Download Photosynthetic acclimation to elevated C02 in

Journal of Experimental Botany, Vol. 48, No. 314, pp. 1681-1689, September 1997
Journal of
Experimental
Botany
Photosynthetic acclimation to elevated C 0 2 in poplar
grown in glasshouse cabinets or in open top chambers
depends on duration of exposure
R. Ceulemans1'4, G. Taylor2, C. Bosac2, D. Wilkins3 and R.T. Besford 35
1
Department of Biology, University of Antwerp, UIA, Universiteitsplein 1, B-2610 Wilrijk, Belgium
2
School of Biological Sciences, University of Sussex, Falmer, Brighton, Sussex BN19QG, UK
3
Horticulture Research International, Worthing Road, Littlehampton, West Sussex BN176LP, UK
Received 15 January 1997; Accepted 10 May 1997
Abstract
The effects of elevated CO 2 were studied on the photosynthetic gas exchange behaviour and leaf physiology
of two contrasting poplar (Populus) hybrids grown and
treated in open top chambers (OTCs in Antwerp,
Belgium) and in closed glasshouse cabinets (GHCs in
Sussex, UK). The CO2 concentrations used in the OTCs
were ambient and ambient +350 fimo\ mol \ while in
the GHCs they were c. 360/miol mol 1 versus 719
//mol mol \ Measurements of photosynthetic gas
exchange were made for euramerican and interamerican poplar hybrids in combination with measurements
of dark respiration rate and Rubisco activity. Significant differences in the leaf anatomy and structure
(leaf mass per area and chlorophyll content) were
observed between the leaves grown in the OTCs and
those grown in the GHCs. Elevated CO 2 stimulated net
photosynthesis in the poplar hybrids after 1 month in
the GHCs and after 4 months in the OTCs, and there
was no evidence of downward acclimation (or downregulation) of photosynthesis when the plants in the
two treatments were measured in their growth C 0 2
concentration. There was also no evidence of downregulation of Rubisco activity and there were even
examples of increases in Rubisco activity. Rubisco
exerted a strong control over the light-saturated rate
of photosynthesis, which was demonstrated by the
close agreement between observed net photosynthetic
rates and those that were predicted from Rubisco
activities and Michaelis-Menten kinetics. After 17
months in elevated CO2 in the OTCs there was a signi-
ficant loss of Rubisco activity for one of the hybrid
clones, i.e. Beaupre, but not for clone Robusta. The
effect of the CO 2 measurement concentration (i.e. the
short-term treatment effect) on net photosynthesis
was always larger than the effect of the growth
concentration in both the OTCs or GHCs (i.e. the longterm growth CO 2 effect), with one exception. For the
interamerican hybrid Beaupre dark respiration rates in
the OTCs were not significantly affected by the elevated CO 2 concentrations. The results suggest that for
rapidly growing tree species, such as poplars, there is
little evidence for downward acclimation of photosynthesis when plants are exposed to elevated CO 2 for up
to 4 months; longer term exposure reveals loss of
Rubisco activity.
Key words: Elevated CO2, Populus, Rubisco, photosynthesis, chlorophyll content.
Introduction
Short-term exposure of C3 plants to elevated atmospheric
CO2 concentrations often stimulates photosynthesis
(Eamus and Jarvis, 1989; Gifford, 1992), producing major
gains in biomass as a result of the improved competitiveness of CO2 over O2 as a substrate for the main C3
photosynthetic enzyme, ribulose-l,5-bisphosphate carboxylase-oxygenase (Rubisco) (Bowes, 1993). Plants
grown in elevated CO2 can show a degree of photosynthetic acclimation (Besford et ai, 1990), i.e. an increase
or more commonly a decrease in photosynthetic perform-
* To whom correspondence should be addressed. Fax: +32 3 8202271. E-mail: rceulemeuia.ua.ac.be
5
Present address: Furlong, The Street, Patching, Worthing, West Sussex BN13 3XF, UK
© Oxford University Press 1997
1682
Ceulemans et al.
ance as compared with plants grown in low (ambient)
concentrations of CO2, when measured under the same
conditions, due to intrinsic changes in the photosynthetic
machinery (Gunderson and Wullschleger, 1994). While
the adaptive response of plants to elevated CO2 can be
positive, negative or indeed negligible (Ceulemans and
Mousseau, 1994), in this paper the term acclimation will
refer only to the negative aspects of plant response to
elevated CO2. Because photosynthesis more often than
not increases with atmospheric CO2 enrichment (Kalina
and Ceulemans, 1997), the approach of comparing rates
of net photosynthesis for leaves or plants measured at
their respective growth CO2 concentration (ambient or
elevated) in many instances yields little information on
acclimation per se if rates remain higher in elevated CO2
(Long and Drake, 1991; Gunderson and Wullschleger,
1994). Therefore, the technique of measuring rates of net
photosynthesis after plants grown at elevated CO2 have
been transferred to a similar ambient CO2 atmosphere,
can be used. If rates of net photosynthesis are comparable
between leaves grown at elevated and ambient CO2, then
an acclimation response can generally be dismissed (Long
et al., 1993; Gunderson and Wullschleger, 1994).
This paper aims (1) to describe the effects of elevated
CO2 on photosynthetic gas exchange behaviour of poplar
hybrids grown and treated both under open top chamber
and glasshouse cabinet conditions, and (2) to relate the
observed differences in photosynthetic CO2 uptake to
underlying biochemical characteristics, such as Rubisco
activity.
Materials and methods
Two experimental methodologies were compared in this study:
an open top chamber (OTC) experiment on the campus of the
University of Antwerp (Belgium) and an indoor glasshouse
cabinet (GHC) experiment at the University of Sussex in
Brighton ( U K ) (Table 1). These two experiments showed great
similarity (same clonal material, identical measurements) as well
as difference and complementarity (contrasting growth and
exposure techniques, different duration of treatment, different
number of replications, and time of measurements). This
allowed the examination of the effects of the growth and
exposure environment on poplar physiology.
Plant materials
In both Antwerp and Sussex, the fast growing, highly productive
interamerican Populus trichocarpa (Torr. & Gray) x P. deltoides
(Bartr. ex Marsh.) clone Beaupre was compared with a slower
growing euramerican P. deltoides (Bartr. ex Marsh.) x P. nigra
(L.) clone. The clone Primo was chosen as the euramerican
hybrid in Sussex, while in Antwerp Robusta, an older standard
reference clone was used. Further details on these clones, such
as parentage, sex, place of origin, and production performance
can be found elsewhere (Ceulemans, 1990). All plants were
grown from hardwood cuttings (25 cm long), which in Antwerp
only were immersed in water for 24 h prior to planting. All
measurements referred to in this paper were made on plants in
their first or second growing season in elevated CO 2 (Table 1).
Open top chambers in Antwerp
Experiments in Antwerp were performed between AprilNovember 1993 and March-November 1994 in open top
chambers (OTCs) constructed on the campus of the University
of Antwerp (Wilrijk, Belgium; 4°24'E, 51°10'N). Four decagonal OTCs with a floor area of 7 m2 and a height of 4 m
(1993) or 6 m (1994) were used. The walls (1 m wide) of the
OTCs were made of acrylic Perspex and the distance between
adjacent chambers was 8-10 m. Incoming air was supplied by
large ventilators (S&P company, type CBM 320-65, Spain) at
a rate of c. 2000 m 3 h" 1 resulting in nearly two air volume
changes every minute. Air was passed through an aluminium
pipe (with vertical adjustment) to a perforated, fibre-structured
polythene annulus 1 m above the ground. Two of the OTCs
received AMBIENT atmospheric CO 2 (c. 350 ^mol mol""') and
two received ELEVATED atmospheric CO 2 , i.e. ambient +
350/^mol mol" 1 . The CO 2 was continuously supplied day and
night from a 3 tonne large tank (L'Oxyhydrique Intern.,
Machelen, Belgium) throughout the entire growing season.
Fifteen cuttings (7-8 plants from each clone) were planted
on 22 April 1993 in each OTC in a circular planting pattern
with an inter-plant distance of 0.6 m (representing a plant
density of more than 21 200 plants ha" 1 ). Before planting the
original, heavy loamy-clay soil within each OTC was excavated
to a depth of 50 cm and replaced with a fertile sandy-loam
horticultural soil. The first (or early) leaves appeared nearly 1
week after planting. Because of the rather late planting a
number of cuttings from each clone did not survive and had to
Table 1. Comparative table showing the similarity and complementarity of the experimental set-up and environmental conditions of the
elevated CO2 studies on poplar in Antwerp (Belgium) and Sussex (Brighton, UK)
Location, site
Antwerp (Belgium)
Sussex (Brighton, UK)
Coordinates of site
Poplar clones
Experimental set-up
CO2 treatments
4J24'E and 51 "lCN
Beaupre and Robusta
Open top chamber
Ambient (c. 350fimol
mol" ') and elevated
(ambient + 350 ^mol mol ~
Two
variable
21 April 1993
10-15 September 1993
April-November 1993
0"05Wand 50"52^
Beaupre and Primo
Glasshouse cabinet
Ambient (c. 360 ^mol mol" ')
and elevated (constant at
719^01 mol"')
Two
constant temperature 20-25 °C
3 and 17 December 1993
10-16 January 1994
December 93-January 94
0.5-0.6 m
Number of replications/treatment
Environmental conditions
Date of planting
Date of photosynthesis measurements
Duration of CO2 treatment
Average height of plants at end of experiment
Photosynthetic responses to CO2 in poplar
be replaced 2 weeks later. Two months after planting the soil
in the OTCs was covered with a layer of woody chips to
maintain soil moisture content and to control weeds. All plants
were irrigated twice a day by a drip irrigation system. On
23 July 1993, 3.751 of a combined nutrient solution was
supplied to each OTC, corresponding to a N supply of nearly
63.5 kg N ha" 1 . A similar amount of N was added the second
year. More details and a complete description of the OTC setup have been previously published (Ceulemans et al., 1995a,
b). Photosynthetic photon flux density (PPFD), as measured at
30min intervals with a Sunfleck Ceptometer (Delta-T-Devices,
Cambridge, UK), amounted to maximum values of
1340 fimol m~ 2 s" 1 at midday during the days of measurement.
Closed glasshouse cabinets in Sussex
Forty dormant cuttings of the clone Primo and 20 of the clone
Beaupre were planted in large, plastic pots (diameter 15 cm,
height 50 cm) containing a 1:1 mix of compost and vermiculite.
The pots were placed into each of four glasshouse cabinets
(GHCs) at the University of Sussex at Brighton (0°05'W,
50°52'N) on 3 December 1993 (clone Primo) and 17 December
1993 (clone Beaupre), respectively. The four cabinets (length
0.86 m, width 0.56 m, height 1.56 m) in the university's
glasshouse represented two experimental CO 2 treatments, i.e.
AMBIENT atmospheric CO 2 (around 360 /xmol m o P 1 ) and
ELEVATED CO 2 . The target CO 2 concentration of the elevated
treatment was a constant 700ftmol mol" 1 , but the mean
concentration
over the entire growth
period
was
719 + 6^mol mol" 1 . The air flow to the cabinets was via an
adapted air conditioning unit, and the constant CO 2 flow was
supplied from a cylinder of pure CO 2 (CP grade; BOC Special
Gases, Surrey, UK). Full details of the exposure and analysis
system were given by Bosac et al. (1995) and Gardner et al.
(1995). Air temperature in the GHCs was maintained at
20-25 °C. Constant artificial light was supplied by high pressure
sodium lamps from 07.00 h GMT until 21.00 h GMT, and
the average PPFD in the glasshouse cabinets was c.
350fimol m" 2 s" 1 (daylight plus artificial light). Plants were
irrigated twice per day with a nutrient solution according to a
modified Lngestad approach as used and described by McDonald
(1989) for the closely related genus, Salix.
Gas exchange measurements
For all CO 2 gas exchange measurements a portable, open gas
exchange system (ADC, type LCA 3, Hoddesdon, UK) and a
Parkinson leaf chamber (PLC type N) were used on intact,
attached leaves. To discriminate between long-term effects (i.e.
OTC or GHC growth effect) and short-term effects (i.e.
measurement conditions) of CO 2 concentrations, measurements
were made in ambient and elevated OTCs or GHCs at LOW
(375 ^mol mol"') and HIGH (700 /xmol m o l " ' ) CO 2 concentrations. In Antwerp net photosynthetic rates were measured in
the OTCs from 10-15 September, 1993 under saturating PPFD
conditions of 1500fimol m" 2 s" 1 with an artificial light source
(Philips sodium lamp). PPFD was monitored with a quantum
photodiode (after Pontailler, 1990). Leaf temperature during
the gas exchange measurements was 25 °C. Mature, fully
expanded leaves of leaf plastochron index (LPf) 7-10 of two
plants were sampled; the relationship between leafage (expressed
in LPI units) and photosynthetic rate, as well as the leaf
longevity and optimal leaf age were known for the clones
studied (Ceulemans and Impens, 1979). Three replicate measurements were made per CO 2 treatment and per clone. To measure
the short-term effect, CO 2 was supplied from a gas cylinder
(L'Oxyhydrique Internationale, Machelen, Belgium) and directly
1683
injected into the leaf cuvette during the photosynthesis measurements. Dark respiration measurements were made prior to
illumination for net photosynthesis measurements.
In the Sussex experiment net photosynthetic rates were
measured from 10—16 January 1994 (i.e. after 1 month in
elevated CO 2 ) in the glasshouse. For the measurements, potted
plants were removed from the growth cabinets and placed
under two artificial light sources (400 W high pressure sodium
lamps, Thermoforce Ltd., Maldon, UK) in the glasshouse.
Saturated net photosynthesis of GHC plants was measured at
a PPFD of 1500 ^onol m""2 s" 1 on mature leaves (LPI of 7-10)
of three plants per clone and per growth cabinet under LOW
(c. 350 ftmol mol" 1 ) and HIGH (700 /xmol mol" 1 ) atmospheric
CO 2 . The air flow for the gas exchange measurements was
directly taken from the closed CO 2 controlled glasshouse
cabinets. No attempt was made to control vapour pressure
difference or temperature during the gas exchange measurements. Leaf temperature during the measurements was close to
29 °C; however, a temperature correction was applied for the
modelled AM (Long, 1991).
Dark respiration rates were measured at the end of the dark
period (between 05.45 h and 07.30 h GMT) on mature leaves
of three plants per clone and per cabinet, i.e. on six plants per
clone and per treatment. The end of night respiration (/?„) was
assumed to be equal to the rate of CO 2 evolution from the
alternative respiratory pathways in the light (i.e. day respiration
or R^), but not to photorespiration in the light, which was
calculated from Rubisco kinetics. The same assumptions were
made in Besford et al. (1985). Therefore R^ is assumed to be
equal to the minimum value of Ra. This value of dark
respiration was then used for the modelling exercise on Rubisco
activity (Besford et al., 1985) as described below. Respiration
data were collected for two reasons: (1) to look for short- and
long-term effects of elevated CO 2 on respiration, and more
importantly (2) to get a figure for respiration which could be
used in the modelling of Aut. Modelling /!„, from Rubisco
values and comparing them to the measured values indicates
where Rn and net photosynthesis measurements are likely to
be robust.
Assays of Rubisco activity and chlorophyll content
In Antwerp leaf discs were sampled for biochemical analysis
around noon on 22 August 1993 and again on 20 July 1994
from recently matured leaves of 3-8 plants per clone and per
OTC (a total of 12 replications per clone and per treatment).
In Sussex samples for biochemical analysis were taken on the
same days and time periods as the gas exchange measurements
(see above). For clones Beaupre and Primo 5 or 6 replicates,
respectively, were taken per cabinet. In all cases (Antwerp and
Sussex) discs of about 2.5 cm2 (c. 50 mg FW) were harvested,
and immediately plunged in liquid nitrogen. Rubisco was
extracted as described by Paulilo et al. (1994) and Wilkins et al.
(1994). After extraction samples were centrifuged at 15000g
for 5 min and stored on ice until assayed (Wilkins et al., 1994).
Rubisco activity was assayed by the coupled-enzyme method of
Lilley and Walker (1974) at 20 °C and pH 8.0 with a 20 min
preincubation period (Besford, 1984).
Rates of PPFD saturated photosynthesis (AM) were predicted
from the kinetics of Rubisco derived from Besford (1984)
adjusted to 29 °C as in Jordan and Ogren (1984), and based on
the equations of Farquhar et al. (1980). In this modelling
approach it was assumed that Aal was limited by fully activated
and RUBP-saturated Rubisco as in Besford et al. (1985, 1990).
No convincing and consistent data exist to suggest a change in
the degree of Rubisco activation in plants grown long term in
1684
Ceulemans et al.
elevated CO2 (Van Oosten et al., 1995). It was further assumed
that after a 20 min activation in Mg2 + and HCOf the in vitro
and in vivo activation states were the same. The maximum in
vitro carboxylation rate in saturating CO2 and RUBP (Kc) was
measured by the Rubisco activity assay described above.
For analysis of chlorophyll content leaf discs of about 50 mg
FW (c. 2.5 cm~2) were harvested in Antwerp and about 100 mg
FW in Sussex. For determination of chlorophyll content the
DMF extraction method (Moran, 1982) was used for the samples
in Antwerp, while the DMSO extraction protocol (after Hiscox
and Israelstam, 1979) was used in Sussex. Chlorophyll absorption was measured spectrophotometrically at wavelengths of
663 and 654 nm. Leaf mass per area (LMA) was determined on
leaf discs sampled from 5-10 leaves, and calculated as the ratio
of leaf area to dry mass of the discs.
a) Euramerican
_35
Open top chamber
Glasshouse cabinet
Ambient Elevated
Ambient Elevated
^f30
f 20
8
£ 15
c
fo 10
Icu 5
Statistical analysis
Results of Rubisco activity, net photosynthetic rates and leaf
mass per area ratios were analysed for statistical differences
between CO2 treatments by a two sample Student's r-test.
Results of chlorophyll analysis were subjected to an analysis of
variance (ANOVA) to examine the effects of treatment and
growth environment (GHC versus OTC), followed by a least
significant differences (LSD) test to identify statisitical
differences.
b) Interamerican
Open top chamber
Glasshouse cabinet
Ambient Elevated
Ambient Elevated
Results
Wef photosynthetic rate
In general, PPFD saturated net photosynthetic rates and
responses of photosynthesis were similar in plants grown
in OTCs and GHCs. In both the OTCs in Antwerp and
the GHCs in Sussex, net photosynthetic rates were significantly enhanced by the elevated CO2 treatment (Fig. 1).
This was evident for the euramerican (clones Primo and
Robusta) as well as for the interamerican (clone Beaupre)
hybrids. For the ambient treatment no differences in
absolute values of PPFD saturated net photosynthesis
were found between OTCs and GHCs, but the photosynthesis of plants in the elevated treatment was always
significantly higher (at P< 0.001) in the OTCs than in
the GHCs. In both the OTCs and the GHCs, the shortterm (i.e. measurement) effect of high CO2 on net photosynthesis was different from the long-term (i.e. growth)
effect. When considering photosynthesis measured at high
(700 ^mol moP 1 ) CO2, values were significantly higher
(at / > <0.01) for the elevated CO2 treatment than for the
ambient treatment in the OTC experiment. However, this
was the converse in the GHC experiment of Sussex: lower
net photosynthesis in the elevated treatment than in the
ambient treatment when measurements were made in high
CO2 (Fig. 1), indicating some acclimation of photosynthesis in this case. However, net photosynthesis measured
under high CO2 (hatched bars on Fig. 1) was found to
be at least twice that measured under low atmospheric
CO2 concentration (open bars on Fig. 1). This was true
for both growth treatments (ambient and elevated) and
for euramerican as well as for interamerican hybrid clones.
Fig. 1. Net photosynthetic rates of euramencan (top figure) and
interamencan (bottom figure) poplar hybrids grown under ambient
(350-360 fimol mol" 1 ) and elevated atmospheric CO 2 concentrations in
open top chambers (OTC, in Antwerp) or in controlled glasshouse
cabinets (GHC, in Sussex). Elevated CO 2 concentrations were ambient
+ 350 ^mol mol~' in the OTCs, respectively, 719/xmol mol* 1 in the
GHCs. Open bars represent measurements made in low CO 2 concentrations (350-375/xmol mol" 1 ) and hatched bars represent measurements
made under high CO 2 concentrations (700 ^±mol mol" 1 ). All data are
mean values of at least ten replications. Vertical bars represent single
standard error of the mean.
The actual measurement conditions were of more importance for net photosynthesis than the experimental growth
(or treatment) conditions, and in this regard all clones
had a very similar response. It should be noted here that
measurements in the GHCs were made only 1 month
after the onset of the elevated CO2 treatment, while those
in the OTCs were made after 4 months of CO2 treatment.
In previous work considerable genotypic differences in
net photosynthesis were observed for the clones studied
here (Ceulemans et al., 1987; Radoglou and Jarvis, 1990).
However, in this study clonal differences both in the
OTCs and the GHCs, were much smaller than observed
previously (Fig. 1), but still significant (at P<0.01). The
PPFD saturated net photosynthetic rate was higher in
euramerican hybrids Primo and Robusta than in the
interamerican hybrid clone Beaupre. The short-term
effects of elevated CO2 on net photosynthesis were slightly
Photosynthetic responses to CO2 in poplar
larger in the euramerican (c. + 160% for clone Robusta)
than in the interamerican hybrids (+140% for clone
Beaupre; Fig. 1).
Dark respiration
Dark respiration rates were not significantly (at P<0.05)
affected by growth in elevated CO 2 , either in the OTC or
in the GHC (Fig. 2). However, for the interamerican
hybrid clone Beaupre growth in elevated CO 2 in the OTC
experiment significantly (at P<0.01) reduced the dark
respiration rate by more than 60% (Fig. 2). For euramerican as well as for interamerican poplars, dark respiration rates in the GHCs were equal to, or a lot lower than
those observed in the OTCs. Dark respiration rates were
found to be higher in the interamerican clone Beaupre
than in the euramerican hybrid clones Primo and
Robusta, but the differences were not significant.
a) Euramerican
Open top chamber
Ambient
Elevated
Glasshouse cabinet
Ambient Elevated
b) Interamerican
Open top chamber
is
Glasshouse cabinet
1
o
34
I'
1
1685
Significant genotypic differences between the euramerican and interamerican hybrids were observed in the
response of dark respiration rate to the short-term CO 2
measurement conditions. In clone Beaupre dark respiration was significantly lower when measurements were
made under high CO 2 (700 ^mol mol" 1 ) than under
low CO 2 (c. 350/^mol mol" 1 ), but no differences were
observed between the measurement conditions with clones
Primo or Robusta (Fig. 2).
Rubisco activity
After exposure to elevated CO 2 for up to 5 weeks in the
GHCs there was no significant difference in the extractable
Rubisco activity from Primo or Beaupre. Also in the
OTCs after exposure to elevated CO 2 for 4 months there
was no significant difference in Rubisco activity obtained
from Beaupre (Table 2). However, there was an increase
in Rubisco activity with elevated CO 2 in clone Robusta.
After extended exposure to elevated CO 2 in the OTCs for
17 months both Beaupre and Robusta contained less
Rubisco activity (Table 2). There was a tendency for the
photosynthesis rates of the OTC grown plants, when
measured at low CO 2 , to be higher after exposure to
elevated CO 2 for 4 months (Fig. 1) which may have been
related to the higher Rubisco activity, but only in clone
Robusta.
The PPFD saturated rate of photosynthesis was predicted from the extractable Rubisco activity assuming
that the Rubisco in vivo is RuBP saturated and fully
activated. With plants grown in the GHC, where photosynthesis measurements and Rubisco analyses were made
on the same leaf, good predictions of photosynthetic
performance were achieved (Table 3). Rubisco kinetics
predicted accurately that leaves grown and measured in
elevated CO 2 would double their PPFD saturated rate of
photosynthesis compared with those grown and measured
in ambient CO 2 . Also the absolute rates were close to
predicted rates (Table 3). With the OTC-grown plants
modelling photosynthetic performance was poor, and in
general photosynthetic rates were underestimated.
However, measurements of Rubisco activity and photosynthesis were carried out on different leaves with a
3-week interval.
Q
Ambient
Elevated
Ambient Elevated
Fig. 2. Dark respiration rates of euramerican (top figure) and interamerican (bottom figure) poplar hybrids grown under ambient (350360 /irnol mol ~') and elevated atmospheric CO 2 concentrations in open
top chambers (OTC, in Antwerp) or in controlled glasshouse cabinets
(GHC, in Sussex). Elevated CO 2 concentrations were ambient
+ 350fimol mol"' in the OTCs, respectively, 719/nmolmol" 1 in the
GHCs. Open bars represent respiration measurements made in low
CO 2 concentrations (350-375^mol mol"') and hatched bars represent
measurements made under high CO 2 concentrations (700 ^mol mol"')
All data are mean values of at least ten replications. Vertical bars
represent single standard error of the mean.
Chlorophyll concentration and leaf mass per area ratio
In the OTC experiment there was a significant (P<0.05)
and consistent decrease in chlorophyll concentrations
(Chi a, Chi b as well as total Chi) in the elevated
treatment as compared to the ambient treatment
(Table 4). However, in the GHC experiment some conflicting results were found: there was a non-significant
decrease in Chi a concentration in clone Beaupre, but for
clone Primo the Chi concentrations significantly (at
/ > <0.01) increased under the elevated CO 2 treatment.
1686
Ceulemans et al.
Table 2. Average Rubisco activity in euramerican (clones Primo and Robusta) and interamerican (clone Beaupre) poplar hybrids
exposed to ambient C02 or elevated C02 concentrations in glasshouse cabinets for 5 weeks (at Brighton) and in open top chambers
for 4 months and 17 months (at Antwerp)
All figures are expressed in ^mol CO 2 m ~2 s ~' and are mean values of 9-13 replications (± standard error). Levels of significance have been indicated.
Hybrid
Date
Ambient CO 2
Elevated CO 2
Glasshouse cabinets
Pnmo and Beaupre*
January 1994
12.7(1 96)
14.8(2.13)
Open top chambers
Beaupre
Beaupre
Robusta
Robusta
August 1993
September 1994
August 1993
September 1994
17.6(3 58)
22.8 (2.27)
12.0(1.60)
14.3 (2.27)
180(2 12)
15.2(2.33)
16 1 (0.95)
122 (1.58)
Significance
/><0.05
ns
•Values were meaned since no significant differences in extractable Rubisco activity were observed between the clones grown in either ambient or
elevated CO 2 .
Table 3. Predicted and measured PPFD saturated net photosynthesis (AUJ of interamerican and euramerican poplar hybrids grown
and treated to ambient and elevated atmospheric CO2 levels in open top chambers (OTC, Antwerp experiment) or glasshouse cabinets
(GHC, Sussex experiment)
Predicted net photosynthesis was obtained from in vitro Rubisco activity measurements (RUBP-saturated carboxylation rate) after Besford et al
(1985). Predicted Aal is based on means of at least nine Rubisco replications. Measured /(„, is based on means of at least ten replications
Hybrid
Exposure system
CO 2 treatment
Predicted AM
(fimol m~ 2 s"')
Measured Aml
(fimol m~2 s~')
Primo and Beaupre*
Pnmo and Beaupre*
Robusta
Robusta
Beaupre
Beaupre
GHC
GHC
OTC
OTC
OTC
OTC
Ambient
Elevated
Ambient
Elevated
Ambient
Elevated
9.9
19.3
8.3
16.9
12.3
19.2
10.6
19.8
12.0
29.5
11.9
28.5
"Mean values are used since no significant differences were observed in extractable Rubisco activity between the two clones grown in either
ambient or elevated CO 2 .
Table 4. Results of chlorophyll analysis on interamerican (clone Beaupre) and euramerican (clones Primo and Robusta) poplar hybrids
grown under ambient and elevated CO2 concentrations in glasshouse cabinets and in open top chambers
All chlorophyll concentrations (Chi a, Chi b and total C h i ) are expressed in jigcm~ 2 and are the means of at least nine replicates (standard error
within brackets). Values followed by the same letter within the same column are not significantly different at P<0.0\.
Hybrid
CO 2 treatment
Chi a
Glasshouse cabinets
Beaupre
Beaupre
Primo
Primo
Ambient
Elevated
Ambient
Elevated
30.6 (1.26)bc
29.6 (0.22)cd
27 3(2.93)d
33.5 (1.71)b
7.37
7.11
6.18
7.60
Open top chambers
Beaupre
Beaupre
Robusta
Robusta
Ambient
Elevated
Ambient
Elevated
42.5 (2.27)a
33.8 (3.27)b
42 0(6.80)a
29.7 (2.76)cd
1051
8.53
10 74
7.67
For both poplar hybrids the values of chlorophyll concentrations (Chi a, Chi b and total Chi) differed significantly
between the OTC and the GHC experiments (Table 4).
Total chlorophyll (Chi a + Chi b) concentrations were
between 35-41 ^g cm" 2 for the plants grown in the GHCs
compared to 37-53 ^.g cm" 2 for plants grown in the
OTCs (Table 4). In the ambient treatment of the GHC
experiment there were also significant differences (at P
Chi b
Total Chi
Chi a/b
(0.18)b
(0.01)c
(0.60)d
(0.27)b
38.0 (1.41 Jc
36.7 (0.22)de
35 1 (3.77)e
41.1 (1.97)bc
4.14
4.13
4.39
4.39
(0.09)b
(004)b
(0.1 l)ab
(0.10)ab
(1.26)a
(3.02)b
(3.02)a
(0.40)b
53.1
42.3
52.7
37.4
4.05
3.96
391
3.87
(0.39)c
(1 90)c
(1.90)c
(0.27)c
(3.20)a
(9.36)b
(9.36)a
(3.04)de
<0.01) in the Chi a and Chi b concentrations between
the two hybrid clones: hybrid clone Primo had lower Chi
a and Chi b values than hybrid clone Beaupre. No
significant changes were observed in the Chi a:Chl b ratio
under the elevated CO2 treatment.
LMA significantly increased under elevated CO2 for
young, expanding leaves of clone Primo in the GHC
experiment, and for mature leaves of both clones (Beaupre
Photosynthetic responses to CO2 in poplar
and Robusta) in the OTC experiment (Table 5).
Differences in LMA between ambient and elevated CO 2
treatments were not significant in the GHC experiment
for clone Beaupre. Significant differences in LMA were
observed between plants in the OTCs and those in the
GHCs as also evidenced in Table 5. LMA values were
always lower, thus thinner leaves, in the GHCs than in
the OTCs.
Discussion
Although there is strong evidence in many tree species
for acclimation (or down-regulation) of photosynthesis
and Rubisco activity when grown long term in elevated
CO 2 (Sage et al., 1989; Van Oosten et al., 1992; Tissue
et al., 1993; Wilkins et al., 1994), this paper shows two
examples where negative acclimation to elevated CO 2 was
not seen after lengthy periods of exposure, although in
one case there was an indication of slight acclimation.
These observations were made on clonal poplar plants
grown for 1 month under elevated CO 2 in GHCs or for
more than 4 months in OTCs. Because acclimation in
crop plants and in trees is often observed by 30 d if not
sooner, the lack of early acclimation in poplar may imply
an unusually high sink strength (Gaudillere and
Mousseau, 1988). Moreover nutrients were most probably
not limiting in these experiments (Ceulemans et al., 19956;
Gardner et al., 1995). Evidence was also found for a
strong control of Rubisco on PPFD saturated photosynthesis since observed and predicted rates of net photosynthesis based on Rubisco activity were close when the
same leaves were used for analysis. Another technique
used in assessing photosynthetic acclimation, was to measure rates of net photosynthesis after plants grown at
ambient and elevated CO 2 had been transferred to a
similar ambient CO 2 atmosphere (Gunderson and
Wullschleger, 1994). With plants in the OTCs the rates
of PPFD saturated net photosynthesis were comparable
between leaves of both clones grown at ambient or
elevated CO 2 ; thus, acclimation during the first few
months can be dismissed. However, there were some
1687
indications of loss of photosynthetic capacity in plants
grown in the GHC experiment since photosynthesis measured in high CO 2 was lower for trees grown in elevated
CO 2 compared with those grown in ambient CO 2 . It does
not seem very likely that this slight acclimation of photosynthesis might be due to root restriction in the pots in
the GHCs since pot volume was far above the 12.5 dm 3
suggested by Arp (1991). After 17 months in elevated
CO 2 there was significant loss of Rubisco activity in the
OTCs, i.e. acclimation to elevated CO 2 concentrations.
This is one of the very few studies in which a good
prediction of photosynthesis has been made from Rubisco
activities in a tree species. As far as is known, there is no
other example in the literature where both net photosynthesis and maximum Vc were measured (as opposed to
calculated) and where there was a good agreement
between predicted and measured
photosynthesis
(Table 3). When the standard errors are included in
Table 3 it becomes clear that there is indeed no significant
difference between predicted and measured rates in half
of the cases. Based on the data it seems evident that the
PPFD saturated rate of photosynthesis at ambient CO 2
concentrations is strongly controlled by carboxylase activity, e.g. in GHC ambient CO2-grown clones Primo and
Beaupre. There is other evidence in the literature for this.
The degree to which photosynthesis is controlled by
Rubisco activity is known as the Rubisco control coefficient (Kacser and Porteous, 1987). In low light the
control coefficient is low (implying low control of photosynthesis by Rubisco), but in saturating light (as in the
measurements made in this study) the control coefficient
approaches 1, implying almost total control over photosynthesis by Rubisco (see also the findings of Krapp et al.
(1994), who found very high control coefficients in genetically engineered tobacco plants in saturating light).
Further literature evidence has been provided for tomato
(Besford et al., 1985, 1990) and for rice (Makino et al.,
1984).
Initial Rubisco activities were not measured for practical reasons. Furthermore, there are also conflicting
reports on the effect of elevated CO 2 on initial activities,
Table 5. Leaf mass per area (gm 2) of euramerican (clones Robusta and Primo) and interamerican (clone Beaupre) poplar hybrids
grown under ambient CO2 or elevated CO2 concentrations in open top chambers and in glasshouse cabinets
All figures are mean values of 9-13 replications and standard erros are within parentheses. ns = non significant; * = significantly different at ^ = 0.05.
Hybrid
Type of leaf
Ambient CO 2
Elevated CO 2
Significance
Glasshouse cabinets
Beauprc
Primo
Young, expanding
Young, expanding
30.7 (2.4)
24.6(1.3)
32.2(4 8)
28.7(0.7)
ns
*
Young, expanding
Mature, fully expanded
Young, expanded
Mature, fully expanded
40.0(2.6)
61.7(4.6)
34.5(7.2)
65.4 (4.7)
45.0(3.4)
82.0 (6.0)
33.9 (2.0)
87.0(6.1)
ns
Open top chambers
Beaupre
Beauprc
Robusta
Robusta
ns
•
1688
Ceulemans et al.
e.g. Woodrow (1994) versus Rowland-Bamford et al.
(1991) and Tissue et al. (1993). These authors found very
different effects of high CO2 on activation state (decreased,
unchanged or increased, respectively). In many studies
initial activation state has been found to be very high
(80-90%); so, the error in assuming a 100% activation
state in the modelling probably has not affected the results
very much.
Major and significant differences were observed in the
morphological characteristics of the leaves that were
produced in the GHCs as compared to the OTCs. Possibly
because of the lower growth irradiance, higher air temperature and higher relative humidity in the growth cabinets,
leaves formed in the GHCs were more like shade-type
leaves with lower leaf mass per area, lower chlorophyll
concentrations and generally lower Rubisco activities
than leaves formed in the OTCs (more sun-type leaves).
This was true for the two contrasting hybrids. Lower
growth irradiance decreases photosynthetic capacity and
Rubisco activity (Besford, 1984; Besford et al., 1990). In
general chlorophyll concentrations are reduced under
elevated CO2, although there is also one report of an
increased chlorophyll concentration in elevated CO2 in
citrus (Koch et al., 1983). The significant difference in
the chlorophyll concentrations between plants in OTCs
and those in GHCs might be explained (1) either by the
different technique that was used in Antwerp and in
Sussex (DMSO technique versus DMF analysis method),
but most likely by (2) the significant difference in leaf
structure and anatomy between the two growth systems
(OTCs and GHCs). The latter is also reflected in their
leaf mass per area ratios, which reflect leaf density and/or
thickness. The lower Chi a and Chi b concentrations in
clone Primo as compared to clone Beaupre (in the ambient
treatment of the GHC experiment) were confirmed by
lower chlorophyll fluorescence values observed for clone
Primo (Kalina and Ceulemans, 1997).
The experimental results presented here for two contrasting poplar hybrids indicate that the elevated CO2
treatment significantly stimulated net photosynthesis in
the leaves of the OTC experiment in Antwerp (sunadapted, heavier or thicker leaves grown under high light)
and in those grown in the cabinets in Brighton (shadeadapted, lighter or thinner leaves grown under low light
conditions). The magnitude of the response of net photosynthesis to the elevated CO2 treatment differed between
OTCs and GHCs, which might be explained by the lower
light intensity in the glasshouse (compare with Visser
et al., 1993). After exposure to elevated CO2 for up to 4
months there was no evidence of a loss in the rate of net
photosynthesis or Rubisco activity which supports the
view that fast growing tree species would show no sign
of acclimation to elevated CO2. However, after extended
exposure to elevated CO2 in OTCs acclimation of Rubisco
was observed.
Acknowledgements
This research was financially supported at the University of
Antwerp (Belgium) by the EC through its Environment R&D
programme
(contract
No. EV57-CT92-0127
as
the
ECOCRAFT research network) and at the University of Sussex
(UK) by the Biotechnology and Biological Sciences Research
Council (BBSRC grant No. PG085/0524). The support from
the British-Flemish Academic Research
Collaboration
Programme (British Council grant No. 26/94) allowed us to
travel easily between our two laboratories and to perform a
series of joint experimental pulse studies. Further financial
support was provided by the Fund for Scientific ResearchFlanders (FWO). We gratefully acknowledge SDL Gardner,
XN Jiang and BY Shao for help with data collection, N Calluy
and F Kockelbergh for technical assistance, as well as the
associate editor and two anonymous reviewers for their critical
comments and useful suggestions on earlier drafts of the
manuscript. RC is a Senior Research Associate of the FWO
(Brussels).
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