Directory UMM :Data Elmu:jurnal:T:Tree Physiology:vol17.1997:
Tree Physiology 17, 319--325
© 1997 Heron Publishing----Victoria, Canada
Effects of elevated CO2 on chloroplast components, gas exchange and
growth of oak and cherry
C. J. ATKINSON,1 J. M. TAYLOR,1 D. WILKINS2 and R. T. BESFORD2
1
Horticulture Research International, East Malling, West Malling, Kent, ME19 6BJ, U.K.
2
Horticulture Research International, Littlehampton, West Sussex, BN17 6LP, U.K.
Received March 7, 1996
Summary Specific chloroplast proteins, gas exchange and
dry matter production in oak (Quercus robur L.) seedlings and
clonal cherry (Prunus avium L. × pseudocerasus Lind.) plants
were measured during 19 months of growth in climate-controlled greenhouses at ambient (350 vpm) or elevated (700 vpm)
CO2. In both species, the elevated CO2 treatment increased the
PPFD saturated-rate of photosynthesis and dry matter production. After two months at elevated CO2, Prunus plants showed
significant increases in leaf (55%) and stem (61%) dry mass
but not in root dry mass. However, this initial stimulation was
not sustained: treatment differences in net assimilation rate (A)
and plant dry mass were less after 10 months of growth than
after 2 months of growth, suggesting acclimation of A to
elevated CO2 in Prunus. In contrast, after 10 months of growth
at elevated CO2, leaf dry mass of Quercus increased (130%)
along with shoot (356%) and root (219%) dry mass, and A was
also twice that of plants grown and measured at ambient CO2.
The amounts of Rubisco and the thylakoid-bound protein
cytochrome f were higher in Quercus plants grown for 19 months
in elevated CO2 than in control plants, whereas in Prunus there
was less Rubisco in plants grown for 19 months in elevated CO2
than in control plants. Exposure to elevated CO2 for 10 months
resulted in increased mean leaf area in both species and increased abaxial stomatal density in Quercus. There was no
change in leaf epidermal cell size in either species in response
to the elevated CO2 treatment. The lack of acclimation of
photosynthesis in oak grown at elevated CO2 is discussed in
relation to the production and allocation of dry matter. We
propose that differences in carbohydrate utilization underlie
the differing long-term CO2 responses of the two species.
Keywords: carbon dioxide, cytochrome f, dry matter, gas exchange, Prunus, Quercus robur, Rubisco.
Introduction
Many reports have detailed the effects of elevated CO2 on
growth and dry matter partitioning of a range of woody perennials (see reviews by Cure and Acock 1986, Eamus and Jarvis
1989, Krupa and Kickert 1989, Ceulemans and Mousseau
1994, Gunderson and Wullschleger 1994, Atkinson 1996). Dry
mass increases induced by elevated CO2 are often accompanied by increases in net assimilation rate (A), at least in the
short term (Long 1991). However, enhancement of A may
disappear after longer-term exposure to elevated CO2.
Several explanations have been proposed to account for
photosynthetic acclimation to elevated CO2, including reduced
activity of sinks for carbohydrate, and the associated problems
of source--sink imbalance (Clough et al. 1981, Thomas and
Strain 1991, Long and Drake 1992). Under elevated CO2,
when A greatly exceeds the capacity for photosynthate utilization, it has been shown that sugars accumulate and modulate
the transcription of chloroplast proteins (Van Oosten and Besford 1994, 1995, Wilkins et al. 1994). Because the amount of
Rubisco can limit the light-saturated rate of A at low CO2
concentrations (von Caemmerer and Farquhar 1981, Besford
et al. 1985), photosynthetic acclimation has also been attributed to a loss of the amount or activity, or both, of Rubisco
(Ceulemans and Mousseau 1994), and various photosynthetic
enzymes and their thylakoid proteins, including cytochrome f
which is an integral part of the cytochrome b6 f complex responsible for electron transfer between photosystem II and
photosystem I (Besford et al. 1990, Van Oosten and Besford
1995).
Because Quercus and Prunus are believed to behave differently in their responses to elevated CO2 (Gunderson et al. 1993,
Wilkins et al. 1994), we first set out to determine whether
allocation of dry matter to different organs in these species is
altered by exposure to elevated CO2 (700 vpm). We then
investigated whether changes in photosynthetic potential in
response to elevated CO2 could explain the differences in dry
mass. To investigate possible mechanisms underlying photosynthetic acclimation to elevated CO2, we compared the contents of Rubisco and cytochrome f in leaves exposed to
ambient CO2 with those of leaves exposed to elevated CO2. We
found that, although both Quercus and Prunus had different
responses to CO2 enrichment, in both species growth responses
to elevated CO2 reflected changes in dry matter production,
whereas carbon allocation responses to elevated CO2 reflected
changes in net assimilation.
320
ATKINSON ET AL.
Materials and methods
Plant culture
Quercus robur L. seedlings were grown individually in
275 cm3 containers from acorns collected in the autumn of
1992, near Nancy, Champenoux (France). Before germination,
the acorns were planted and placed in cold storage at 2 °C for
1 month and then transferred to two temperature-controlled
greenhouses (see Wilkins et al. 1994). In one greenhouse CO2
was held constantly at 350 vpm (ambient), whereas in the other
it was held at 700 vpm (elevated). In mid-summer, the plants
were repotted in 5 dm3 pots at which time they were inoculated
with the ectomycorrhizal fungus Thelephora terrestris
Echr. Fr. (supplied by INRA-Nancy).
Cherry plants of the clone ‘‘Colt,’’ a cross between two wild
Prunus species (P. avium L. × P. pseudocerasus Lind.), were
obtained by micropropagation. After one month of hardeningoff, the micropropagated plants were transferred in February
to the greenhouses in which the Quercus seedlings were growing. In mid-summer, the plants were repotted in 15 dm3 pots.
For both species, the pots were free-draining to minimize
accumulation of ABA and other substances from the roots that
might inhibit photosynthesis. Fewer plants of Prunus were
available at the start of the experiments, but on three subsequent occasions (in April, August and October of the first
year), approximately 40 additional micropropagated plants
were transferred to the greenhouses, after hardening-off. Some
of the Quercus and Prunus plants were used for destructive
analysis, and some plants were grown for a further two years
at their respective CO2 concentrations.
Both greenhouses were ventilated at 25 °C. Air temperature
was continuously monitored with thermographs, and statistical
analyses showed no significant difference in air temperature
between the greenhouses. To minimize the risk of confounding
effects that possible differences in mineral nutrition might
have on A (see Eamus and Jarvis 1989, Sinclair 1992, Ceulemans and Mousseau 1994, Wilkins et al. 1994), from March
onward, all plants were fertilized weekly with nutrient solution
(stock solution contained dm --3: KNO3, 43 g; MgSO4.7H2O,
22 g; NH4NO3, 40 g; NH4H2PO4, 7 g; MnSO4.H2O, 0.2 g; plus
micronutrients and chelated iron) diluted 200-fold with calcium-rich tap water. Osmocote slow-release fertilizer
(15,11,13, N,P,K) was applied in June.
The analysis of variance was based on the variation within a
single greenhouse for each treatment, differences between the
greenhouses were then compared. Where appropriate, the significance of the differences is shown along with the standard
error of the difference of means.
Leaf gas exchange
Leaf gas exchange rates (net assimilation (A), transpiration (E)
and leaf conductance to water vapor (g)) were measured on
several occasions during the growing season. Two portable gas
exchange systems were used (LCA2, Analytical Development
Corp. Ltd., Hoddesdon, U.K., and Ciras-1, PP Systems,
Hitchin, U.K.). During measurements, PPFD was supplemented to ensure saturation of A (> 600 µmol m --2 s --1). The
LCA2 was used to measure gas exchange at the CO2 concentration in which the plants were grown. A minimum of ten
plants, with three leaves of similar physiological age per plant,
was used for each measurement. Although A varied with plant
age and time of year, the general pattern of treatment differences was similar (data not shown). To investigate the causes
of the treatment differences in A, it was measured at a PPFD of
860 µmol m --2 s--1 in plants from both treatments at both CO2
concentration. The leaves were not preconditioned to the
measurement CO2 concentration.
Extraction of Rubisco and cytochrome f and
immunodetection by Western blotting
In September 1994, after 19 months in the greeenhouses,
young, fully expanded leaves of Prunus and Quercus were
analyzed for Rubisco large subunit (LSU) and thylakoid bound
cytochrome f as described by Mehta et al. (1992), Besford et al.
(1993) and Van Oosten and Besford (1995). To avoid confounding of the results by leaf-level differences in light acclimation, we assayed leaves from full sun and from shade
(< 50% full sun). Western blotting was carried out as described
by Besford (1990) and Van Oosten and Besford (1995). Treatment comparisons were based on separate leaf extracts, loaded
on an equal leaf area basis, from at least three plants per
treatment; each sample was blotted twice and typical blots are
presented (Figure 1).
Leaf cell number and stomatal density
Stomatal density and leaf epidermal cell number per unit area
were quantified from epidermal impressions of the abaxial
surface using a silicone-based dental product (Provil M, Bayer,
Lever Kusen, Germany). During the growing season, leaf impressions were taken of the central region around the midrib of
the youngest fully expanded leaf from each of ten plants.
Positive impressions were made with nail varnish and viewed
with a projection light microscope at low magnification (45×).
For each leaf impression, three fields of view were selected for
analysis and the mean calculated.
Distribution and production of dry matter, and leaf mineral
analysis
The allocation of dry mass was used to describe the relationship between leaf canopy, stem development and wood production (radial growth). Main stem extension growth
(branches not included) was determined in relation to radial
wood production. For Quercus in particular, the phenological
development of the main stem was quantified with respect to
the number of growth flushes produced. Total plant leaf area
and mean individual leaf area were measured with a leaf area
meter (Model LI-3050A, Li-Cor, Inc., Lincoln, NE) on ten
leaves from each of ten Quercus plants per treatment, and on
20 leaves from each of five Prunus plants per treatment.
Total dry mass was quantified for leaves and wood. Samples
from each of 20 plants of Quercus and Prunus were dried at
80 °C to constant weight. Roots were washed free of soil by
hand, detached root loss was minimized by collecting root
washings in graded sieves. Fine roots (< 2 mm in diameter)
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF CO2 ON OAK AND CHERRY GROWTH
321
Figure 1. Immunodetection after
SDS-PAGE and electroblotting
of the large subunit of Rubisco
(LSU) in the soluble phase and
cytochrome f in the thylakoid
preparations from Prunus and
Quercus grown in ambient or elevated CO2. Molecular weights of
marker proteins are indicated at
the banding positions (kDa) in
lanes 1 and 12, unshaded young
fully expanded leaves were used
except in lanes 7 and 9 where
shaded leaves were analyzed. A,
ambient CO2; E, elevated CO2
samples were loaded on the basis of equal leaf area.
were separated from coarse roots (> 2 mm diameter), and cut
to a length of 2--5 cm. The length of coarser roots was measured by hand with a ruler. Fine root length was determined with
a root length scanner (Comair, Commonwealth Aircraft Corp.
Ltd., Melbourne, Australia). A calibration curve was used to
convert counts to root length, i.e., actual root length = --0.2246
+ 0.9655 (L) + 0.00123 (L2) where L is the estimated root
length from the scan.
Leaf mineral status was determined on oven-dried material
from 20 plants each of Quercus and Prunus, by a standard
Kjeldhal digestion procedure. Nitrogen and phosphorus were
analyzed colorimetrically with an auto-analysis system (Technicon Instruments Corp., Tarrytown, NY), and calcium, magnesium and potassium were analyzed by atomic absorption
and emission spectroscopy.
Results
Leaf development, gas exchange, and chloroplast proteins
Light-saturated A of Quercus plants grown for 10 months at
elevated CO2 and measured at the growth CO2 concentration,
was significantly greater (158%) than in plants grown and
measured in ambient CO2 (Table 1). Both g and E increased
slightly in Quercus leaves grown in elevated CO2 and measured at ambient CO2, whereas partial stomatal closure occurred
in Quercus leaves grown in ambient CO2 and measured at the
elevated CO2 concentration. Net assimilation rate increased
twofold for Prunus plants grown at ambient CO2 and measured
at the elevated CO2 concentration, compared to Prunus plants
grown and measured at ambient CO2 concentration. However,
A of Prunus grown and measured at elevated CO2 was not
Table 1. Gas exchange characteristics (assimilation, transpiration, leaf conductance and WUE) measured at 20 °C under light-saturating conditions
for young, fully expanded leaves of Quercus robur and Prunus avium × pseudocerasus exposed for 10 months to either ambient or elevated CO2.
Superscript M denotes measurement CO2 concentration, superscript G denotes growth CO2 concentration.
Assimilation µmol m --2 s --1
Transpiration mmol m --2 s --1
Leaf conductance mmol m --2 s --1
Water use efficiency × 103 (A/E)
AmbM
ElevM
AmbM
ElevM
AmbM
ElevM
AmbM
ElevM
9.9
14.2
1.08
1.15
0.75
1.14
88.6
102.4
61.7
94.8
5.08
4.77
13.53
12.45
Quercus
AmbientG
5.5
ElevatedG
5.6
SED1
1.1
P growth CO2
ns
P measurement CO2 ***
Prunus
AmbientG
6.6
ElevatedG
1.4
SED
1.0
***
P growth CO2
P measurement CO2 ***
1
0.13
ns
ns
12.2
5.3
1.28
0.64
13.9
ns
ns
1.08
0.65
0.11
***
ns
104.0
47.5
0.61
ns
***
87.4
49.4
10.8
***
ns
5.09
2.20
11.35
8.48
0.64
***
***
SED = Standard error of difference of means; significant differences (P) are shown by the symbols: ns = not significant, * = 0.05, ** = 0.01 and
*** = 0.001.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
322
ATKINSON ET AL.
significantly different from that of Prunus plants grown and
measured at ambient CO2. Stomatal conductance did not increase when Prunus leaves grown in elevated CO2 were measured at ambient CO2, and there was only a small reduction in
g when ambient-grown Prunus leaves were measured at elevated CO2 (Table 1).
The amount of the large subunit of Rubisco (LSU) was less
in Prunus plants grown in elevated CO2 than in Prunus plants
grown in ambient CO2, whereas no appreciable treatment
differences were observed in Quercus seedlings (Figure 1). In
Prunus, there was no effect of elevated CO2 on the amount of
cytochrome f (although the amount appeared to be enhanced
by shading), whereas cytochrome f content was increased in
Quercus plants grown in elevated CO2 compared with Quercus
plants grown in ambient CO2 (Figure 1).
Mean individual leaf area in Quercus and Prunus increased
(P < 0.001) in response to elevated CO2, from 20.8 to 26.8 cm2
and from 13.6 to 25.4 cm2, respectively. The elevated CO2
treatment increased the number of stomata per unit of abaxial
surface between 2 and 3 times in Quercus (data not shown),
whereas the treatment had no effect on stomatal density in
Prunus. Leaf epidermal cell density was higher for Quercus
than for Prunus, indicating a smaller epidermal cell size for
oak leaves, but no CO2 response was evident for either species.
(Table 3). Shoot length of the third flush increased 296% by
the elevated CO2 treatment.
Because Prunus species grow faster than Quercus species,
the amount of dry mass produced by Prunus plants was determined after 2 and 10 months of growth. Analysis of 2-monthold Prunus plants showed significant increases in the dry mass
of shoots (61%) and leaves (55%) in response to elevated CO2,
but not in roots (Table 4). Although small increases in dry mass
in response to elevated CO2 were still evident in 10-month-old
Prunus plants, none of the treatment effects were significant
(Table 4). In both species, there were linear relationships
between leaf area and stem diameter that were independent of
CO2 concentration (data not shown).
Leaf mineral content (N, P, K, Ca and Mg) in Quercus and
Prunus was measured in early August and in October, because
differences between dates were slight in both species only the
August data are presented in Table 5. The only statistically
significant effect of elevated CO2 was to lower the foliar
concentrations of potassium in Quercus and calcium in Prunus
(Table 5).
Discussion
Acclimation of assimilation as an explanation of differences
in dry matter production
Dry matter production and distribution and leaf mineral
analysis
Prunus plants were less responsive to elevated CO2 than Quercus seedlings. Compared to the ambient CO2 treatment, the
After 10 months, there were significant increases in the accumulated dry mass of leaves (131%), wood (main stem and all
branches by 356%) and roots (219%) of Quercus seedlings
growth in elevated CO2 compared to seedlings grown at ambient CO2 (Table 2). Main stem extension growth was examined
for each growth flush, and during the first year most of the trees
in both treatments produced around three flushes (Table 3).
Although the length of the first flush did not differ between
treatments, significant treatment differences in stem radial
growth (wood production) were apparent in all three flushes
Table 2. Total dry mass of shoots and roots, leaf area production and
total root and shoot length (including main stem plus all branches) of
Quercus robur seedlings after 10 months of exposure to either ambient
or elevated CO2.
Ambient CO2
Elevated CO2
SED1
P
4.76
11.00
1.52
***
640
1177
186
**
Shoot dry mass (g) 3.62
16.52
3.74
***
Shoot length (cm) 46.7
110.5
26.1
*
Root dry mass (g) 6.93
22.14
5.42
*
Root length (m)
155
38
*
Leaf dry mass (g)
2
Leaf area (cm )
Table 3. Main stem length (cm), stem cross-sectional area (mm2) and
radial growth (mm2 day --1 × 103) of Quercus robur seedlings measured
or calculated within three stem flushes over a 4-month period of
growth between 6 months and 10 months of exposure to either ambient
or elevated CO2.
Flush one
1
SED = Standard error of difference of means; significant differences
(P) are shown by the symbols: ns = not significant, * = 0.05, ** =
0.01 and *** = 0.001.
Flush three
12.1 (39)
16.1 (53)
1.5
**
11.3 (28)
44.7 (45)
2.9
***
12.6
28.5
1.9
***
6.5
17.9
1.4
***
Main stem length1 (cm)
Ambient CO2
Elevated CO2
SED2
P
13.1 (71)
12.4 (70)
0.6
ns
Stem cross-sectional area (mm2)
Ambient CO2
25.7
Elevated CO2
48.7
SED
3.0
P
***
Mean radial growth of stem (mm2 day --1 × 103)
152
74
Ambient CO2
Elevated CO2
577
299
SED
47
26
P
***
***
1
46
Flush two
2
55
157
15
***
Main stem length not including branches. The figures in brackets
refer to the number of seedlings with one, two or three stem flushes.
SED = Standard error of difference of means; significant differences
(P) are shown by the symbols: ns = not significant, * = 0.05, ** = 0.01
and *** = 0.001.
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF CO2 ON OAK AND CHERRY GROWTH
Table 4. Total dry mass of shoots and roots, leaf area production and
total root and shoot length (including main stem and branches) of
Prunus avium × pseudocerasus exposed for either 2 or 10 months to
either ambient or elevated CO2.
Ambient CO2
Elevated CO2
SED1
P
After a 2-month exposure
Leaf dry mass (g)
2.89
4.47
0.64
*
Leaf area (cm2)
459
636
83
*
Shoot dry mass (g) 2.27
3.65
0.53
*
Total shoot length
(cm)
37.1
53.8
6.5
*
Root dry mass (g) 6.77
6.98
1.06
ns
Root length (m)
129
13
ns
112
157
21
ns
15600
19800
2200
ns
Shoot dry mass (g) 182
311
53
ns
Total shoot length
(cm)
546
867
154
ns
Root dry mass2 (g):
< 2 mm
50.9
61.2
9.1
ns
> 2 mm
42.6
54.8
15.3
ns
Root length (m):
< 2 mm
686
714
132
ns
> 2 mm
5.4
7.5
1.1
ns
111
After a 10-month exposure
Leaf dry mass (g)
2
Leaf area (cm )
2
1
2
SED = Standard error of difference of means; significant differences
(P) are shown by the symbols: ns = not significant, * = 0.05, ** = 0.01
and *** = 0.001.
Root dry mass and root length were partitioned with respect to root
diameter.
323
elevated CO2 treatment caused a 27% enhancement in dry
mass production in Prunus after two months and 51% after
10 months compared to a dry mass gain of more than 200%
after 10 months in Quercus seedlings. The limited response of
Prunus to elevated CO2 may be associated with photosynthetic
acclimation (Besford et al. 1990, Gunderson and Wullschleger
1994, Wilkins et al. 1994). After 10 months of growth in
elevated CO2, A, measured at elevated CO2 for Prunus, was not
significantly different from that of plants grown and measured
at ambient CO2; whereas, A of Prunus plants grown at elevated
CO2 and measured at ambient CO2 was significantly lower by
about 80%, than A of plants grown and measured at ambient
CO2. Because the area of individual Prunus leaves in the
elevated CO2 treatment was about 87% greater than the area of
leaves in the ambient CO2, we also expressed A on a per leaf
basis. Net assimilation rate of leaves grown in elevated CO2
and measured at ambient CO2 was only about 40% that of
leaves grown in ambient CO2 on a per leaf basis, which suggests that photosynthetic acclimation had taken place.
Net assimilation rate of Quercus grown and measured at
elevated CO2 was over twice that of seedlings grown and
measured at ambient CO2, whereas A of seedlings grown at
elevated CO2 and measured at ambient CO2 was similar to A of
seedlings grown and measured at ambient CO2. Thus, in contrast to Prunus, Quercus seedlings exhibited no loss of photosynthetic activity even after 10 months in elevated CO2. A
comparison of A measured at elevated CO2 of Quercus seedlings grown in ambient and elevated CO2, indicated that there
was some gain in photosynthetic capacity in response to the
elevated CO2 treatment (Table 1).
It has been suggested that light-saturated A at low CO2
concentrations is limited by RuBP-saturated Rubisco activity
(e.g., von Caemmerer and Farquhar 1981, Besford et al. 1985),
whereas at high CO2 concentrations A may become increasingly dependent on RuBP regeneration through, for example,
light harvesting and photosynthetic electron transport capacity
Table 5. Mineral nutrient concentration (% dry mass basis) in leaves of Quercus robur after 6 months exposure to ambient or elevated CO2 and
Prunus avium × pseudocerasus after two months of growth at either ambient or elevated CO2.
Nitrogen
Phosphorus
Potassium
Calcium
Magnesium
Quercus
Ambient CO2
Elevated CO2
SED1
P
2.93
2.71
0.11
ns
0.23
0.21
0.04
ns
1.31
0.97
0.29
**
1.45
1.30
0.31
ns
0.37
0.38
0.05
ns
Prunus
Ambient CO2
Elevated CO2
SED
P
3.10
2.92
0.15
ns
0.39
0.34
0.03
ns
2.96
2.90
0.14
ns
3.13
2.88
0.09
*
0.47
0.47
0.01
ns
1
SED = Standard error of difference of means; significant differences (P) are shown by the symbols: ns = not significant, * = 0.05, ** = 0.01 and
*** = 0.001.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
324
ATKINSON ET AL.
(von Caemmerer and Farquhar 1981). Leaves of Quercus
plants grown at elevated CO2 contained increased amounts of
thylakoid-bound cytochrome f (a potential rate limiting step in
photoelectron transport) with no apparent change in the
amount of Rubisco LSU. In previous work with Prunus avium
grown in elevated CO2 for two years, both Rubisco LSU and
cytochrome f amounts were lower in plants grown in elevated
CO2 than in plants grown in ambient CO2 (Wilkins et al. 1994)
and the amounts of Rubisco decreased before changes in
composition of thylakoid membranes were observed (Van
Oosten and Besford 1995). In our study, Prunus grown in
elevated CO2 showed a reduction in the amount of LSU, but
not of cytochrome f.
It has not yet been demonstrated unequivocally that the
apparent difference in response between Quercus and Prunus
is not an experimental artifact. There is some evidence that
photosynthetic acclimation is less likely to occur in fieldgrown trees than in container-grown trees in a greenhouse
(Gunderson et al. 1993). In our study, the physical effects of
root restriction were minimized by increasing pot size twice in
the first year and by using large, deep pots. Furthermore, recent
work with Prunus avium has shown that root restriction does
not necessarily have a negative affect on the CO2-induced
stimulation of dry matter production (Kerstiens and Hawes
1994).
An absence of acclimation of A has been observed in several
Quercus species after long-term exposure to elevated CO2
(Gunderson et al. 1993, Ceulemans and Mousseau 1994),
although there are exceptions. Of the temperate deciduous tree
species examined by Bunce (1992), only Quercus robur
showed photosynthetic acclimation. No conclusion can be
reached for Prunus, because there have been few studies of the
effects of elevated CO2 on this species (see Bazzaz et al. 1990,
Wilkins et al. 1994). However, we found no evidence that
environmentally-based factors could explain the differences
between Quercus and Prunus. In the elevated CO2 treatment,
Quercus roots represented 45% of total dry mass, whereas in
Prunus only 20% of total dry mass was partitioned to roots.
Acclimation of A may be an effective mechanism for restricting the rate of aerial growth and thereby restoring a more even
distribution of dry mass between root and shoot. The maintenance of this ratio is an important component of the ability of
the root to sustain the shoot, particularly with respect to the
supply of water and nutrients. Low nutrient status can impose
a limitation on A especially in plants grown in elevated CO2
(Norby et al. 1992). In our experiments, the possibility of soil
nutrient depletion, at elevated CO2, influencing leaf nitrogen
and A was minimized by weekly applications of nutrient solution. Mineral analyses indicated that the loss of photosynthetic
capacity in Prunus grown at elevated CO2 was not the result of
foliar nutrient deficiency.
Dry matter allocation and sink development influences on
assimilation
The elevated CO2 treatment had a pronounced effect on the
development of Quercus shoots. The main stem of Quercus
developed through a series of growth flushes (Hanson et al.
1986). Shoot length of the first flush did not differ with respect
to CO2 treatment, but later extension growth, particularly of the
third flush, was significantly different for trees grown at elevated CO2. Radial growth (wood production) was significantly
greater in all three flushes in plants grown at elevated CO2.
These findings highlight the effects of elevated CO2 on carbon
allocation and meristematic function with respect to apical
growth and cambial production, which in turn will have implications for the development of the tree canopy to maturity. The
switch to a more indeterminate pattern of Quercus shoot
growth in response to elevated CO2 is important, because the
stimulation of new apical meristems creates new sinks for
carbohydrate utilization, which may explain why photosynthetic capacity was maintained in the elevated CO2 treatment.
In conclusion, we found important differences in the behavior of two woody species to elevated CO2. Quercus robur did
not show photosynthetic acclimation, whereas Prunus did.
This difference appeared to be associated with the ability of
Quercus to maintain its photosynthetic potential as a result of
a stable content of Rubisco. A stable Rubisco content in leaves
exposed to elevated CO2 may reflect the ability of Quercus to
develop new sinks for carbohydrate utilization.
Acknowledgments
This work was sponsored by the European Commission (Climate
Change Impacts, Project EV5V-CT92-0093) and the Ministry of Agriculture, Fisheries and Food, U.K. We are grateful to Drs. J.D. Quinlan, B.H. Howard and Prof. H.G. Jones for their comments, to Martin
Ridout for statistical advice, the greenhouse staff at HRI-Littlehampton for their assistance with growing the plants and for technical help
from Ann Lucas and Lorraine Taylor (HRI-East Malling).
References
Atkinson, C.J. 1996. Global changes in atmospheric carbon dioxide:
The influence on terrestrial vegetation. In Plant Response to Air
Pollution. Eds. M. Iqbal and M. Yunus. John Wiley and Sons Ltd.,
Sussex, U.K., pp 99--133.
Bazzaz, F.A., J.S. Coleman and S.R. Morse. 1990. Growth responses
of major co-occurring tree species of the Northeastern United States
to elevated CO2. Can. J. For. Res. 20:1479--1484.
Besford, R.T. 1990. The greenhouse effect: Acclimation of tomato
plants growing in high CO2, relative changes in Calvin Cycle
enzymes. J. Plant Physiol. 136:458--463.
Besford, R.T., L.J. Ludwig and A.C. Withers. 1990. The greenhouse
effect: Acclimation of tomato plants growing in high CO2 photosynthesis and ribulose-1,5-bisphosphate carboxylase protein. J. Exp.
Bot. 41:925--931.
Besford, R.T., C.M. Richardson, J.L. Campos and A.F. Tiburcio. 1993.
Effect of polyamines on stabilization of molecular complexes in
thylakoid membranes of osmotically stressed oat leaves. Planta
189:201--206.
Besford, R.T., A.C. Withers and L.J. Ludwig. 1985. Ribulose bisphosphate carboxylase activity and photosynthesis during leaf development in the tomato. J. Exp. Bot. 36:1530--1541.
Bunce, J.A. 1992. Stomatal conductance, photosynthesis and respiration of temperate deciduous tree seedlings grown outdoors at an
elevated concentration of carbon dioxide. Plant Cell Environ.
15:541--549.
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF CO2 ON OAK AND CHERRY GROWTH
Ceulemans, R. and M. Mousseau. 1994. Effects of elevated atmospheric CO2 on woody plants. New Phytol. 127:425--446.
Clough, J.M., M.M. Peet and P.J. Kramer. 1981. Effects of high
atmospheric CO2 and sink size on rates of photosynthesis of a
soybean cultivar. Plant Physiol. 67:1007--1010.
Cure, J.D. and B. Acock. 1986. Crop response to carbon dioxide
doubling: A literature survey. Agric. For. Meteorol. 38:127--145.
Eamus, D. and P.G. Jarvis. 1989. The direct effects of an increase in
the global atmospheric CO2 concentration on natural and commercial temperate trees and forests. Adv. Ecol. Res. 19:1--55.
Gunderson, C.A., R.J. Norby and S.D. Wullschleger. 1993. Foliar gas
exchange responses of two deciduous hardwoods during 3 years of
growth in elevated CO2: No loss of photosynthetic enhancement.
Plant Cell Environ. 16:797--807.
Gunderson, C.A. and S.D. Wullschleger. 1994. Photosynthetic acclimation in trees to rising atmospheric CO2: A broader perspective.
Photosynth. Res. 39:369--388.
Hanson, P.J., R.E. Dickson, J.G. Isebrands, T.R. Crow and R.K. Dixon.
1986. A morphological index of Quercus seedling ontogeny for use
in studies of physiology and growth. Tree Physiol. 2:273--281.
Kerstiens, G. and C.V. Hawes. 1994. Response of growth and carbon
allocation to elevated CO2 in young cherry (Prunus avium L.)
saplings in relation to root environment. New Phytol. 128:607-614.
Krupa, S.V. and R.N. Kickert. 1989. The greenhouse effect: Impacts
of ultraviolet-B (UV-B) radiation, carbon dioxide (CO2), and ozone
(O3) on vegetation. Environ. Pollut. 61:263--393.
Long, S.P. 1991. Modification of the response of photosynthetic productivity to rising temperature by atmospheric CO2 concentrations:
Has its importance been underestimated? Plant Cell Environ.
14:729--739.
325
Long, S.P. and B.G. Drake. 1992. Photosynthetic CO2 assimilation and
rising atmospheric CO2 concentration. In Crop Photosynthesis:
Spatial and Temporal Determinants. Eds. N.R. Baker and H. Thomas.
The Hague: Elsevier Science Publishers B.V., pp 69--103.
Mehta, R.A., T.W. Fawcett, D. Porath and A.K. Mattoo. 1992. Oxidative stress causes rapid membrane translocation and in vivo degradation of ribulose-1,5-bisphosphate carboxylase oxygenase. J. Biol.
Chem. 267:2810--2816.
Norby, R.J., C.A. Gunderson, S.D. Wullschleger, E.G. O’Neill and
M.K. McCracken. 1992. Productivity and compensatory responses
of yellow-poplar trees in elevated CO2. Nature 357:322--324.
Sinclair, T.R. 1992. Mineral nutrition and plant growth response to
climate change. J. Exp. Bot. 43:1141--1146.
Thomas, R.B. and B.R. Strain. 1991. Root restriction as a factor in
photosynthetic acclimation of cotton seedlings grown in elevated
carbon dioxide. Plant Physiol. 96:627--634.
von Caemmerer, S. and G.D. Farquhar. 1981. Some relationships
between the biochemistry of photosynthesis and the gas exchange
of leaves. Planta 153:376--387.
Van Oosten, J.-J. and R.T. Besford. 1994. Sugar feeding mimics effect
of acclimation to high CO2-rapid down regulation of RuBisCo
small subunit transcripts but not of the large subunit transcripts.
J. Plant Physiol. 143:306--312.
Van Oosten, J.-J. and R.T. Besford. 1995. Some relationships between
gas-exchange, biochemistry and molecular biology of photosynthesis during leaf development of tomato plants after transfer to different carbon dioxide concentrations. Plant Cell Environ.
18:1253--1266.
Wilkins, D., J.-J. Van Oosten and R.T. Besford. 1994. Effects of
elevated CO2 on growth and chloroplast proteins in Prunus avium.
Tree Physiol. 14:769--779.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
© 1997 Heron Publishing----Victoria, Canada
Effects of elevated CO2 on chloroplast components, gas exchange and
growth of oak and cherry
C. J. ATKINSON,1 J. M. TAYLOR,1 D. WILKINS2 and R. T. BESFORD2
1
Horticulture Research International, East Malling, West Malling, Kent, ME19 6BJ, U.K.
2
Horticulture Research International, Littlehampton, West Sussex, BN17 6LP, U.K.
Received March 7, 1996
Summary Specific chloroplast proteins, gas exchange and
dry matter production in oak (Quercus robur L.) seedlings and
clonal cherry (Prunus avium L. × pseudocerasus Lind.) plants
were measured during 19 months of growth in climate-controlled greenhouses at ambient (350 vpm) or elevated (700 vpm)
CO2. In both species, the elevated CO2 treatment increased the
PPFD saturated-rate of photosynthesis and dry matter production. After two months at elevated CO2, Prunus plants showed
significant increases in leaf (55%) and stem (61%) dry mass
but not in root dry mass. However, this initial stimulation was
not sustained: treatment differences in net assimilation rate (A)
and plant dry mass were less after 10 months of growth than
after 2 months of growth, suggesting acclimation of A to
elevated CO2 in Prunus. In contrast, after 10 months of growth
at elevated CO2, leaf dry mass of Quercus increased (130%)
along with shoot (356%) and root (219%) dry mass, and A was
also twice that of plants grown and measured at ambient CO2.
The amounts of Rubisco and the thylakoid-bound protein
cytochrome f were higher in Quercus plants grown for 19 months
in elevated CO2 than in control plants, whereas in Prunus there
was less Rubisco in plants grown for 19 months in elevated CO2
than in control plants. Exposure to elevated CO2 for 10 months
resulted in increased mean leaf area in both species and increased abaxial stomatal density in Quercus. There was no
change in leaf epidermal cell size in either species in response
to the elevated CO2 treatment. The lack of acclimation of
photosynthesis in oak grown at elevated CO2 is discussed in
relation to the production and allocation of dry matter. We
propose that differences in carbohydrate utilization underlie
the differing long-term CO2 responses of the two species.
Keywords: carbon dioxide, cytochrome f, dry matter, gas exchange, Prunus, Quercus robur, Rubisco.
Introduction
Many reports have detailed the effects of elevated CO2 on
growth and dry matter partitioning of a range of woody perennials (see reviews by Cure and Acock 1986, Eamus and Jarvis
1989, Krupa and Kickert 1989, Ceulemans and Mousseau
1994, Gunderson and Wullschleger 1994, Atkinson 1996). Dry
mass increases induced by elevated CO2 are often accompanied by increases in net assimilation rate (A), at least in the
short term (Long 1991). However, enhancement of A may
disappear after longer-term exposure to elevated CO2.
Several explanations have been proposed to account for
photosynthetic acclimation to elevated CO2, including reduced
activity of sinks for carbohydrate, and the associated problems
of source--sink imbalance (Clough et al. 1981, Thomas and
Strain 1991, Long and Drake 1992). Under elevated CO2,
when A greatly exceeds the capacity for photosynthate utilization, it has been shown that sugars accumulate and modulate
the transcription of chloroplast proteins (Van Oosten and Besford 1994, 1995, Wilkins et al. 1994). Because the amount of
Rubisco can limit the light-saturated rate of A at low CO2
concentrations (von Caemmerer and Farquhar 1981, Besford
et al. 1985), photosynthetic acclimation has also been attributed to a loss of the amount or activity, or both, of Rubisco
(Ceulemans and Mousseau 1994), and various photosynthetic
enzymes and their thylakoid proteins, including cytochrome f
which is an integral part of the cytochrome b6 f complex responsible for electron transfer between photosystem II and
photosystem I (Besford et al. 1990, Van Oosten and Besford
1995).
Because Quercus and Prunus are believed to behave differently in their responses to elevated CO2 (Gunderson et al. 1993,
Wilkins et al. 1994), we first set out to determine whether
allocation of dry matter to different organs in these species is
altered by exposure to elevated CO2 (700 vpm). We then
investigated whether changes in photosynthetic potential in
response to elevated CO2 could explain the differences in dry
mass. To investigate possible mechanisms underlying photosynthetic acclimation to elevated CO2, we compared the contents of Rubisco and cytochrome f in leaves exposed to
ambient CO2 with those of leaves exposed to elevated CO2. We
found that, although both Quercus and Prunus had different
responses to CO2 enrichment, in both species growth responses
to elevated CO2 reflected changes in dry matter production,
whereas carbon allocation responses to elevated CO2 reflected
changes in net assimilation.
320
ATKINSON ET AL.
Materials and methods
Plant culture
Quercus robur L. seedlings were grown individually in
275 cm3 containers from acorns collected in the autumn of
1992, near Nancy, Champenoux (France). Before germination,
the acorns were planted and placed in cold storage at 2 °C for
1 month and then transferred to two temperature-controlled
greenhouses (see Wilkins et al. 1994). In one greenhouse CO2
was held constantly at 350 vpm (ambient), whereas in the other
it was held at 700 vpm (elevated). In mid-summer, the plants
were repotted in 5 dm3 pots at which time they were inoculated
with the ectomycorrhizal fungus Thelephora terrestris
Echr. Fr. (supplied by INRA-Nancy).
Cherry plants of the clone ‘‘Colt,’’ a cross between two wild
Prunus species (P. avium L. × P. pseudocerasus Lind.), were
obtained by micropropagation. After one month of hardeningoff, the micropropagated plants were transferred in February
to the greenhouses in which the Quercus seedlings were growing. In mid-summer, the plants were repotted in 15 dm3 pots.
For both species, the pots were free-draining to minimize
accumulation of ABA and other substances from the roots that
might inhibit photosynthesis. Fewer plants of Prunus were
available at the start of the experiments, but on three subsequent occasions (in April, August and October of the first
year), approximately 40 additional micropropagated plants
were transferred to the greenhouses, after hardening-off. Some
of the Quercus and Prunus plants were used for destructive
analysis, and some plants were grown for a further two years
at their respective CO2 concentrations.
Both greenhouses were ventilated at 25 °C. Air temperature
was continuously monitored with thermographs, and statistical
analyses showed no significant difference in air temperature
between the greenhouses. To minimize the risk of confounding
effects that possible differences in mineral nutrition might
have on A (see Eamus and Jarvis 1989, Sinclair 1992, Ceulemans and Mousseau 1994, Wilkins et al. 1994), from March
onward, all plants were fertilized weekly with nutrient solution
(stock solution contained dm --3: KNO3, 43 g; MgSO4.7H2O,
22 g; NH4NO3, 40 g; NH4H2PO4, 7 g; MnSO4.H2O, 0.2 g; plus
micronutrients and chelated iron) diluted 200-fold with calcium-rich tap water. Osmocote slow-release fertilizer
(15,11,13, N,P,K) was applied in June.
The analysis of variance was based on the variation within a
single greenhouse for each treatment, differences between the
greenhouses were then compared. Where appropriate, the significance of the differences is shown along with the standard
error of the difference of means.
Leaf gas exchange
Leaf gas exchange rates (net assimilation (A), transpiration (E)
and leaf conductance to water vapor (g)) were measured on
several occasions during the growing season. Two portable gas
exchange systems were used (LCA2, Analytical Development
Corp. Ltd., Hoddesdon, U.K., and Ciras-1, PP Systems,
Hitchin, U.K.). During measurements, PPFD was supplemented to ensure saturation of A (> 600 µmol m --2 s --1). The
LCA2 was used to measure gas exchange at the CO2 concentration in which the plants were grown. A minimum of ten
plants, with three leaves of similar physiological age per plant,
was used for each measurement. Although A varied with plant
age and time of year, the general pattern of treatment differences was similar (data not shown). To investigate the causes
of the treatment differences in A, it was measured at a PPFD of
860 µmol m --2 s--1 in plants from both treatments at both CO2
concentration. The leaves were not preconditioned to the
measurement CO2 concentration.
Extraction of Rubisco and cytochrome f and
immunodetection by Western blotting
In September 1994, after 19 months in the greeenhouses,
young, fully expanded leaves of Prunus and Quercus were
analyzed for Rubisco large subunit (LSU) and thylakoid bound
cytochrome f as described by Mehta et al. (1992), Besford et al.
(1993) and Van Oosten and Besford (1995). To avoid confounding of the results by leaf-level differences in light acclimation, we assayed leaves from full sun and from shade
(< 50% full sun). Western blotting was carried out as described
by Besford (1990) and Van Oosten and Besford (1995). Treatment comparisons were based on separate leaf extracts, loaded
on an equal leaf area basis, from at least three plants per
treatment; each sample was blotted twice and typical blots are
presented (Figure 1).
Leaf cell number and stomatal density
Stomatal density and leaf epidermal cell number per unit area
were quantified from epidermal impressions of the abaxial
surface using a silicone-based dental product (Provil M, Bayer,
Lever Kusen, Germany). During the growing season, leaf impressions were taken of the central region around the midrib of
the youngest fully expanded leaf from each of ten plants.
Positive impressions were made with nail varnish and viewed
with a projection light microscope at low magnification (45×).
For each leaf impression, three fields of view were selected for
analysis and the mean calculated.
Distribution and production of dry matter, and leaf mineral
analysis
The allocation of dry mass was used to describe the relationship between leaf canopy, stem development and wood production (radial growth). Main stem extension growth
(branches not included) was determined in relation to radial
wood production. For Quercus in particular, the phenological
development of the main stem was quantified with respect to
the number of growth flushes produced. Total plant leaf area
and mean individual leaf area were measured with a leaf area
meter (Model LI-3050A, Li-Cor, Inc., Lincoln, NE) on ten
leaves from each of ten Quercus plants per treatment, and on
20 leaves from each of five Prunus plants per treatment.
Total dry mass was quantified for leaves and wood. Samples
from each of 20 plants of Quercus and Prunus were dried at
80 °C to constant weight. Roots were washed free of soil by
hand, detached root loss was minimized by collecting root
washings in graded sieves. Fine roots (< 2 mm in diameter)
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF CO2 ON OAK AND CHERRY GROWTH
321
Figure 1. Immunodetection after
SDS-PAGE and electroblotting
of the large subunit of Rubisco
(LSU) in the soluble phase and
cytochrome f in the thylakoid
preparations from Prunus and
Quercus grown in ambient or elevated CO2. Molecular weights of
marker proteins are indicated at
the banding positions (kDa) in
lanes 1 and 12, unshaded young
fully expanded leaves were used
except in lanes 7 and 9 where
shaded leaves were analyzed. A,
ambient CO2; E, elevated CO2
samples were loaded on the basis of equal leaf area.
were separated from coarse roots (> 2 mm diameter), and cut
to a length of 2--5 cm. The length of coarser roots was measured by hand with a ruler. Fine root length was determined with
a root length scanner (Comair, Commonwealth Aircraft Corp.
Ltd., Melbourne, Australia). A calibration curve was used to
convert counts to root length, i.e., actual root length = --0.2246
+ 0.9655 (L) + 0.00123 (L2) where L is the estimated root
length from the scan.
Leaf mineral status was determined on oven-dried material
from 20 plants each of Quercus and Prunus, by a standard
Kjeldhal digestion procedure. Nitrogen and phosphorus were
analyzed colorimetrically with an auto-analysis system (Technicon Instruments Corp., Tarrytown, NY), and calcium, magnesium and potassium were analyzed by atomic absorption
and emission spectroscopy.
Results
Leaf development, gas exchange, and chloroplast proteins
Light-saturated A of Quercus plants grown for 10 months at
elevated CO2 and measured at the growth CO2 concentration,
was significantly greater (158%) than in plants grown and
measured in ambient CO2 (Table 1). Both g and E increased
slightly in Quercus leaves grown in elevated CO2 and measured at ambient CO2, whereas partial stomatal closure occurred
in Quercus leaves grown in ambient CO2 and measured at the
elevated CO2 concentration. Net assimilation rate increased
twofold for Prunus plants grown at ambient CO2 and measured
at the elevated CO2 concentration, compared to Prunus plants
grown and measured at ambient CO2 concentration. However,
A of Prunus grown and measured at elevated CO2 was not
Table 1. Gas exchange characteristics (assimilation, transpiration, leaf conductance and WUE) measured at 20 °C under light-saturating conditions
for young, fully expanded leaves of Quercus robur and Prunus avium × pseudocerasus exposed for 10 months to either ambient or elevated CO2.
Superscript M denotes measurement CO2 concentration, superscript G denotes growth CO2 concentration.
Assimilation µmol m --2 s --1
Transpiration mmol m --2 s --1
Leaf conductance mmol m --2 s --1
Water use efficiency × 103 (A/E)
AmbM
ElevM
AmbM
ElevM
AmbM
ElevM
AmbM
ElevM
9.9
14.2
1.08
1.15
0.75
1.14
88.6
102.4
61.7
94.8
5.08
4.77
13.53
12.45
Quercus
AmbientG
5.5
ElevatedG
5.6
SED1
1.1
P growth CO2
ns
P measurement CO2 ***
Prunus
AmbientG
6.6
ElevatedG
1.4
SED
1.0
***
P growth CO2
P measurement CO2 ***
1
0.13
ns
ns
12.2
5.3
1.28
0.64
13.9
ns
ns
1.08
0.65
0.11
***
ns
104.0
47.5
0.61
ns
***
87.4
49.4
10.8
***
ns
5.09
2.20
11.35
8.48
0.64
***
***
SED = Standard error of difference of means; significant differences (P) are shown by the symbols: ns = not significant, * = 0.05, ** = 0.01 and
*** = 0.001.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
322
ATKINSON ET AL.
significantly different from that of Prunus plants grown and
measured at ambient CO2. Stomatal conductance did not increase when Prunus leaves grown in elevated CO2 were measured at ambient CO2, and there was only a small reduction in
g when ambient-grown Prunus leaves were measured at elevated CO2 (Table 1).
The amount of the large subunit of Rubisco (LSU) was less
in Prunus plants grown in elevated CO2 than in Prunus plants
grown in ambient CO2, whereas no appreciable treatment
differences were observed in Quercus seedlings (Figure 1). In
Prunus, there was no effect of elevated CO2 on the amount of
cytochrome f (although the amount appeared to be enhanced
by shading), whereas cytochrome f content was increased in
Quercus plants grown in elevated CO2 compared with Quercus
plants grown in ambient CO2 (Figure 1).
Mean individual leaf area in Quercus and Prunus increased
(P < 0.001) in response to elevated CO2, from 20.8 to 26.8 cm2
and from 13.6 to 25.4 cm2, respectively. The elevated CO2
treatment increased the number of stomata per unit of abaxial
surface between 2 and 3 times in Quercus (data not shown),
whereas the treatment had no effect on stomatal density in
Prunus. Leaf epidermal cell density was higher for Quercus
than for Prunus, indicating a smaller epidermal cell size for
oak leaves, but no CO2 response was evident for either species.
(Table 3). Shoot length of the third flush increased 296% by
the elevated CO2 treatment.
Because Prunus species grow faster than Quercus species,
the amount of dry mass produced by Prunus plants was determined after 2 and 10 months of growth. Analysis of 2-monthold Prunus plants showed significant increases in the dry mass
of shoots (61%) and leaves (55%) in response to elevated CO2,
but not in roots (Table 4). Although small increases in dry mass
in response to elevated CO2 were still evident in 10-month-old
Prunus plants, none of the treatment effects were significant
(Table 4). In both species, there were linear relationships
between leaf area and stem diameter that were independent of
CO2 concentration (data not shown).
Leaf mineral content (N, P, K, Ca and Mg) in Quercus and
Prunus was measured in early August and in October, because
differences between dates were slight in both species only the
August data are presented in Table 5. The only statistically
significant effect of elevated CO2 was to lower the foliar
concentrations of potassium in Quercus and calcium in Prunus
(Table 5).
Discussion
Acclimation of assimilation as an explanation of differences
in dry matter production
Dry matter production and distribution and leaf mineral
analysis
Prunus plants were less responsive to elevated CO2 than Quercus seedlings. Compared to the ambient CO2 treatment, the
After 10 months, there were significant increases in the accumulated dry mass of leaves (131%), wood (main stem and all
branches by 356%) and roots (219%) of Quercus seedlings
growth in elevated CO2 compared to seedlings grown at ambient CO2 (Table 2). Main stem extension growth was examined
for each growth flush, and during the first year most of the trees
in both treatments produced around three flushes (Table 3).
Although the length of the first flush did not differ between
treatments, significant treatment differences in stem radial
growth (wood production) were apparent in all three flushes
Table 2. Total dry mass of shoots and roots, leaf area production and
total root and shoot length (including main stem plus all branches) of
Quercus robur seedlings after 10 months of exposure to either ambient
or elevated CO2.
Ambient CO2
Elevated CO2
SED1
P
4.76
11.00
1.52
***
640
1177
186
**
Shoot dry mass (g) 3.62
16.52
3.74
***
Shoot length (cm) 46.7
110.5
26.1
*
Root dry mass (g) 6.93
22.14
5.42
*
Root length (m)
155
38
*
Leaf dry mass (g)
2
Leaf area (cm )
Table 3. Main stem length (cm), stem cross-sectional area (mm2) and
radial growth (mm2 day --1 × 103) of Quercus robur seedlings measured
or calculated within three stem flushes over a 4-month period of
growth between 6 months and 10 months of exposure to either ambient
or elevated CO2.
Flush one
1
SED = Standard error of difference of means; significant differences
(P) are shown by the symbols: ns = not significant, * = 0.05, ** =
0.01 and *** = 0.001.
Flush three
12.1 (39)
16.1 (53)
1.5
**
11.3 (28)
44.7 (45)
2.9
***
12.6
28.5
1.9
***
6.5
17.9
1.4
***
Main stem length1 (cm)
Ambient CO2
Elevated CO2
SED2
P
13.1 (71)
12.4 (70)
0.6
ns
Stem cross-sectional area (mm2)
Ambient CO2
25.7
Elevated CO2
48.7
SED
3.0
P
***
Mean radial growth of stem (mm2 day --1 × 103)
152
74
Ambient CO2
Elevated CO2
577
299
SED
47
26
P
***
***
1
46
Flush two
2
55
157
15
***
Main stem length not including branches. The figures in brackets
refer to the number of seedlings with one, two or three stem flushes.
SED = Standard error of difference of means; significant differences
(P) are shown by the symbols: ns = not significant, * = 0.05, ** = 0.01
and *** = 0.001.
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF CO2 ON OAK AND CHERRY GROWTH
Table 4. Total dry mass of shoots and roots, leaf area production and
total root and shoot length (including main stem and branches) of
Prunus avium × pseudocerasus exposed for either 2 or 10 months to
either ambient or elevated CO2.
Ambient CO2
Elevated CO2
SED1
P
After a 2-month exposure
Leaf dry mass (g)
2.89
4.47
0.64
*
Leaf area (cm2)
459
636
83
*
Shoot dry mass (g) 2.27
3.65
0.53
*
Total shoot length
(cm)
37.1
53.8
6.5
*
Root dry mass (g) 6.77
6.98
1.06
ns
Root length (m)
129
13
ns
112
157
21
ns
15600
19800
2200
ns
Shoot dry mass (g) 182
311
53
ns
Total shoot length
(cm)
546
867
154
ns
Root dry mass2 (g):
< 2 mm
50.9
61.2
9.1
ns
> 2 mm
42.6
54.8
15.3
ns
Root length (m):
< 2 mm
686
714
132
ns
> 2 mm
5.4
7.5
1.1
ns
111
After a 10-month exposure
Leaf dry mass (g)
2
Leaf area (cm )
2
1
2
SED = Standard error of difference of means; significant differences
(P) are shown by the symbols: ns = not significant, * = 0.05, ** = 0.01
and *** = 0.001.
Root dry mass and root length were partitioned with respect to root
diameter.
323
elevated CO2 treatment caused a 27% enhancement in dry
mass production in Prunus after two months and 51% after
10 months compared to a dry mass gain of more than 200%
after 10 months in Quercus seedlings. The limited response of
Prunus to elevated CO2 may be associated with photosynthetic
acclimation (Besford et al. 1990, Gunderson and Wullschleger
1994, Wilkins et al. 1994). After 10 months of growth in
elevated CO2, A, measured at elevated CO2 for Prunus, was not
significantly different from that of plants grown and measured
at ambient CO2; whereas, A of Prunus plants grown at elevated
CO2 and measured at ambient CO2 was significantly lower by
about 80%, than A of plants grown and measured at ambient
CO2. Because the area of individual Prunus leaves in the
elevated CO2 treatment was about 87% greater than the area of
leaves in the ambient CO2, we also expressed A on a per leaf
basis. Net assimilation rate of leaves grown in elevated CO2
and measured at ambient CO2 was only about 40% that of
leaves grown in ambient CO2 on a per leaf basis, which suggests that photosynthetic acclimation had taken place.
Net assimilation rate of Quercus grown and measured at
elevated CO2 was over twice that of seedlings grown and
measured at ambient CO2, whereas A of seedlings grown at
elevated CO2 and measured at ambient CO2 was similar to A of
seedlings grown and measured at ambient CO2. Thus, in contrast to Prunus, Quercus seedlings exhibited no loss of photosynthetic activity even after 10 months in elevated CO2. A
comparison of A measured at elevated CO2 of Quercus seedlings grown in ambient and elevated CO2, indicated that there
was some gain in photosynthetic capacity in response to the
elevated CO2 treatment (Table 1).
It has been suggested that light-saturated A at low CO2
concentrations is limited by RuBP-saturated Rubisco activity
(e.g., von Caemmerer and Farquhar 1981, Besford et al. 1985),
whereas at high CO2 concentrations A may become increasingly dependent on RuBP regeneration through, for example,
light harvesting and photosynthetic electron transport capacity
Table 5. Mineral nutrient concentration (% dry mass basis) in leaves of Quercus robur after 6 months exposure to ambient or elevated CO2 and
Prunus avium × pseudocerasus after two months of growth at either ambient or elevated CO2.
Nitrogen
Phosphorus
Potassium
Calcium
Magnesium
Quercus
Ambient CO2
Elevated CO2
SED1
P
2.93
2.71
0.11
ns
0.23
0.21
0.04
ns
1.31
0.97
0.29
**
1.45
1.30
0.31
ns
0.37
0.38
0.05
ns
Prunus
Ambient CO2
Elevated CO2
SED
P
3.10
2.92
0.15
ns
0.39
0.34
0.03
ns
2.96
2.90
0.14
ns
3.13
2.88
0.09
*
0.47
0.47
0.01
ns
1
SED = Standard error of difference of means; significant differences (P) are shown by the symbols: ns = not significant, * = 0.05, ** = 0.01 and
*** = 0.001.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
324
ATKINSON ET AL.
(von Caemmerer and Farquhar 1981). Leaves of Quercus
plants grown at elevated CO2 contained increased amounts of
thylakoid-bound cytochrome f (a potential rate limiting step in
photoelectron transport) with no apparent change in the
amount of Rubisco LSU. In previous work with Prunus avium
grown in elevated CO2 for two years, both Rubisco LSU and
cytochrome f amounts were lower in plants grown in elevated
CO2 than in plants grown in ambient CO2 (Wilkins et al. 1994)
and the amounts of Rubisco decreased before changes in
composition of thylakoid membranes were observed (Van
Oosten and Besford 1995). In our study, Prunus grown in
elevated CO2 showed a reduction in the amount of LSU, but
not of cytochrome f.
It has not yet been demonstrated unequivocally that the
apparent difference in response between Quercus and Prunus
is not an experimental artifact. There is some evidence that
photosynthetic acclimation is less likely to occur in fieldgrown trees than in container-grown trees in a greenhouse
(Gunderson et al. 1993). In our study, the physical effects of
root restriction were minimized by increasing pot size twice in
the first year and by using large, deep pots. Furthermore, recent
work with Prunus avium has shown that root restriction does
not necessarily have a negative affect on the CO2-induced
stimulation of dry matter production (Kerstiens and Hawes
1994).
An absence of acclimation of A has been observed in several
Quercus species after long-term exposure to elevated CO2
(Gunderson et al. 1993, Ceulemans and Mousseau 1994),
although there are exceptions. Of the temperate deciduous tree
species examined by Bunce (1992), only Quercus robur
showed photosynthetic acclimation. No conclusion can be
reached for Prunus, because there have been few studies of the
effects of elevated CO2 on this species (see Bazzaz et al. 1990,
Wilkins et al. 1994). However, we found no evidence that
environmentally-based factors could explain the differences
between Quercus and Prunus. In the elevated CO2 treatment,
Quercus roots represented 45% of total dry mass, whereas in
Prunus only 20% of total dry mass was partitioned to roots.
Acclimation of A may be an effective mechanism for restricting the rate of aerial growth and thereby restoring a more even
distribution of dry mass between root and shoot. The maintenance of this ratio is an important component of the ability of
the root to sustain the shoot, particularly with respect to the
supply of water and nutrients. Low nutrient status can impose
a limitation on A especially in plants grown in elevated CO2
(Norby et al. 1992). In our experiments, the possibility of soil
nutrient depletion, at elevated CO2, influencing leaf nitrogen
and A was minimized by weekly applications of nutrient solution. Mineral analyses indicated that the loss of photosynthetic
capacity in Prunus grown at elevated CO2 was not the result of
foliar nutrient deficiency.
Dry matter allocation and sink development influences on
assimilation
The elevated CO2 treatment had a pronounced effect on the
development of Quercus shoots. The main stem of Quercus
developed through a series of growth flushes (Hanson et al.
1986). Shoot length of the first flush did not differ with respect
to CO2 treatment, but later extension growth, particularly of the
third flush, was significantly different for trees grown at elevated CO2. Radial growth (wood production) was significantly
greater in all three flushes in plants grown at elevated CO2.
These findings highlight the effects of elevated CO2 on carbon
allocation and meristematic function with respect to apical
growth and cambial production, which in turn will have implications for the development of the tree canopy to maturity. The
switch to a more indeterminate pattern of Quercus shoot
growth in response to elevated CO2 is important, because the
stimulation of new apical meristems creates new sinks for
carbohydrate utilization, which may explain why photosynthetic capacity was maintained in the elevated CO2 treatment.
In conclusion, we found important differences in the behavior of two woody species to elevated CO2. Quercus robur did
not show photosynthetic acclimation, whereas Prunus did.
This difference appeared to be associated with the ability of
Quercus to maintain its photosynthetic potential as a result of
a stable content of Rubisco. A stable Rubisco content in leaves
exposed to elevated CO2 may reflect the ability of Quercus to
develop new sinks for carbohydrate utilization.
Acknowledgments
This work was sponsored by the European Commission (Climate
Change Impacts, Project EV5V-CT92-0093) and the Ministry of Agriculture, Fisheries and Food, U.K. We are grateful to Drs. J.D. Quinlan, B.H. Howard and Prof. H.G. Jones for their comments, to Martin
Ridout for statistical advice, the greenhouse staff at HRI-Littlehampton for their assistance with growing the plants and for technical help
from Ann Lucas and Lorraine Taylor (HRI-East Malling).
References
Atkinson, C.J. 1996. Global changes in atmospheric carbon dioxide:
The influence on terrestrial vegetation. In Plant Response to Air
Pollution. Eds. M. Iqbal and M. Yunus. John Wiley and Sons Ltd.,
Sussex, U.K., pp 99--133.
Bazzaz, F.A., J.S. Coleman and S.R. Morse. 1990. Growth responses
of major co-occurring tree species of the Northeastern United States
to elevated CO2. Can. J. For. Res. 20:1479--1484.
Besford, R.T. 1990. The greenhouse effect: Acclimation of tomato
plants growing in high CO2, relative changes in Calvin Cycle
enzymes. J. Plant Physiol. 136:458--463.
Besford, R.T., L.J. Ludwig and A.C. Withers. 1990. The greenhouse
effect: Acclimation of tomato plants growing in high CO2 photosynthesis and ribulose-1,5-bisphosphate carboxylase protein. J. Exp.
Bot. 41:925--931.
Besford, R.T., C.M. Richardson, J.L. Campos and A.F. Tiburcio. 1993.
Effect of polyamines on stabilization of molecular complexes in
thylakoid membranes of osmotically stressed oat leaves. Planta
189:201--206.
Besford, R.T., A.C. Withers and L.J. Ludwig. 1985. Ribulose bisphosphate carboxylase activity and photosynthesis during leaf development in the tomato. J. Exp. Bot. 36:1530--1541.
Bunce, J.A. 1992. Stomatal conductance, photosynthesis and respiration of temperate deciduous tree seedlings grown outdoors at an
elevated concentration of carbon dioxide. Plant Cell Environ.
15:541--549.
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF CO2 ON OAK AND CHERRY GROWTH
Ceulemans, R. and M. Mousseau. 1994. Effects of elevated atmospheric CO2 on woody plants. New Phytol. 127:425--446.
Clough, J.M., M.M. Peet and P.J. Kramer. 1981. Effects of high
atmospheric CO2 and sink size on rates of photosynthesis of a
soybean cultivar. Plant Physiol. 67:1007--1010.
Cure, J.D. and B. Acock. 1986. Crop response to carbon dioxide
doubling: A literature survey. Agric. For. Meteorol. 38:127--145.
Eamus, D. and P.G. Jarvis. 1989. The direct effects of an increase in
the global atmospheric CO2 concentration on natural and commercial temperate trees and forests. Adv. Ecol. Res. 19:1--55.
Gunderson, C.A., R.J. Norby and S.D. Wullschleger. 1993. Foliar gas
exchange responses of two deciduous hardwoods during 3 years of
growth in elevated CO2: No loss of photosynthetic enhancement.
Plant Cell Environ. 16:797--807.
Gunderson, C.A. and S.D. Wullschleger. 1994. Photosynthetic acclimation in trees to rising atmospheric CO2: A broader perspective.
Photosynth. Res. 39:369--388.
Hanson, P.J., R.E. Dickson, J.G. Isebrands, T.R. Crow and R.K. Dixon.
1986. A morphological index of Quercus seedling ontogeny for use
in studies of physiology and growth. Tree Physiol. 2:273--281.
Kerstiens, G. and C.V. Hawes. 1994. Response of growth and carbon
allocation to elevated CO2 in young cherry (Prunus avium L.)
saplings in relation to root environment. New Phytol. 128:607-614.
Krupa, S.V. and R.N. Kickert. 1989. The greenhouse effect: Impacts
of ultraviolet-B (UV-B) radiation, carbon dioxide (CO2), and ozone
(O3) on vegetation. Environ. Pollut. 61:263--393.
Long, S.P. 1991. Modification of the response of photosynthetic productivity to rising temperature by atmospheric CO2 concentrations:
Has its importance been underestimated? Plant Cell Environ.
14:729--739.
325
Long, S.P. and B.G. Drake. 1992. Photosynthetic CO2 assimilation and
rising atmospheric CO2 concentration. In Crop Photosynthesis:
Spatial and Temporal Determinants. Eds. N.R. Baker and H. Thomas.
The Hague: Elsevier Science Publishers B.V., pp 69--103.
Mehta, R.A., T.W. Fawcett, D. Porath and A.K. Mattoo. 1992. Oxidative stress causes rapid membrane translocation and in vivo degradation of ribulose-1,5-bisphosphate carboxylase oxygenase. J. Biol.
Chem. 267:2810--2816.
Norby, R.J., C.A. Gunderson, S.D. Wullschleger, E.G. O’Neill and
M.K. McCracken. 1992. Productivity and compensatory responses
of yellow-poplar trees in elevated CO2. Nature 357:322--324.
Sinclair, T.R. 1992. Mineral nutrition and plant growth response to
climate change. J. Exp. Bot. 43:1141--1146.
Thomas, R.B. and B.R. Strain. 1991. Root restriction as a factor in
photosynthetic acclimation of cotton seedlings grown in elevated
carbon dioxide. Plant Physiol. 96:627--634.
von Caemmerer, S. and G.D. Farquhar. 1981. Some relationships
between the biochemistry of photosynthesis and the gas exchange
of leaves. Planta 153:376--387.
Van Oosten, J.-J. and R.T. Besford. 1994. Sugar feeding mimics effect
of acclimation to high CO2-rapid down regulation of RuBisCo
small subunit transcripts but not of the large subunit transcripts.
J. Plant Physiol. 143:306--312.
Van Oosten, J.-J. and R.T. Besford. 1995. Some relationships between
gas-exchange, biochemistry and molecular biology of photosynthesis during leaf development of tomato plants after transfer to different carbon dioxide concentrations. Plant Cell Environ.
18:1253--1266.
Wilkins, D., J.-J. Van Oosten and R.T. Besford. 1994. Effects of
elevated CO2 on growth and chloroplast proteins in Prunus avium.
Tree Physiol. 14:769--779.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com