Tree Physiology 19, 807--814
© 1999 Heron Publishing----Victoria, Canada
Long-term effects of elevated carbon dioxide concentration and
provenance on four clones of Sitka spruce (Picea sitchensis). II.
Photosynthetic capacity and nitrogen use efficiency
MAURO CENTRITTO1 and PAUL G. JARVIS2
1
Istituto di Biochimica ed Ecof isiologia Vegetale, Consiglio Nazionale delle Ricerche, via Salaria km 29.300, 00016 Monterotondo Scalo (Roma), Italy
2
Institute of Ecology and Resource Management, University of Edinburgh, Darwin Building, King’s Buildings, Mayfield Road, Edinburgh, EH9 3JU,
Scotland
Received November 17, 1998
Summary Four clones of Sitka spruce (Picea sitchensis
(Bong.) Carr.) from two provenances, at 53.2° N (Skidegate a
and Skidegate b) and at 41.3° N (North Bend a and North
Bend b), were grown for three growing seasons in ambient
(~350 µmol mol −1) and elevated (~700 µmol mol−1) CO2
concentrations. The clones were grown in stress-free conditions (adequate nutrition and water) to assess the effect of
elevated [CO2] on tree physiology. Growth in elevated [CO2]
significantly increased instantaneous photosynthetic rates of
the clonal Sitka spruce saplings by about 62%. Downward
acclimation of photosynthesis (A) was found in all four clones
grown in elevated [CO2]. Rubisco activity and total chlorophyll
concentration were also significantly reduced in elevated
[CO2]. Provenance did not influence photosynthetic capacity.
Best-fit estimates of Jmax (maximum rate of electron transport),
Vcmax (RuBP-saturated rate of Rubisco) and Amax (maximum
rate of assimilation) were derived from responses of A to
intercellular [CO2] by using the model of Farquhar et al. (1980).
At any leaf N concentration, the photosynthetic parameters
were reduced by growth in elevated [CO2]. However, the ratio
between Jmax and Vcmax was unaffected by CO2 growth concentration, indicating a tight coordination in the allocation of N
between thylakoid and soluble proteins. In elevated [CO2], the
more southerly clones had a higher initial N use efficiency
(more carbon assimilated per unit of leaf N) than the more
northerly clones, so that they had more N available for those
processes or organs that were most limiting to growth at a
particular time. This may explain the initial higher growth
stimulation by elevated [CO2] in the North Bend clones than
in the Skidegate clones.
Keywords: acclimation, chlorophyll, electron transport, elevated [CO2 ], photosynthesis, Rubisco.
Introduction
Predicted changes in atmospheric [CO2 ] are expected to increase photosynthetic rates in C3 plants both by increasing the
rate of carbon fixation and by reducing photorespiratory loss
of carbon. Generally, photosynthetic rates of woody plants are
increased by a doubling in CO2 concentration (Eamus and
Jarvis 1989, Amthor 1995). Gunderson and Wullschleger
(1994), in surveying studies of 39 tree species, reported an
average increase of photosynthetic rates of 44% when measured at the growth [CO2] concentrations. Ceulemans and
Mousseau (1994) observed that, in short-term experiments,
elevated [CO2 ] stimulates photosynthesis on average about
40% in conifers and 61% in broad-leaved species. However,
long-term growth in elevated [CO2] often results in a variable
decrease (depending on the species) in the amounts of photosynthetic pigments and enzymes, for instance, amount and
activation state of Rubisco, and, thus, acclimation of the photosynthetic apparatus (Long and Drake 1992). Apparently, this
decrease may occur even when the supply of nitrogen is adequate and rooting volume is large (Drake et al. 1997). Acclimatory depression of photosynthetic capacity has come to the
fore as a process that regulates source--sink interactions (Sheen
1994).
This study reports the effects of stress-free (adequate nutrition, water, pot space) growth in a CO2 concentration of 700
µmol mol −1 on gas exchange, and carbon and nitrogen relationships of four clones of Sitka spruce (Picea sitchensis
(Bong.) Carr.) of two provenance after three years of CO2
exposure. It has been shown in a companion paper (Centritto
et al. 1999) that the dry mass of all four clones was increased
by elevated [CO2 ], but that the more northerly clones, despite
being grown at a latitude close to their latitudinal provenance,
were significantly less responsive than the more southerly
clones. To understand whether these differences in growth
resulted from a direct effect of elevated [CO2 ] on photosynthesis, the photosynthetic capacity of the clones was analyzed in
relation to leaf nitrogen concentration. Clones from two provenances were used because in the temperate and northerly
regions the contribution of clones to the fitness of the populations is relevant (Callaghan et al. 1992). However, climate
change is thought to give rise to mass migration of plants,
thereby modifying composition of plant communities by
changing the intraspecific as well as interspecific competitive
performances. Understanding specific ways in which different
808
CENTRITTO AND JARVIS
genotypes respond to elevated [CO2] is necessary to predict the
likely impact of global change on vegetation.
Materials and methods
Saplings of four clones of Sitka spruce (Picea sitchensis
(Bong.) Carr.) from two provenances, at 53.2° N (Skidegate a
and Skidegate b) and at 41.3° N (North Bend a and North
Bend b), were grown for three growing seasons in open top
chambers (OTCs) with ambient [CO2] (~350 µmol mol −1) or
elevated [CO2] (ambient + ~350 µmol mol −1). The OTCs were
located at the Institute of Terrestrial Ecology (ITE), Bush
Estate, near Edinburgh, U.K. The saplings were potted in
standard potting compost, watered every other day to pot water
capacity and regularly fertilized in both growing seasons following Ingestad’s principles (Ingestad and Ågren 1992). Full
details of the growth conditions, and of the statistical analysis
used to test the data are described in a companion paper
(Centritto et al. 1999).
Gas exchange measurements were made in a glasshouse at
the University of Edinburgh, on the central section of currentyear branches with a portable gas exchange system (ADCLCA-3, Analytical Development Co. Ltd., Hoddesdon, U.K.)
equipped with a conifer leaf chamber (PLC-3, Analytical Development Co. Ltd., Hoddesdon, U.K.). To enable measurements of PPFD-saturated photosynthetic rates, illumination of
the leaf cuvette by natural sunlight was supplemented with
artificial light (provided by a white fluorescent lamp) to maintain PPFD incident on the needles above 1700 µmol m −2 s −1.
The planar area of needles on each sample was determined by
removing all the needles enclosed by the leaf cuvette and
passing them through a leaf area meter (LI 3000, Li-Cor, Inc.,
Lincoln, NE). Instantaneous leaf CO2 assimilation rates (A)
were measured between 1100 and 1300 h at the end of July
1993 on 20 saplings (five plants per clone) per [CO2] treatment, at the growth CO2 concentrations. Rates of short-term
(10 min) PPFD-saturated (1700--2000 µmol m −2 s −1) CO2
assimilation were measured at leaf internal CO2 concentrations
(A--Ci) ranging from 40 to 1200 µmol mol −1. Observations
were made in August 1993 between 1000 and 1700 h, on
twelve saplings (three per clone) per [CO2 ] treatment. The
initial slope of the A--C i curves is an estimate of the carboxylation efficiency (Vcmax , RuBP-saturation rate of Rubisco),
whereas the maximum rate of assimilation (Amax ) (the net CO2
assimilation rate under conditions of PPFD and CO2 saturation) is indicative of the role of RuBP regeneration, and is
related to electron transport under conditions of PPFD-saturation (Jmax ). Parameters Jmax , Vcmax , and Amax , were estimated by
fitting the C3 photosynthesis model of Farquhar et al. (1980) to
individual A--Ci response curves following the procedure outlined by Wullschleger (1993).
Total Rubisco activity was assayed spectrophotometrically
by a coupled enzyme method (Besford 1984, Van Oosten et al.
1995). Five needles from one plant from each clone (i.e., 100
needles per [CO2 ] treatment) were sampled in July 1993 for
Rubisco activity assays. The needles were removed from midway along current-year branches. Needle mass and area were
rapidly measured before plunging the needles in liquid nitrogen.
Needle concentrations of chlorophylls a, b, and a + b were
measured on different needles sampled as described above.
The concentration of chlorophylls was measured in intact leaf
tissues by immersion in N,N-dimethylformamide (DMF) following the techniques described by Porra et al. (1989). The
saplings were sampled once a month from March to October
in the second growing season, and from February to October
in the third growing season. Needles were removed from
midway down a current-year branch and their area rapidly
measured before they were plunged in liquid nitrogen.
Samples for determination of sugar and starch concentrations of roots and current-year needles were taken at each
harvest during the second and third growing seasons (see
Centritto et al. 1999). The numbers of root and leaf samples
taken were 24 per [CO2] treatment (six per clone) on Day 381,
40 per [CO2] treatment (10 per clone) on Day 551, and 20 per
[CO2 ] treatment (five per clone) on Days 719 and 972. Soluble
sugars were measured by high pressure liquid chromatography
(HPLC). Five cm3 of NaOH solution (0.0025 mol m3) was
added to freeze-dried, ground tissue of the sapling samples
ranging in mass between 0.049 and 0.051 g. The samples were
incubated for 15 minutes in a water bath at 30 °C. The solutions
were then centrifuged for 15 minutes at 3000 g, and the
supernatant collected and vacuum filtered using a 0.2 µm
nitrocellulose filter (Whatman International Ltd., Maidstone,
U.K.). The amount of soluble sugars present in the sample was
determined by HPLC (Dionex DX 500, Dionex Corp., Sunnyvale, CA). Starch concentration was determined by the iodometric method (Allen 1989).
Results
Elevated CO2 significantly (P < 0.001) increased CO2 assimilation rate of Sitka spruce saplings by about 62% at the end
of July of the third growing season, when measured at the
growth CO2 concentration (Table 1). Stimulation occurred in
all clones, and was higher, though not significantly, in the
Skidegate a and b clones (about 95% and 76%, respectively)
than in the North Bend clones (about 43%) (Table 2). The
relationship between PPFD-saturated CO2 assimilation rate
and leaf internal CO2 concentration was used to ascertain the
Table 1. Total Rubisco activity (on a leaf area basis), measured with
saturating CO2 in vitro, and assimilation rate (A) of the Sitka spruce
saplings (all clones) in ambient or elevated [CO2]. Data are means of
20 plants per [CO2 ] treatment ± 1 SEM. Measurements of A were
made at the growth CO2 concentrations, with a mean temperature of
24.9 ± 0.28 °C (1 SEM) in saturating PPFD (> 1700 µmol m − 2 s −1)
between 1100 and 1300 h.
Elevated
Ambient
Significance: CO2
TREE PHYSIOLOGY VOLUME 19, 1999
Rubisco (µmol m −2 s − 1)
A (µmol m − 2 s −1)
27.88 ± 1.54
43.72 ± 3.13
P < 0.001
16.83 ± 0.44
10.09 ± 0.37
P < 0.001
EFFECTS OF CO2 AND PROVENANCE ON SPRUCE PHYSIOLOGY
809
Table 2. Assimilation rate (A) measured at the growth CO2 concentrations, Amax (from A--C i curves), and total Rubisco activity in vitro (on a leaf
area basis), of the four Sitka spruce clones. Data are means of three to five plants per treatment ± 1 SEM. The significance levels (* = P < 0.05,
** = P < 0.01, *** = P < 0.001) apply to the within-clone difference in response to [CO2].
A (µmol m −2 s − 1)
Skidegate a
Skidegate b
North Bend a
North Bend b
A max (µmol m −2 s − 1)
Rubisco (µmol m − 2 s −1)
Elevated
Ambient
Elevated
Ambient
Elevated
Ambient
18.33 ± 0.99
16.33 ± 0.72
15.88 ± 0.49
16.19 ± 0.67
9.39 ± 0.52 ***
9.27 ± 0.11 **
11.15 ± 0.23 ***
11.29 ± 1.28 **
21.97 ± 1.24
19.86 ± 1.19
19.50 ± 1.17
19.23 ± 0.96
27.28 ± 0.69 **
26.86 ± 0.95 *
26.01 ± 0.92 *
27.14 ± 0.57 **
29.97 ± 1.99
28.10 ± 2.13
27.51 ± 4.41
25.94 ± 2.90
52.41 ± 7.48 **
36.23 ± 2.77 *
45.48 ± 6.35 *
40.75 ± 5.33 *
biochemical limitation to photosynthesis. These A--Ci measurements showed some downward acclimation of photosynthesis in the saplings grown in elevated [CO2] (Figure 1). The
decrease in Amax in the elevated [CO2] treatment was about
25% (P < 0.001). A downward acclimation of Amax was observed in all four clones grown in elevated [CO2 ] (Table 2).
There were no differences in Amax among clones in either
elevated or ambient [CO2].
The A--C i response curves also showed that ambient [CO2]grown saplings had higher carboxylation efficiencies than
elevated [CO2 ]-grown saplings (Figure 1). Carboxylation efficiency is generally interpreted as a limitation by Rubisco
activity, which was significantly reduced in vitro by 36% by
elevated [CO2 ] (Table 1). All clones in elevated [CO2] showed
downward acclimation of carboxylation efficiency (data not
shown). In parallel, Rubisco activity in vitro significantly decreased in response to long-term growth in elevated [CO2] by
about 22% in Skidegate b, 36% in North Bend b, 39% in North
Bend a, and 43% in Skidegate a (Table 2). However, there were
no significant differences in Rubisco activity among the clones
in either [CO2] treatment.
Total chlorophyll concentration was significantly lower at
all sampling times in saplings grown in elevated [CO2] com-
pared with saplings grown in ambient [CO2 ] in both growing
seasons (Table 3).
Figure 2 shows needle and root sugar concentrations per unit
of dry mass of the Sitka spruce saplings harvested at the
beginning and end of both the second and third growing seasons. The sugar concentration was greater in both needles
(Figure 2a) and roots (Figure 2b) of plants grown in elevated
[CO2 ] compared with plants grown in ambient [CO2 ], but the
differences were significant only in needles harvested on Day
382. There was a significant increase in starch concentration at
the end of both the second and third growing season (Days 551
and 972, respectively) in needles (Figure 3a) and roots (Figure
3b) of saplings grown in elevated [CO2] compared with ambient [CO2 ]-grown saplings.
Figure 4 shows the relationships between leaf N concentration and mean total dry mass of the saplings in both ambient
and elevated [CO2 ]. Growth in different CO2 concentrations
affected the nitrogen concentrations when the saplings were
the same size, as demonstrated by the linear relationships
between mean total dry mass and nitrogen concentration of
leaves (R 2 = 0.926 for the elevated [CO2 ] saplings, and R 2 =
0.993 for the ambient [CO2 ] saplings). Growth in elevated
[CO2 ] also affected foliar nitrogen concentration of the four
Table 3. Total chlorophyll concentration (mg cm − 2), measured at
monthly intervals in the second and third growing seasons, in needles
of Sitka spruce saplings grown in ambient or elevated [CO2 ]. Data are
means of 20 plants per treatment ± 1 SEM. Abbreviation: ns = not
significant.
Figure 1. The relationship between net CO2 assimilation rate (A) of
Sitka spruce saplings of the four clones and intercellular CO2 concentration (Ci ) in saturating PPFD (> 1700 µmol m −2 s − 1). The measurements were made on shoots of twelve saplings per [CO2] treatment.
Mean Amax , averaged across the four clones, was statistically different
between treatments (P < 0.001).
February
March
April
May
June
July
August
September
October
Second growing season
Third growing season
Elevated
Ambient
Elevated
-77.53 ± 2.93
70.53 ± 2.93
52.09 ± 1.61
73.28 ± 3.99
66.44 ± 3.05
75.70 ± 9.36
80.14 ± 4.07
79.75 ± 5.46
-91.27 ± 6.62 107.90 ± 5.51
87.98 ± 3.83 78.66 ± 6.51 83.95 ± 4.88
80.98 ± 3.83 105.53 ± 6.96 111.04 ± 7.16
57.68 ± 2.10 111.90 ± 6.43 122.73 ± 7.39
84.46 ± 4.08 33.18 ± 2.56 36.14 ± 1.72
76.76 ± 3.53 31.15 ± 2.91 41.21 ± 2.92
96.01 ± 7.14 40.44 ± 3.95 50.71 ± 3.73
85.54 ± 4.52 33.45 ± 3.22 51.20 ± 4.31
86.49 ± 3.66 37.80 ± 3.50 50.70 ± 2.97
Statistical significance:
CO2
Time
Interaction
P < 0.001
P < 0.001
ns
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
Ambient
P < 0.001
P < 0.001
ns
810
CENTRITTO AND JARVIS
Figure 2. Needle (a) and root
(b) sugar concentrations per
unit of dry mass in the Sitka
spruce saplings grown in ambient or elevated [CO2], plotted
against time since the beginning of the experiment. Data
are means of 20 to 40 plants
per treatment ± 1 SEM. The
significance level (** = P <
0.01) shows the difference in
sugar concentration in response to the [CO2] treatments.
Figure 3. Needle (a) and root
(b) starch concentrations per
unit of dry mass in the Sitka
spruce saplings grown in ambient or elevated [CO2], plotted
against time since the beginning of the experiment. Data
are means of 20 to 40 plants
per treatment ± 1 SEM. The
significance levels (* = P <
0.05, ** = P < 0.01, *** = P <
0.001) show the difference in
starch concentration in response to the [CO2] treatments.
Figure 4. Linear relationships between the combined mean foliar
nitrogen concentration and total dry mass of the Sitka spruce saplings
grown in ambient or elevated [CO2]. Data are means of 20 to 40 plants
per treatment ± 1 SEM.
clones when the saplings were the same size (Figure 5). There
was a trend to produce more biomass with a lower foliar N
concentration in the more southerly clones when they were
younger. Foliar N concentration decreased to similar values in
all four clones as they grew larger, although the North Bend
clones maintained a greater total dry mass than the Skidegate
clones.
There was a positive linear relationship between best-fit
estimates of Jmax, Vcmax , and Amax and leaf nitrogen concentration when expressed on a leaf area basis in both elevated and
ambient [CO2] (Figure 6). However, saplings grown in elevated [CO2] had lower values of all three parameters per unit
Figure 5. Relationships between foliar nitrogen concentration and total
dry mass of the four clones of Sitka spruce grown in elevated [CO2 ].
Data are means of five to 10 plants per treatment ± 1 SEM.
of foliar nitrogen on a leaf area basis than saplings grown in
ambient [CO2 ]. The values of Vcmax and Jmax were similar in
magnitude to those reported by Walcroft et al. (1997) for Pinus
radiata D. Don, when based on projected leaf area. A strong
positive, linear correlation was also observed between the
best-fit estimates of Jmax and Vcmax (Figure 7); i.e., Jmax =
2.39Vcmax .
Discussion
The four clones of Sitka spruce were grown in stress-free
conditions (adequate nutrition and water) to assess the effect
of elevated [CO2 ] on tree physiology, thus ruling out any
TREE PHYSIOLOGY VOLUME 19, 1999
EFFECTS OF CO2 AND PROVENANCE ON SPRUCE PHYSIOLOGY
Figure 6. Linear relationships between best-fit estimates of the photosynthetic parameters derived from individual A--C i curves (using the
model of Farquhar et al. 1980) and foliar nitrogen concentration on
a leaf area basis; (a) J max (R 2 = 0.533 for the elevated [CO2 ] plants,
R 2 = 0.553 for the ambient [CO2 ] plants), (b) Vcmax (R 2 = 0.523 for the
elevated [CO2 ] plants, R 2 = 0.510 for the ambient [CO2] plants), and
(c) A max (R 2 = 0.569 for the elevated [CO2 ] plants, R 2 = 0.502 for the
ambient [CO2] plants).
Figure 7. Linear relationship (R 2 = 0.934) between the maximum rate
of electron transport (J max ) and the maximum rate of carboxylation
(Vcmax ), derived as best-fit estimates from individual A--C i curves.
effects that insufficient water or nutrient supply might have
caused. Recently, Drake et al. (1997) have stressed the importance of available nutrients in determining the extent of the
stimulation of A in elevated [CO2]; in a review of eight experiments the average stimulation dropped from 57% when nitrogen availability was high to 23% when nitrogen availability
was low. Long-term growth in elevated [CO2] increased instan-
811
taneous photosynthetic rates of the clonal Sitka spruce saplings by about 62% (Table 1). Similar increases in assimilation
rate were found in juvenile foliage of nine-year-old Pinus
taeda L. trees exposed for the second year to a doubling of CO2
concentration (Murthy et al. 1997). In two-year-old Sitka
spruce clones, Townend (1993) found a significant effect of
growth in elevated [CO2] (600 µmol mol −1) on the response of
A to PPFD, but did not quantify this increase. Lucas (1998)
found that A of 3-year-old Sitka spruce seedlings was 35%
higher in elevated [CO2] (700 µmol mol −1) than in ambient
[CO2 ]. Barton (1997) found that A of both current-year and
1-year-old shoots of mature Sitka spruce doubled in response
to elevated [CO2] (700 µmol mol −1) in a branch bag experiment.
The more southerly clones (North Bend a and b) showed an
increase in assimilation rate of about 43%, which is close to
the average increase for conifers reported in the survey by
Gunderson and Wullschleger (1994), whereas the increases in
photosynthetic rates of the more northerly clones (Skidegate a
and b) were well above the average (Table 2). However, the
Skidegate a and b clones produced significantly less total dry
mass than the North Bend clones in elevated [CO2 ] at the end
of the third growing season (Centritto et al. 1999). Either the
increases in instantaneous assimilation rates of each clone,
measured at the beginning of August in the third growing
season, were not representative of the average increase over the
entire growth period studied, or there were large losses of
carbon in respiration, volatilization, root exudation and fine
root turnover in the more northerly clones. These processes
were not studied in this work, but they can account for a large
proportion of assimilates lost (Wang et al. 1998). Furthermore,
other important factors, including mutual leaf shading, leaf
area per plant and respiratory costs, affect growth responses.
Oleksyn et al. (1992) found that, in Pinus sylvestris L., root
respiration accounted for about two-thirds of total respiratory
cost. At the beginning of the growth period, the Skidegate
clones allocated proportionally more dry mass to the roots
compared with the North Bend clones (data not shown), which
might have increased whole-plant respiratory losses and led to
an initially smaller growth stimulation by [CO2 ] than in the
North Bend clones.
Long-term growth in elevated [CO2] may result in reduced
amounts of photosynthetic pigments and enzymes (Eamus and
Jarvis 1989). However, Arp (1991) showed that downward
acclimation of photosynthetic capacity and size of pot were
highly correlated, and that constrained rooting caused by inadequate pot volume may cause downward acclimation of
photosynthetic capacity in elevated [CO2 ]. In contrast, plants
rooted in the ground show little acclimation of photosynthetic
capability (Liu and Teskey 1995, Curtis and Wang 1998). A
three-year exposure to elevated [CO2] of branches of mature
Sitka spruce in a stand did not cause acclimation of A in
current-year needles, but caused some downward acclimation
in one-year-old needles (Barton 1997). There were no significant changes in the A--Ci relationship measured in branches
exposed to elevated [CO2 ] of 22-year-old Pinus taeda trees in
all the three years of growth, indicating that elevated [CO2]
(ambient + 330 µmol mol −1) did not alter the photosynthetic
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
812
CENTRITTO AND JARVIS
capacity of the foliage when adequate sinks were available
(Teskey 1997). There were no significant differences in A--C i
curves between Betula pendula Roth plants grown in elevated
(700 µmol mol −1) and ambient [CO2 ] in Ingestad units with
steady-state nutrition (Pettersson and McDonald 1992). In the
present experiment, the Sitka spruce saplings were supplied
with free access to nutrients, and dry mass allocation was
identical when the plants were the same size suggesting that
they were not pot limited (Centritto et al. 1999). Yet, downward
acclimation of photosynthetic capacity occurred (Figure 1) in
each clone (Table 3), as was also found by Lucas (1998) in
three Sitka spruce clones and Barton (1997) in 2-year-old
seedlings of Sitka spruce.
The A--C i response curves showed both a decrease in Amax,
which is a function of photosynthetic electron transport and
regeneration of RuBP, and in carboxylation efficiency, which
is limited by Rubisco activity. The latter observation is consistent with the in vitro Rubisco activities, which decreased in
elevated [CO2]-grown saplings (Table 1). This decline of photosynthesis was in agreement with the downward acclimation
of Rubisco (Van Oosten et al. 1995), which may result from
both lowered enzyme activation and enzyme amount (Tissue
et al. 1993, Vu et al. 1997).
Nitrogen concentration was affected by growth in elevated
[CO2] (Centritto et al. 1999). Reduction in leaf N concentration in elevated [CO2] was only partially caused by starch
dilution, because starch concentration was not always affected
(Figure 3a). There are several processes by which elevated
[CO2] can lead to decreased leaf N concentration. These include inhibition of photorespiration and reduced amounts of
photosynthetic pigments and enzymes. Rubisco is the largest
pool of nitrogen in leaves (Drake et al. 1997), and its content
can be reduced by about 35% in elevated [CO2] before there is
co-limitation of A (Long and Drake 1992). Carbon assimilation rate of the four clones was increased by about 51% in
elevated [CO2] (Table 2) despite a reduction in Vcmax per unit
of leaf nitrogen (Figure 6b). Thus, because less Rubisco was
required in elevated [CO2] (Table 1), increased carbon assimilation per unit of leaf N led to increased nitrogen use efficiency
(NUE).
The chlorophyll--protein complexes also constitute major
pools of N in leaves (Evans 1997), and are usually decreased
by elevated [CO2 ] (Drake et al. 1997), as found here (Table 3).
Wullschleger (1993) found that, in a retrospective analysis of
the A--Ci curves from a large number of C3 species, the carboxylation and light-harvesting capabilities were closely coupled. Coordinated regulation of the biochemical limitations to
photosynthesis has also been shown in elevated [CO2]. Van
Oosten et al. (1995) reported that downward acclimation of the
light-harvesting complexes in tomato plants grown in elevated
[CO2] followed the decline in carboxylation capacity. In our
study, a strong positive linear relationship between the best-fit
estimates of Jmax and Vcmax was also found (Figure 7). This may
indicate that the decline in chlorophyll concentration (on a leaf
area basis) and the downward acclimation of Jmax per unit of
leaf N (also expressed on a leaf area basis) in elevated [CO2]
(Figure 6a), resulted from the tight coordination of the activities of thylakoid proteins and soluble proteins to match each
other. The ability to maintain a constant ratio between the
carboxylation and light-harvesting activities across a wide
range of environmental conditions (Wullschleger 1993) originates from a functional balance in the allocation of N between
thylakoid and soluble proteins. This allows optimization of
resource allocation (Evans 1997) and leads to a more efficient
use of N (Von Caemmerer and Farquhar 1981).
Furthermore, inhibition of photorespiration in plants grown
in elevated [CO2] may also reduce the amount of nitrogen
required per unit of dry mass produced (Conroy and Hocking
1993). Thus, changes in the biochemistry of photosynthesis
and photorespiration, which usually occur when N uptake does
not keep pace with carbon uptake (Jarvis 1995), may be regarded as an optimization process that involves reallocation of
nitrogen away from non-limiting components to more limiting
processes or organs (i.e., additional or larger sinks for the extra
carbon assimilated), and consequently leading to increased
NUE.
The lower N concentration per unit of leaf area in elevated
[CO2 ] may account for the acclimation of the photosynthetic
capability of the four clones, despite free access to nitrogen
and the large rooting volume (10 dm3), although, as Drake et
al. (1997) pointed out, downward acclimation of A is the
exception rather than the rule when the rooting volume exceeds 10 dm3. However, a recent study on the interactive effects of elevated [CO2] (700 µmol mol −1) and N supply in
field-grown rice has shown that reduced foliage concentration
of N (even if calculated as a percentage of structural dry mass)
was always associated with increased [CO2 ] (Ziska et al.
1996). This is a common observation in many species and is
hard to explain. In the Sitka spruce saplings, leaf N concentration was clearly lower when the plants were the same size in
the two [CO2 ] treatments (Figure 4), indicating that growth in
elevated [CO2] increased the dry mass produced per unit of
nitrogen taken up. Increased growth per unit of plant nitrogen
and phosphorus was also noted with seedlings of Pinus ponderosa Dougl. ex Laws. grown in 700 µmol mol −1 of CO2
(DeLucia et al. 1997).
Van Oosten and Besford (1996) have described a molecular
model for photosynthetic acclimation. The model invokes metabolite regulation of gene expression, which probably occurs
when the production of new assimilates is larger than the
capacity to handle them. This can involve a source--sink imbalance leading to feedback effects on photosynthesis by endproduct accumulation (Stitt 1991). The cytoplasmatic pool of
glucose may provide a regulatory signal for coarse control,
which determines the amount of photosynthetic systems by
repressing photosynthetic gene expression (rbcS, cab-7, cab3C, rca), thereby triggering a cascade of reactions that lead to
acclimation of the photosynthetic apparatus. However, sugar
concentration in needles of the Sitka spruce saplings was not
increased significantly at the beginning or end of the third
growing season (Figure 2), when photosynthetic acclimation
in elevated [CO2 ] was detected (Figure 1). This finding seems
to conflict with the model put forward by Van Oosten and
Besford (1996). However, Paul and Driscoll (1997) have recently shown that loss of photosynthetic activity is better
correlated with an increase in the C/N ratio than with carbohy-
TREE PHYSIOLOGY VOLUME 19, 1999
EFFECTS OF CO2 AND PROVENANCE ON SPRUCE PHYSIOLOGY
drate status. Our results accord with this view. The higher leaf
C/N ratio in needles at the end of both the second and third
growing seasons (Days 719 and 972), as a result of similar
sugar concentrations (Figure 2a) but lower nitrogen concentrations (Centritto et al. 1999), may explain photosynthetic downward acclimation in elevated [CO2]. This result is consistent
with findings in several other studies. For instance, A of two
full-sib families of Pinus ponderosa seedlings was lower in
elevated [CO2 ] than in ambient [CO2 ] after about 39 days from
germination, but the C/N ratio of the needles had already
increased in elevated [CO2 ] (Grulke et al. 1993).
Reductions in Rubisco activity and chlorophyll concentration led to improved nitrogen use efficiency in elevated [CO2],
because Amax , Jmax , and Vcmax scaled linearly with leaf N concentration (Figure 6), and the ratio between Jmax and Vcmax was
not changed by growth in elevated [CO2] (Figure 7). Wullschleger (1993) has shown that these two parameters describing photosynthetic capacity are species-specific. Furthermore,
despite their importance in mechanistic models predicting the
effects of global change on growth processes, there are few
values of these parameters for different species in the literature
(Walcroft et al. 1997). As far as we know, there are no published values of Jmax and Vcmax, determined from A--Ci curves, in
relation to leaf N concentration for Picea sitchensis (Bong.)
Carr. grown in elevated [CO2 ].
In conclusion, the amount of N needed for growth was less
when Sitka spruce saplings growing in elevated [CO2] were the
same size as saplings in ambient [CO2 ] (Figure 4), and this is
likely to result in an advantage for plants growing in a nitrogen-limited environment. Provenance did not significantly influence either photosynthetic capacity or A measured at the
growth CO2 concentrations in the clonal Sitka spruce saplings
(Table 2), although, the more northerly clones were significantly less responsive to elevated [CO2] than the southerly
clones (Centritto et al. 1999). Genetic differences in growth
response to elevated [CO2] were already evident after the first
year of growth, and they were magnified over time becoming
significant after three full growing seasons. The more southerly clones had higher initial NUE than the more northerly
clones (Figure 5), showing that they had more N available for
those processes or organs that were most limiting to growth.
Clonal provenance affected growth in elevated [CO2 ] and plant
nitrogen use efficiency may have played an important role.
This finding is particularly important for northern countries
where N is the most limiting resource and will, therefore, affect
Sitka spruce growth as the atmospheric CO2 concentration
rises, unless compensated by wet and dry N deposition.
Acknowledgments
This research was done within the EU Project ECOCRAFT (Contract
No. ENV4-CT95-0077) ‘‘The likely impact of rising CO2 and temperature on European forests.’’ Mauro Centritto was supported by an
agreement between Consiglio Nazionale delle Ricerche and British
Council. We would like to thank Dr R. Besford (then at Horticulture
Research International Institute, Littlehampton, U.K.) for the analyses
of Rubisco activity.
813
References
Allen, S.E. 1989. Chemical analysis of ecological materials. 2nd Edn.
Blackwell Science, Oxford, 384 p.
Amthor, J.S. 1995. Terrestrial higher-plant response to increasing
atmospheric [CO2] in relation to the global carbon cycle. Global
Change Biol. 1:243--274.
Arp, W.J. 1991. Effects of source--sink relations on photosynthetic
acclimation to elevated CO2. Plant Cell Environ. 14:869--875.
Barton, C.V.M. 1997. Effects of elevated atmospheric carbon dioxide
concentrations on growth and physiology of Sitka spruce (Picea
sitchensis (Bong.) Carr). PhD Thesis, Univ. Edinburgh, 203 p.
Besford, R.T. 1984. Some properties of ribulose bisphosphate carboxylase extracted from tomato leaves. J. Exp. Bot. 35:495--504.
Callaghan, T.V., B.Å. Carlsson, I.S. Jónsdóttir, B.M. Svensson and
S. Jonasson. 1992. Clonal plants and environmental change: introduction to the proceedings and summary. Oikos 63:341--347.
Centritto, M., H.S.J. Lee and P.G. Jarvis. 1999. Long-term effect of
elevated carbon dioxide concentrations and provenance on four
clones of Sitka spruce (Picea sitchensis). I. Plant growth, allocation
and ontogeny. Tree Physiol. 19:799--806.
Ceulemans, R. and M. Mousseau. 1994. Effects of elevated atmospheric CO2 on woody plants. New Phytol. 127:425--446.
Conroy, J. and P. Hocking. 1993. Nitrogen nutrition of C3 plants at
elevated atmospheric CO2 concentrations. Physiol. Plant. 89:570-576.
Curtis, P.S. and X. Wang. 1998. A meta-analysis of elevated CO2
effects on woody plant mass, form and physiology. Oecologia
113:299--313.
De Lucia, E.H., R.M. Callaway, E.M. Thomas and W.H. Schlesinger.
1997. Mechanisms of phosphorus acquisition for ponderosa pine
seedlings under high CO2 and temperature. Ann. Bot. 79:111--120.
Drake, B.G., M.A. Gonzàlez-Meler and S.P. Long. 1997. More efficient plants: a consequence of rising atmospheric CO2? Annu. Rev.
Plant Physiol. Plant Mol. Biol. 48:607--637.
Eamus, D. and P.G. Jarvis. 1989. The direct effects of increase in the
global atmospheric CO2 concentration on natural and commercial
temperate trees and forest. Adv. Ecol. Res. 19:1--55.
Evans, J.R. 1997. Developmental constraints on photosynthesis: effects
of light and nutrition. In Photosynthesis and the Environment. Ed.
N.R. Baker. Kluwer Academic Publishers, Dordrecht, pp 281--304.
Farquhar, G.D., S. Von Caemmerer and J.A. Berry. 1980. A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta 149:78--90.
Grulke, N.E., J.L. Hom and S.W. Roberts. 1993. Physiological adjustment of two full-sib families of ponderosa pine to elevated CO2.
Tree Physiol. 104:391--401.
Gunderson, C.A. and S.D. Wullschleger. 1994. Photosynthetic acclimation in trees to rising atmospheric CO2: a broader perspective.
Photosynth. Res. 39:369--388.
Ingestad, T. and G.I. Ågren. 1992. Theories and methods on plant
nutrition and growth. Physiol. Plant. 84:177--184.
Jarvis, P.G. 1995. The role of temperate trees and forests in CO2
fixation. Vegetatio 21:157--174.
Liu, S. and R.O. Teskey. 1995. Responses of foliar gas exchange to
long-term elevated CO2 concentration in mature loblolly pine trees.
Tree Physiol. 15:351--359.
Long, S.P. and B.G. Drake. 1992. Photosynthetic CO2 assimilation and
rising atmospheric CO2 concentrations. In Crop Photosynthesis:
Spatial and Temporal Determinants. Eds. N.R. Baker and H.
Thomas. Elsevier Science Publishers B.V., Amsterdam, pp 69--103.
Lucas, M. 1998. Allocation in tree seedlings. PhD Thesis, Univ.
Edinburgh, 300 p.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
814
CENTRITTO AND JARVIS
Murthy, R., S.J. Zarnoch and P.M. Dougherty. 1997. Seasonal trends
of light-saturated net photosynthesis and stomatal conductance of
loblolly pine trees grown in contrasting environments of nutrition,
water and carbon dioxide. Plant Cell Environ. 20:558--568.
Oleksyn, J., M.G. Tjoelker and P.B. Reich. 1992. Whole-plant CO2
exchange of seedlings of two Pinus sylvestris L. provenances grown
under stimulated photoperiodic conditions of 50° and 60° N. Trees
6:225--231.
Paul, M.J. and S.P. Driscoll. 1997. Sugar repression of photosynthesis:
the role of carbohydrates in signalling nitrogen deficiency through
source:sink imbalance. Plant Cell Environ. 20:110--116.
Pettersson, R. and A.J.S. McDonald. 1992. Effects of elevated carbon
dioxide concentration on photosynthesis and growth of small birch
plants (Betula pendula Roth.) at optimal nutrition. Plant Cell Environ. 15:911--919.
Porra, R.J., W.A. Thompson and P.E. Kriedemann. 1989. Determination of accurate extinction coefficients and simultaneous equations
for assaying chlorophylls a and b extracted with four different
solvents: verification of the concentration of chlorophyll standards
by atomic absorption spectroscopy. Biochim. Biophys. Acta
975:384--394.
Sheen, J. 1994. Feedback-control of gene expression. Photosynth.
Res. 39:427--438.
Stitt, M. 1991. Rising CO2 levels and their potential significance for
carbon flow in photosynthetic cells. Plant Cell Environ. 14:741-762.
Teskey, R.O. 1997. Combined effects of elevated CO2 and air temperature on carbon assimilation of Pinus taeda trees. Plant Cell Environ.
20:373--380.
Tissue, D.T., R.B. Thomas and B.R. Strain. 1993. Long-term effects
of elevated CO2 and nutrients on photosynthesis and Rubisco in
loblolly pine seedlings. Plant Cell Environ. 16:859--865.
Townend, J. 1993 Effects of elevated carbon dioxide and drought on
the growth and physiology of clonal Sitka spruce plants (Picea
sitchensis (Bong.) Carr.). Tree Physiol. 13:389--399.
Van Oosten, J.-J. and R.T. Besford. 1996. Acclimation of photosynthesis to elevated CO2 through feedback regulation of gene expression:
climate of opinion. Photosynth. Res. 48:353--365.
Van Oosten, J.-J., D. Wilkins and R.T. Besford. 1995. Acclimation of
tomato to different carbon dioxide concentrations. Relationship
between biochemistry and gas exchange during leaf development.
New Phytol. 130:357--367.
Von Caemmerer, S. and G.D. Farquhar. 1981. Some relationships
between the biochemistry of photosynthesis and gas exchange of
leaves. Planta 159:376--387.
Vu, J.C.V., L.H. Allen, Jr., K.J. Boote and G. Bowes. 1997. Effects of
elevated CO2 and temperature on photosynthesis and Rubisco in
rice and soybean. Plant Cell Environ. 20:68--76.
Walcroft, A.S., D. Whitehead, W.B. Silvester and F.M. Kelliher. 1997.
The response of photosynthetic model parameters to temperature
and nitrogen concentration in Pinus radiata D. Don. Plant Cell
Environ. 20:1338--1348.
Wang, Y.-P., A. Rey and P.G. Jarvis. 1998. Carbon balance of young
birch trees grown in ambient and elevated atmospheric CO2 concentrations. Global Change Biol. 4:797--808.
Wullschleger, S.D. 1993. Biochemical limitations to carbon assimilation in C3 plants----A retrospective analysis of the A--Ci curves from
109 species. J. Exp. Bot. 44:907--920.
Ziska, L.H., W. Weerakoon, O.S. Namuco and R. Pamplona. 1996. The
influence of nitrogen on the of elevated CO2 response in fieldgrown rice. Aust. J. Plant Physiol. 23:45--52.
TREE PHYSIOLOGY VOLUME 19, 1999