norway spruce. The ET

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102 SALLAS, LUOMALA, UTRIAINEN, KAINULAINEN AND HOLOPAINEN

Table 4. Effects of elevated [CO2] (EC) and temperature (ET) on the Rubisco/Chlorophyll ratio (mg mg –1), Rubisco/Soluble protein ratio (%),
chlorophyll concentration (mg g –1 fm), Chl a/b ratio and soluble protein concentration (mg g –1 fm) in current-year and 1-year-old needles of
Scots pine and Norway spruce, and summary of analysis of variance of the treatment effects (***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; and ns = not
significant). Values are means of 3–6 replicate samples (± SEM) (where no SEM is indicated, only one sample was analyzed).

Control EC ET EC + ET CO2 T CO2 × T

Rubisco/Chlorophyll
Scots pine
Current-year 2.24 ± 0.48 2.32 ± 0.52 1.76 ± 0.20 1.69 ± 0.17 ns ns ns
One-year-old 4.98 ± 1.04 3.96 ± 0.44 2.24 ± 0.35 1.81 ± 0.22 ns *** ns
Norway spruce
Current-year 1.51 ± 0.34 3.11 1.39 ± 0.34 1.13 ± 0.47 ns ** ns
One-year-old 4.02 ± 1.05 4.72 ± 0.82 1.48 ± 0.19 1.90 ± 0.33 ns ns ns
Rubisco/Soluble protein (%)
Scots pine
Current-year 7.03 ± 1.56 5.19 ± 1.08 7.25 ± 0.93 5.83 ± 0.92 ns ns ns
One-year-old 19.24 ± 1.07 16.20 ± 1.43 12.86 ± 2.55 8.43 ± 0.78 * *** ns
Norway spruce
Current-year 2.46 ± 0.45 2.02 3.38 ± 1.53 3.77 ± 0.45 ns ns ns
One-year-old 13.16 ± 3.89 24.66 ± 6.09 8.91 ± 1.31 15.62 ± 4.25 ns ns ns
Chlorophyll (mg g –1 fm)
Scots pine
Current-year 0.63 ± 0.05 0.50 ± 0.04 0.64 ± 0.05 0.55 ± 0.04 * ns ns
One-year-old 1.18 ± 0.16 1.30 ± 0.14 0.97 ± 0.09 0.84 ± 0.11 ns * ns
Norway spruce
Current-year 0.45 ± 0.07 0.26 ± 0.04 0.55 ± 0.09 0.34 ± 0.03 ** ns ns
One-year-old 0.66 ± 0.07 0.57 ± 0.05 0.95 ± 0.23 0.83 ± 0.10 ns * ns
Chl a/b
Scots pine
Current-year 3.74 ± 0.11 4.04 ± 0.11 3.49 ± 0.05 3.86 ± 0.04 *** * ns
One-year-old 4.04 ± 0.22 4.03 ± 0.12 3.50 ± 0.07 3.59 ± 0.06 ns *** ns
Norway spruce
Current-year 4.59 ± 0.66 3.95 ± 0.26 3.59 ± 0.20 4.30 ± 0.57 ns ns ns
One-year-old 3.79 ± 0.15 4.40 ± 0.15 3.35 ± 0.07 3.60 ± 0.10 ** *** ns
Soluble protein (mg g –1 fm)
Scots pine
Current-year 19.7 ± 0.4 22.1 ± 1.8 16.6 ± 0.7 16.7 ± 1.4 ns ** ns
One-year-old 27.6 ± 2.8 31.4 ± 3.2 18.4 ± 2.2 17.1 ± 1.3 ns *** ns
Norway spruce
Current-year 23.3 ± 3.5 15.7 ± 1.7 20.5 ± 1.8 12.5 ± 2.9 ** ns ns
One-year-old 20.5 ± 2.7 13.9 ± 3.7 13.4 ± 1.9 10.7 ± 1.6 ns ns ns

treatment increased the amount of cytoplasmic lipid bodies in
both species, whereas EC increased the swelling of chloro-
plast thylakoids in Scots pine only (data not shown).
Mono- and sesquiterpenes
In current-year needles of Scots pine, EC decreased the con-
centrations of total terpenes and six of the nine monoterpenes
analyzed (tricyclene, α-pinene, camphene, sabinene, β-pinene
and myrcene; Table 6). The ET treatment increased the con-
centrations of the same six monoterpenes and total terpenes,
and decreased the concentration of bornyl acetate. There was a
significant interactive effect of EC + ET on needle concentra-
tions of some individual terpenes. In Scots pine stems, EC in-
creased concentrations of 3-carene, limonene and terpinolene,
which were unaffected in the needles, and total terpenes.
There were significant CO2 × T interaction effects on concen-
trations of 3-carene and total terpenes in stems.
In current-year needles of Norway spruce, EC decreased
concentrations of most individual terpenes and total terpenes
(Table 7), whereas ET increased concentrations of all mono-
terpenes, except bornyl acetate, and total terpenes. In Norway
spruce stems, EC increased the concentration of terpinolene
and ET increased the concentrations of total terpenes and
monoterpenes, except 3-carene. There was a significant inter-
active CO2 × T effect on stem concentrations of some individ-
ual terpenes and total terpenes.
Resin acids
In current-year Scots pine needles, EC decreased concentra-
tions of some resin acids and total resin acids (Table 6),

TREE PHYSIOLOGY VOLUME 23, 2003

 TEMPERATURE AND CO2 EFFECTS ON GROWTH AND DEFENSE 103

phenolics in current-year needles of Scots pine (Figure 2). In

Norway spruce needles, EC decreased total phenolic concen-
tration (P = 0.038), and there was a significant two-way inter-
action between CO2 and T on total phenolic concentration (P =
0.001).

Discussion

Growth and physiology

Figure 1. Effects of elevated [CO2] and temperature on maximal pho-
tochemical efficiency of PSII (Fv /Fm) in current-year and 1-year-old
needles of (a) Scots pine and (b) Norway spruce seedlings.
whereas ET increased concentrations of some resin acids.
There was a significant interactive effect of CO2 × T on needle
concentrations of levopimaric + palustric acids. In Scots pine
stems, EC decreased the concentrations of some resin acids,
and ET increased the concentration of sandaracopimaric acid.
In current-year needles of Norway spruce, EC decreased
concentrations of some resin acids and total resin acids (Ta-
ble 7), but increased the concentration of abietic acid. The ET
treatment increased concentrations of most resin acids and to-
tal resin acids in needles. There was a significant interactive
effect of CO2 × T on concentrations of most resin acids and to-
tal resin acids in needles. In Norway spruce stems, ET in-
creased concentrations of all resin acids and total resin acids.
Total phenolics
The treatments had no effect on the concentration of total
Net photosynthesis of trees is generally enhanced by elevated
[CO2], and the enhancement is maintained for long periods
(Norby et al. 1999). Mortensen (1994) reported that elevated
[CO2] increased the dry mass of nine conifer species by be-
tween 0% and about 50%, with Pinus sylvestris and Picea
abies showing large differences between provenances. We
found no significant effect of EC on dry mass of either species,
whereas ET increased dry mass of both species, indicating
that, in boreal conditions, an increase in temperature alone
could greatly enhance dry matter production. Similarly,
Tjoelker et al. (1998) reported that growth responses of North
American boreal tree species to elevated [CO2] were minimal
at low temperatures, but increased toward optimal growth tem-
peratures. In contrast to Scots pine, final height and stem di-
ameter of Norway spruce were higher in the ET treatment than
in the EC + ET treatment. Both the ET-induced increase in
stem biomass and the EC-induced reduction in SLA in Nor-
way spruce seedlings might be associated with greater
transpirational stress than in Scots pine seedlings, perhaps re-
flecting the need for increased water-use efficiency. Eamus
(1991) reported that plant water-use efficiency is substantially
increased by elevated [CO2] even at elevated air temperatures.
Although EC had no significant effect on Rubisco activity, it
increased starch accumulation in chloroplasts of current-year
needles of both species. This increased starch accumulation
probably does not reflect increased photosynthesis. Thylakoid
swelling and increased numbers of plastoglobuli, which we
observed in Scots pine needles in response to EC, have also
been observed in senescing leaves, and are considered to be
non-specific stress symptoms of chloroplasts (e.g., Cave et al.
1981, Bowes 1991, Woodward et al. 1991, Pritchard et al.

Table 5. Effects of elevated [CO2] (EC) and temperature (ET) on chloroplast starch and number of plastoglobuli in current-year (C) needle meso-
phyll cells of Scots pine and Norway spruce seedlings. Values are means ± SE. Means followed by different letters are significantly different
(ANOVA; Tukey’s HSD test, P < 0.05). Significance of main effects: **, P < 0.01; *, P < 0.05; (*), P < 0.1; and ns, not significant.

Treatment Starch grain area (µm2) Starch (% of chloroplast area) Number of plastoglobuli
Scots pine Norway spruce Scots pine Norway spruce Scots pine Norway spruce
Control 20.30 ± 2.84 a 36.00 ± 1.82 a 62.40 ± 3.96 a 77.05 ± 4.02 a 10.25 ± 1.45 ab 10.88 ± 2.63 a
EC 27.85 ± 0.62 b 44.90 ± 1.72 a 76.90 ± 2.77 b 90.12 ± 0.77 b 14.88 ± 1.27 b 3.88 ± 2.26 a
ET 18.94 ± 1.43 a 38.68 ± 3.83 a 59.76 ± 9.18 a 83.74 ± 2.34 ab 9.38 ± 1.14 ab 6.38 ± 1.07 a
EC + ET 25.96 ± 2.23 ab 38.55 ± 2.06 a 80.11 ± 3.45 b 82.36 ± 3.27 ab 8.38 ± 2.03 a 9.75 ± 2.77 a
Main effects
CO2 ** * ** (*) ns ns
T ns ns ns ns * ns
CO2 × T ** ns * ns (*) ns

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104 SALLAS, LUOMALA, UTRIAINEN, KAINULAINEN AND HOLOPAINEN

Table 6. Effects of elevated [CO2] (EC) and temperature (ET) on mean mono- and sesquiterpene concentrations (µg g –1 fm) and mean resin acid
concentrations (µg g –1 dm) in current-year needles and stems of Scots pine seedlings, and summary of analysis of variance of treatment effects on
concentrations of secondary metabolites (***, P = 0.001; **, P ≤ 0.01; *, P ≤ 0.05; and ns = not significant).

Leaf constituent Control EC ET EC + ET CO2 T CO2 × T

Mono- and sesquiterpenes

Needles
Tricyclene 9 3 14 12 *** *** **
α-Pinene 506 185 705 423 *** ** ns
Camphene 40 12 57 48 *** *** **
Sabinene 26 9 33 22 ** * ns
β-Pinene 25 9 33 22 ** * ns
Myrcene 25 14 26 23 * * ns
3-Carene 82 231 84 154 ns ns ns
Limonene 31 28 36 35 ns ns ns
Terpinolene 8 17 8 12 ns ns ns
Bornyl acetate 19 5 17 24 ns ns *
Total monoterpenes 797 513 978 781 ** ** ns
β-Caryophyllene 23 12 31 21 *** ** ns
Stems
α-Pinene 828 889 877 803 ns ns ns
Camphene 15 17 17 14 ns ns ns
Sabinene 71 58 139 103 ns ns ns
β-Pinene 70 58 138 103 ns ns ns
Myrcene 340 226 327 227 ns ns ns
3-Carene 333 1996 669 1131 ** ns *
Limonene 842 869 1234 738 * ns ns
Terpinolene 37 217 81 132 ** ns ns
Bornyl acetate 9 9 19 12 ns ns ns
Total monoterpenes 2585 4394 3540 3298 ** ns *
β-Caryophyllene 34 46 32 29 ns ns ns
Resin acids
Needles
Sandaracopimaric 151 140 174 133 ns ns ns
Isopimaric 23 21 36 15 ** ns ns
Levopimaric + palustric 456 164 478 339 *** * *
Dehydroabietic 1501 1428 1398 1152 ns ns ns
Abietic 971 988 1056 972 ns ns ns
Neoabietic 429 193 514 303 *** * ns
Total resin acids 3536 2945 3660 2917 * ns ns
Stems
Pimaric 119 137 151 268 ns ns ns
Sandaracopimaric 401 368 540 399 * * ns
Isopimaric 759 573 1342 411 ** ns ns
Levopimaric + palustric 678 519 906 365 ns ns ns
Dehydroabietic 3877 3143 4285 3061 ** ns ns
Abietic 6584 6826 9780 9049 ns ns ns
Neoabietic 348 376 580 258 ns ns ns
Total resin acids 12765 11942 17584 13810 ns ns ns

1997, Saxe et al. 1998).
In both species, concentrations of chlorophyll and Rubisco
and Rbc/Chl and Rbc/Prot ratios were lower in current-year
needles than in 1-year-old needles. In Scots pine seedlings,
concentrations of N and Rubisco are generally lower in
1-year-old needles than in current-year needles (Utriainen and
Holopainen 2001). However, this difference may also be asso-
ciated with the varying growth conditions in the previous sea-
son in the nursery, where the 1-year-old needles were formed,
compared with the controlled conditions in the growth cham-
bers, where the current-year needles developed.
In 1-year-old needles of both species, ET decreased the
amount and activity of Rubisco. In Scots pine, the ET-induced
reduction in Rubisco was associated with decreased concen-
trations of chlorophyll and soluble proteins. In both species,
ET also decreased the N concentration of current-year need-
les. It is possible that the ET-induced stimulation of growth led
to an increased demand for N and to increased N allocation

TREE PHYSIOLOGY VOLUME 23, 2003

 TEMPERATURE AND CO2 EFFECTS ON GROWTH AND DEFENSE 105

Table 7. Effects of elevated [CO2] (EC) and temperature (ET) on mean mono- and sesquiterpene concentrations (µg g –1 fm) and mean resin acid
concentrations (µg g –1 dm) in current-year needles and stems of Norway spruce seedlings, and summary of analysis of variance of treatment ef-
fects on concentrations of secondary metabolites (***, P = 0.001; **, P ≤ 0.01; *, P ≤ 0.05; and ns = not significant).

Leaf constituent Control EC ET EC + ET CO2 T CO2 × T

Mono- and sesquiterpenes

Needles
Tricyclene 34 29 61 36 ** ** ns
α-Pinene 196 174 364 244 * *** ns
Camphene 312 256 559 343 * *** ns
Sabinene 18 13 61 32 ** *** ns
β-Pinene 34 32 61 44 ns ** ns
Myrcene 95 64 181 91 ** ** ns
Limonene 272 292 525 324 ns * ns
Eucalyptol 100 73 292 147 ** *** ns
Terpinolene 14 12 25 16 * ** ns
Camphor 18 8 124 55 ns *** ns
Borneol 104 52 186 95 * ** ns
α-Terpineol 27 18 96 46 ** *** ns
Bornyl acetate 526 479 842 630 ns ns ns
Total monoterpenes 1784 1530 3429 2138 * *** ns
β-Caryophyllene 11 11 14 11 ns ns ns
α-Humulene 11 10 15 11 ns ns ns
Stems
α-Pinene 217 131 421 450 ns *** *
Camphene 20 14 30 53 ns *** **
Sabinene 12 15 46 41 ns ** ns
β-Pinene 279 146 478 646 ns *** **
Myrcene 37 29 88 98 ns *** ns
3-Carene 100 150 351 325 ns ns ns
Limonene 337 286 782 881 ns *** ns
Terpinolene 13 15 47 44 * *** ns
Total monoterpenes 1026 791 2262 2561 ns *** *
Resin acids
Needles
Sandaracopimaric 120 118 357 137 * ** **
Isopimaric 53 47 120 59 ** *** *
Levopimaric + palustric 50 215 101 37 ns *** ***
Dehydroabietic 1158 848 2687 1648 *** *** ns
Abietic 127 138 288 149 * *** **
Total resin acids 1512 1382 3557 2033 * *** *
Stems
Pimaric 11 19 57 85 ns *** ns
Sandaracopimaric 361 349 745 791 ns *** ns
Isopimaric 1089 1285 2389 2117 ns *** ns
Levopimaric + palustric 352 64 794 761 ns * ns
Dehydroabietic 3728 2988 5641 5130 ns *** ns
Abietic 1048 1094 1637 1666 ns * ns
Neoabietic 40 12 86 89 ns * ns
Total resin acids 6630 5811 11350 10638 ns *** ns

from older needles to the growing parts of the plant. In
1-year-old Scots pine needles, the decreases in Rbc/Chl and
Rbc/Prot indicate that relatively more N was allocated from
Rubisco than from other proteins; however, the decreases in
the concentrations of Chl and soluble proteins indicate that N
was also allocated from other N-containing compounds. In
1-year-old Norway spruce needles, ET decreased Rbc/Chl but
not Rbc/Prot, indicating that N remobilization from Rubisco
was less than in Scots pine. Because elevated [CO2] has fre-
quently been observed to decrease the amount or activity of
Rubisco in older foliage (Medlyn et al. 1999, Norby et al.
1999, Stitt and Krapp 1999, Laitinen et al. 2000), photosyn-
thetic acclimation could be explained as the result of N defi-
ciency-induced senescence or ontogenetic drift (Stitt and
Krapp 1999). However, we found that EC had little effect on
Rubisco or N concentrations of needles, whereas ET had a

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106 SALLAS, LUOMALA, UTRIAINEN, KAINULAINEN AND HOLOPAINEN
Figure 2. Effects of elevated [CO2] and temperature on total phenolic
concentration (mg g –1 dm) in needles and stems of Norway spruce
and Scots pine seedlings.
large impact on N-containing compounds and increased N al-
location from 1-year-old needles. The EC + ET treatment had
no significant interaction effect on Rubisco, Chl or soluble
proteins, and so the values of these variables in the EC + ET
treatment were close to the additive effects of EC and ET.
Elevated [CO2] generally increases the growth and light-sat-
urated leaf photosynthetic rates (A sat) of C3 plants more in
warm climates than in cool climates (Long 1991). The opti-
mum temperature of A sat increases with increasing atmo-
spheric [CO2] and intercellular [CO2], because of the effect of
elevated [CO2] on the balance between carboxylation and oxy-
genation by Rubisco (Cannell and Thornley 1998). We ob-
served this temperature-dependent CO2 growth response in
both species, with seedlings accumulating the most biomass in
the combined EC + ET treatment. Similarly, Kellomäki and
Wang (1996) reported higher assimilation enhancement ratios
in response to elevated [CO2] for Scots pine trees growing at
temperatures 2–4 °C above ambient compared with trees
growing at ambient temperature. The optimum temperature
for photosynthesis at high irradiance for Scots pine and Nor-
way spruce in northern Europe is 12–15 °C (Troeng and
Linder 1982, Lindroth et al. 1998, Lundmark et al. 1998). This
optimum was exceeded in our ET treatment (23/16 °C).
Secondary metabolites
The reduction in terpene concentrations in response to EC is in
agreement with results for loblolly pine (Williams et al. 1994).
Similarly, Constable et al. (1999) found reductions in β-pinene
concentrations in previous-year needles of ponderosa pine at
elevated [CO2], and reductions in α- and β-pinene and total
monoterpene concentrations in current-year needles of
Douglas-fir at elevated [CO2] at both growth temperatures
(control and +4 °C). However, foliar terpenoids of Scots pine
trees grown for 3 years in elevated [CO2] showed no signifi-
TREE PHYSIOLOGY VOLUME 23, 2003
cant response to CO2 (Kainulainen et al. 1998), whereas α-
pinene increased in response to elevated [CO2] in Scots pine
saplings (Heyworth et al. 1998) and seedlings (Sallas et al.
2001). In the studies reporting no effect of elevated [CO2] on
foliar terpenoids, sampling was conducted late in the autumn
when growth had ceased and reduced sink strength may have
made more resources available for terpenoid synthesis. In de-
ciduous trees, which lack terpene storage structures, elevated
[CO2] down-regulated synthesis of the most volatile mono-
terpenes (α-pinene, β-pinene and sabinene), but not the less
volatile limonene (Loreto et al. 2001), and this might also be
the case in actively growing pine seedlings.
Increased carbohydrate accumulation in leaves may result
in feedback inhibition of photosynthesis. Decreased photosyn-
thetic capacity can occur within days after initial exposure to
elevated [CO2] (Ward and Strain 1999). The decrease, which is
accompanied by depletion of substrates for secondary metabo-
lite production, could account for the decline in foliar terpen-
oid concentrations in response to EC. Faster responses of
terpenoids to [CO2] compared with phenolics may be associ-
ated with the rapid turnover of terpenes (Reichardt et al. 1991,
cf. Gershenzon et al. 1993), because resin acids, which are
terpenoids with high molecular weights that do not undergo
rapid turnover, decrease less than terpenes in response to EC.
An alternative explanation for the decrease in foliar terpen-
oid concentrations in response to EC might be low nitrogen
concentrations, especially in Norway spruce seedlings, which
suffered from a slight N deficiency and accumulated more
starch than Scots pine in all treatments. Both Pinus and Picea
species have elaborate resin-producing structures, the forma-
tion of which is limited by N (Björkman et al. 1991). Kain-
ulainen et al. (1996) found more resin ducts in mature needles
of N-fertilized Scots pine than in unfertilized trees, and con-
centrations of total resin acids and some individual resin acids
in mature needles increased with increasing N fertilization.
Despite incipient N deficiency in the Norway spruce seed-
lings, the EC-induced increase in dry mass was higher in Nor-
way spruce than in Scots pine, and there was an EC-induced
reduction in needle phenolic concentration in Norway spruce.
Peñuelas et al. (1996) also found an inverse relationship be-
tween the responses to elevated [CO2] of foliar total phenolics
and biomass of pine trees (Pinus eldarica L.). Phenylprop-
anoids and proteins share a common precursor (phenylal-
anine), and therefore any environmental factor that affects
plant growth and protein synthesis can also affect the avail-
ability of phenylalanine for phenylpropanoid synthesis (Kor-
icheva et al. 1998). In Norway spruce, phenolic production
may have decreased in response to increased growth in EC, be-
cause major groups of phenolics are produced via phenyl-
alanine and therefore their production competes with protein
synthesis (Haukioja et al. 1998).
An explanation for the increase in monoterpene concentra-
tion in response to ET is that there is an increase in terpene
production at high temperatures, which compensates for in-
creased terpene emission at elevated temperature (Guenther et
al. 1991). Emitted terpenes may protect plants from heat stress
by increasing the thermotolerance of photosynthesis (Singsaas
 TEMPERATURE AND CO2 EFFECTS ON GROWTH AND DEFENSE
et al. 1997). Monoterpenes may be more effective than iso-
prene (Delfine et al. 2000) in protecting membranes from heat
denaturation (Sharkey and Singsaas 1995).
Conclusions
Seedling biomass growth was promoted by ET, but EC had
only slight effects on the growth parameters measured, sup-
porting the prediction of Farrar and Williams (1991) that ele-
vated [CO2] has little effect on the growth of plants adapted to
low temperatures. Both species accumulated the most biomass
in the combined EC + ET treatment (cf. Long 1991). Increased
stem biomass in response to ET and reduced SLA in response
to EC in Norway spruce seedlings may be associated with
greater transpirational stress compared with Scots pine and
could reflect the need for higher water-use efficiency. The
EC-induced thylakoid swelling and increased numbers of
plastoglobuli that we observed in Scots pine needles are con-
sidered to be nonspecific stress symptoms. Although EC had
little effect on Rubisco or N concentration of needles, ET de-
creased the amount and activity of Rubisco of 1-year-old need-
les and the N concentration of current-year needles in both
species. Increased growth in response to ET probably led to in-
creased demand for N and to increased N allocation from the
1-year-old needles to the growing parts of the plant.
The EC treatment had no effect on total phenolic concentra-
tion in needles of Scots pine seedlings and reduced total phe-
nolic concentration in needles of Norway spruce seedlings,
which refutes our hypothesis. Increased growth may account
for the EC-induced decline in total phenolics in Norway
spruce needles. Terpenoids were more responsive to EC and
ET than total phenolics. Generally, EC reduced terpenoid con-
centrations, which is contrary to the source–sink balance hy-
pothesis but supports the suggestion of Loreto et al. (2001)
that terpenoid enzymatic activities in foliage are down-regu-
lated by elevated [CO2]. Elevated temperature increased terp-
enoid concentrations in accordance with our hypothesis. Thus,
although there was a general trend toward increasing concen-
trations of carbon-based secondary compounds in response to
EC, it was restricted to particular compounds such as soluble
phenolics and condensed tannins (Peñuelas and Estiarte
1998). We conclude that, in the boreal species Scots pine and
Norway spruce, elevated [CO2] had less effect on the studied
parameters than elevated temperature.
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