Journal of Experimental Botany, Vol. 47, No. 304, pp. 1755-1761, November 1996
Journal of
Experimental
Botany
Growth at elevated C0 2 leads to down-regulation of
photosynthesis and altered response to high
temperature in Quercus suber L. seedlings
T. Faria1, D. Wilkins2, R. T. Besford2, M. Vaz\ J. S. Pereira1 and M. M. Chaves 13
1
2
1nstituto Superior de Agronomia, Tapada da Ajuda, P-1399 Lisboa codex, Portugal
Horticulture Research International, Worthing Road, Littlehampton, West Sussex BN176LP, UK
Received 16 October 1995; Accepted 24 June 1996
Abstract
Introduction
The effects of growth at elevated CO2 on the response
to high temperatures in terms of carbon assimilation
(net photosynthesis, stomatal conductance, amount
and activity of Rubisco, and concentrations of total
soluble sugars and starch) and of photochemistry (for
example, the efficiency of excitation energy captured
by open photosystem II reaction centres) were studied
in cork oak (Quercus suber L.). Plants grown in elevated CO2 (700 ppm) showed a down-regulation of
photosynthesis and had lower amounts and activity of
Rubisco than plants grown at ambient CO2 (350 ppm),
after 14 months in the greenhouse. At that time plants
were subjected to a heat-shock treatment (4 h at 45 °C
in a chamber with 80% relative humidity and 800-1000
//mol m" 2 s~1 photon flux density). Growth in a CO2enriched atmosphere seems to protect cork oak leaves
from the short-term effects of high temperature.
Elevated CO2 plants had positive net carbon uptake
rates during the heat shock treatment whereas plants
grown at ambient C0 2 showed negative rates.
Moreover, recovery was faster in high CO2-grown
plants which, after 30 min at 25 °C, exhibited higher
net carbon uptake rates and lower decreases in
photosynthetic capacity (AmoJ as well as in the efficiency of excitation energy captured by open photosystem II reaction centres (FJF^ than plants grown
at ambient CO2. The stomata of elevated CO2 plants
were also less responsive when exposed to high
temperature.
In recent years there has been great interest in the
ecophysiological consequences of elevated CO2 in the
atmosphere. It was shown that this may lead to a shortterm increase in the photosynthesis of C3 plants by
stimulating the carboxylation reaction. However, in the
long-term, this effect may be partly lost as a result of
down-regulation in photosynthesis (Gunderson and
Wullschleger, 1994). This down-regulation of photosynthesis seems to be associated with an over-production of
assimilates relative to sink demand and may, therefore,
be associated with an accumulation of soluble sugars in
the leaves (van Oosten and Besford, 1994; van Oosten
et al., 1994). Krapp et al. (1993) argued that it is not the
presence of high concentrations of carbohydrates per se,
but the metabolic events associated with these concentrations that are responsible for changes in gene expression,
enzyme activity and protein content which lead to a
negative feedback on the assimilation rate. The downregulation of photosynthesis at elevated CO2 has been
found to be associated with a decrease in the amount and
the activity of Rubisco (Besford, 1990; van Oosten et al.,
1992). Recent molecular studies in tomato plants grown
under high CO2 showed that when down-regulation of
photosynthesis by low sink demand occurred, the abundance of rbcS transcripts derived from a nuclear genefamily coding for the small subunit of Rubisco was
dramatically reduced, whereas the decline in rbcL RNA
transcripts from the chloroplast gene coding for the large
subunit of Rubisco was less pronounced (van Oosten and
Besford, 1994). Wilkins et al. (1994) showed that the loss
of Rubisco protein was accompanied by decreases in the
contents of chlorophyll and of thylakoid membrane proteins D t , D2 and cytochrome f.
Key words: Elevated CO2, temperature, acclimation, photosynthesis, Quercus suber L.
'To whom correspondence should be addressed. Fax: +351 1 3645000. E-mail: mchaves®isa.utl.pt
© Oxford University Press 1996
1756
Faria et al.
High temperatures and high light intensities are commonly associated as stress factors with a strong impact
on plant metabolism. Among all cell functions, photosynthesis is believed to be one of the most heat-sensitive. As
CO2 is the main sink for absorbed radiation, leaves
subjected to excess of energy may experience some photoinhibition when gas diffusion is restricted by stomatal
closure under heat stress (Demmig-Adams et al., 1989).
However, the extent to which a given photon flux will
actually cause measurable inhibition of carbon uptake
will depend on several internal and external factors,
namely the structure and acclimation of the pigment
systems, the activity of different defence mechanisms, and
the superimposition of other stress factors (Krause, 1994).
It is thought that the primary site of heat damage is
associated with the components of the photosynthetic
system located in the thylakoid membranes, most probably photosystem II (PSII). Protection against excess of
energy can be achieved by an increase in the dissipation
of excess excitation energy, which is accompanied by a
decrease in the quantum yield of PSII (Genty et al, 1989).
In regions with a Mediterranean type of climate, high
temperature stress is a common occurrence during the
summer and very little is known about the effects of
elevated CO2 in plant responses in such environments.
The aim of this work was to study the effects of acclimation to elevated CO2 in the response of Quercus suber L.
(cork oak) leaves to high temperature stress. Heat stress
effects were studied in terms of the regulation of carbon
assimilation (including stomatal conductance, amount
and activity of Rubisco, total soluble sugars) and photochemistry (efficiency of excitation energy capture by open
photosystem II reaction centres, Fv/Fm), in plants grown
for 14 months in elevated CO2.
Materials and methods
Plant material and experimental conditions
Seedlings of Quercus suber L were grown in 10 1 pots filled
with a nursery soil mixture. At 6 months old the seedlings were
transferred from the open-air conditions to greenhouses with
homogeneous descending forced-air convection (0.3 m s" 1 ),
two different CO2 concentrations (elevated CO2, 700 ppm and
ambient CO2, 350 ppm) and natural light (c. 25% mean
reduction in relation to the open-air conditions was observed
in sunny days with maximum values of about 1500 and 600
^mol m~2 s"1 PAR in summer and winter months, respectively).
Air temperature was maintained near the average ambient
values for each month in Lisbon (maximal temperatures of
28 °C in August and minima of 7.2 °C in January). To avoid
effects of the microenvironment, the plants were rotated inside
and between greenhouses every week. After 14 months in the
greenhouses the plants were subjected to a heat-shock treatment
at growth CO2 concentration (4 h at 45 °C in a chamber with
80% relative humidity and 800-1000 funol m" 2 s" 1 PAR). Air
temperature in the chamber increased from 25 °C to 45 "C at a
constant rate of 10°C h" 1 and was maintained at 45 °C for 4
h; thereafter temperature returned to 25 "C (Fig. 1). Leaf
BO
25
A
e
e.s
ZA
Tim*, hours
Fig. 1. The heat-shock treatment was performed in a chamber with
80% relative humidity and 800-1000 ^mol m'1 s" 1 PAR Temperature
in the chamber increased from 25 °C to 45 °C at a constant rate of
10°C h " 1 and was maintained at 45°C for 4 h; thereafter plants were
returned to 25 °C. Measurements were made with a minicuvette system
at temperatures (1) 25°C, (2) 30°C, (3, 4) 45"C, and (5, 6) 25°C.
temperature (measured with thin copper-constantan thermocouples attached to the leaves) changed, following the same
pattern as air temperature, from c. 26 °C to 45 °C with nonsignificant differences between plants from both treatments.
All measurements were taken in fully expanded 6-month-old
leaves (entirely developed in the greenhouses) from 4-6 different
trees per treatment. Individual leaf samples were frozen in
liquid nitrogen and stored at -80 °C until biochemical analysis.
Methods
Specific leaf area (SLA) was calculated as the ratio between the
area and the dry weight (DW) (obtained after 48 h at 80 °C)
of leaf discs of 0.229 cm2. Disc fresh weight was also determined
at the time of collection.
Assimilation rate (A) and stomatal conductance (g,) of
ambient and elevated CO2-grown plants were always measured
at two CO2 concentrations, 350 ppm and 700 ppm using a
Compact Minicuvette System (Heinz Walz, Germany) with the
Bypass Humidity Control Unit to measure transpiration with a
vapour pressure deficit in the air of the cuvette of 1.9 ± 0.1 kPa
and a photosynthetic photon flux density (PPFD) of 1200
l
Maximal photosynthetic capacity ( ^ J , i.e. oxygen evolution
at CO2 and light saturation, was determined in a leaf-disc
oxygen electrode (Hansatech Ltd, Kings Lynn, UK), at 25 °C
and 45°C with a PPFD of 1200 Mmol m~2 s"1 and saturating
CO2 concentration (10%CO2 provided by mixing air and CO2),
immediately after detaching the leaves. The A^^ measurements
were done at 25 °C in leaves of control plants, i.e. plants that
did not suffer the 45 °C heat shock and in heated plants, i.e.
plants immediately after they were subjected to the 45 °C heat
shock. AmMX measurements at 45 °C were done in leaves of the
control plants only.
Chlorophyll afluorescencewas measured in attached leaves
of control and heated plants using a pulse amplitude modulation
fiuorimeter (PAM 2000, Walz, Effeltrich, Germany). The
efficiency of excitation energy captured by open photosystem II
(PSII) reaction centres in dark-adapted leaves (30 min) was
estimated by thefluorescenceratio (Fy/Fm).
Elevated CO2 and temperature in cork oak
Heat tolerance limits were determined by measurements of
the heat-induced increase in the basal fluorescence of chlorophyll
a (Fo) as described in Bilger et al. (1984) in leaves of control
plants, by subjecting the leaves to a continuous increase of
temperature (c. 1.5 °C min" 1 ).
Leaf chlorophyll concentration was determined as described
in Arnon (1949) and expressed per unit dry weight and on an
area basis. The concentrations of total soluble leaf carbohydrates
(sucrose, glucose and fructose) and starch were determined as
described in Stitt et al. (1989).
The extracts for Rubisco analysis were prepared as described
by Wilkins et al. (1994) from leaf samples of control and heated
plants, which were immediately frozen in liquid nitrogen and
stored at -80 °C. Total 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. Western
blotting for the large subunit (LSU) of Rubisco protein was
carried out as described by Besford (1990) using extracts
prepared as above. The blots were probed with a polyclonal
Rubisco antibody raised to tomato. Treatment comparisons
were made with separate Rubisco extracts from at least three
plants per treatment and each sample was blotted twice. The
soluble protein content was determined after Bradford (1976),
by using the commercial Bio-Rad protein assay.
Data are shown as means + standard deviation. Student's t
test was used for statistical comparison of means.
Results
Leaf chemical composition and structure were modified
by the CO 2 treatments. The pools of total soluble sugars
and starch accumulated in leaves of elevated CO 2 plants
was higher than in ambient CO2-grown plants by 44%
(soluble sugars) and 58% (starch) (Table 1). The concentration of total chlorophyll showed a small increase in
elevated CO 2 plants on leaf area basis (Table 1), but it
decreased significantly on a dry weight basis as a result
of the decrease in SLA In elevated CO 2 plants. The
Table 1. Morphological and physiological characterization of
leaves of cork oak (Quercus suber L.) plants grown for 14 months
either al ambient CO2 or at elevated CO2
Standard error of at least 5 replicates. Comparison between treatment
means were made using Student's I test. Means significantly different
are marked with * or *• if P<0.05 or P<0.0\, respectively.
Parameter
Starch concentration
(nmol g"1 FW)
Soluble sugars content
(jxmol g"1 FW)
Protein content
(mgg" 1 FW)
Chlorophyll content
(/imol g"1 DW)
Chlorophyll content
((imol cm"2)
FW/DW ratio
Specific leaf area
(SLA) (cm2 g"1)
Leaf thickness
Ambient CO2-grown
plants
Elevated CO2-grown
plants
11.33±3.25"
27.16±9.27**
591.32 ±39.20**
848.84 ±57.80**
6.34±0.08**
4.70±0.20»*
53.8 ±0.96*
51.1 ± 1.02*
710.71 ±1.03*
750.39 ±3 59*
4.80 ±0.14*
75.71 ±1.54
4.34±0.11*
68.08 ±2.70
208.57±0.64*
202.12±0.01*
1757
changes in SLA may result either from effects on leaf
thickness (represented by the ratio fresh weight/area) or
changes in tissue density (represented by the ratio of dry
to fresh weight) and may be interpreted according to the
equation:
I/SLA = (FW/\eaf area) x (D W/FW)
where FW stands for fresh weight (Dijkstra, 1989). These
data suggested that SLA decreased (non-significantly) in
elevated CO 2 plants, mainly as a result of the increase in
the DW/FW ratio implying a denser tissue, since leaf
thickness was even slightly decreased (Table 1). This
increase in tissue density probably resulted from the
accumulation of starch and explains the lower chlorophyll
concentration on a leaf dry weight basis in elevated CO 2
plants, in spite of the higher concentration on a leaf
area basis.
The heat tolerance limit measured by the continuous
rise in Fo (illustrated for one leaf per treatment in Fig. 2)
in leaves of control plants was 45.1 ±0.4 °C for plants
grown at elevated CO 2 and 45.5 ± 0.6 °C for plants grown
at ambient CO 2 . No significant differences were found
between plants from either treatment.
Carbon assimilation rates (A) measured at plant growth
CO 2 concentrations were not significantly different in
plants grown at elevated CO 2 and at ambient CO 2 at
25 °C and 30 °C (Fig. 3a). However, during the heat shock
treatment at 45 °C, elevated CO 2 plants had a positive
net carbon assimilation rate whereas ambient CO 2 plants
had carbon assimilation rates near zero. Stomatal conductance (gs) measured at the same temperatures
remained almost unaltered in plants grown at elevated
CO 2 whereas in plants grown at ambient CO 2 a significant
decrease in g, (c. 50%) was observed at 45 CC as compared
lo g, at 25 °C (Fig. 3b).
Recovery of A after 30 min at 25 °C was more pronounced in plants grown in elevated CO 2 than in plants
grown at ambient CO 2 . After 24 h the recovery of A and
g, was complete in plants from both treatments
1
.S
Fig. 2. Example of a heat-induced increase in basal fluorescence of
chlorophyll a (Fo) as described in Bilger et al. (1984) for one leaf of
cork oak (Quercus suber L.) of ambient CO2-grown plants (dashed line)
and elevated CO2-grown plants (solid line).
1758 Faria etal.
14
25
3 0 45 45 25
Temperature,°C
25
14O
25
3 0 45 45 25
Temperature, °C
Fig. 3. Changes in (a) net photosynthesis (A), (b) stomatal conductance
(£«) with temperature in leaves of cork oak (Quercus suber L.) plants
grown for 14 months either at ambient CO 2 , at 350 ppm (solid bars) or
elevated CO 2 , at 700 ppm (open bars) with constant light (1300 ^mol
m " 2 s" 1 PAR) and 1.9±0.1 kPa. Measurements were taken for (I*)
25 X , (2*) 30 °C, (3*, 4*), 45 °C, and (5», 6*) 25 °C. Error bars
represent the SE of at least 4 replicates (*, see Fig. 1 for details).
(Fig. 3a, b). The pattern shown in this figure was not
substantially modified when ambient CO2-grown plants
were measured at 700 ppm and elevated CO2-grown
plants were measured at 350 ppm (Table 2).
Plants grown at elevated CO 2 had an / ( m i at 25 °C
c. 40% lower than plants grown at ambient CO 2 (Fig. 4a).
In contrast, the effect of the heat shock treatment on
AmBX measured at 25 °C was more pronounced in plants
grown at ambient CO 2 than in plants grown at elevated
CO 2 (Fig. 4a). On the other hand, Amtx measured at
45 °C in leaves of controls (plants that did not suffer the
4 h 45 °C heat shock) was positive in plants grown at
elevated CO 2 , whereas it was negative in plants grown at
ambient CO 2 (Fig. 4b).
Table 3 shows that plants grown at elevated CO 2 had
about 66% less total Rubisco activity than plants grown
at ambient CO 2 and that after the heat-shock, there was
no decrease in the activity of this enzyme in plants from
either treatment. Immunodetection, after SDS-PAGE, of
the large subunit (LSU) of Rubisco extracted from the
leaves of control and heated plants, indicated that ambient
CO 2 plants (lanes 1C, 3C, 5C, 7H, 9H, and 11H in Fig. 5)
had higher Rubisco contents than plants grown at elevated CO 2 (lanes 2C, 4C, 6C, 8H, 10H, and 12H in Fig. 5).
The comparison of lanes \C (control plants grown at
ambient CO 2 ) with 7H (heated plant grown at ambient
CO 2 ) and 2C (control plant grown at elevated CO 2 ) with
8H (heated plant grown at elevated CO 2 ) also shows that
there was no degradation of the LSU of Rubisco after
the heat-shock in plants from either treatment. The
soluble protein concentration in leaves of elevated CO 2
plants was significantly lower than in plants grown at
ambient CO 2 (Table 1).
The maximal efficiency of excitation energy capture by
open PSII reaction centres (FJFm) did not change under
elevated CO 2 (Fig. 6). After heat shock there was a slight
but significant decrease of the Fv/Fm: 12% for ambient
CO 2 plants and 6% for elevated CO 2 plants (Fig. 6).
Discussion
The fact that plants grown at either ambient or elevated
CO 2 had similar net photosynthesis rates when measured
at their growth CO 2 concentrations (Fig. 3a), suggests
that the development of leaves in elevated CO 2 led to a
down-regulation of photosynthesis in Quercus suber. This
is consistent with the decrease of c. 40% observed in
maximal photosynthetic capacity of leaves grown at elevated CO 2 as compared with leaves grown at ambient
CO2 m 2 s ') and stomatal conductance (g,, mmol H2O in
Table 2. Net photosynthesis
cork oak (Quercus suber L.) leaves grown at ambient CO2 and elevated CO2
2
s ') measured during heat-shock, of
The CO 2 concentration during measurements was 700 ppm for ambient CO2-grown plants and 350 ppm for elevated CO2-grown plants. Mean
values and standard error of 5 replicates.
Temperature
Ambient CO2-grown plants
(700 ppm CO 2 during measurement)
A
25
30
45
45
25
25
10.34±3.41
8.71 ±1.01
-0.23 ±0.04
-0.41 ±0.20
n.d.
11.71±2.17
Elevated CO2-grown plants
(350 ppm CO 2 during measurement)
g,
85.25±9.18
83.54±2.62
46.31 ±6.10
43.13± 11.20
n.d.
106.56± 12.40
10.05 ±0.96
8 12±1.02
0.91 ±0.03
0.91 ±0.05
n.d.
9.48±I.I3
80.13±10.11
73.77±6.42
59.72 ±3.20
60.53 ±6.42
n.d.
86.18±7.01
Elevated C02 and temperature in cork oak
1759
3O
H
C
H
of leaves of cork oak (Quercus
suber L.)
Fig.
capacity
g 4. Photosynthetic
y
p y (,^
Q
) g grown in ambient CO2-grown plants (solid bars) and elevated CO2grown plants (open bars) with a photosynthetic photon flax density (PPFD) of 1200 j*mol m'2 s~l and saturating CO2 (10%): (a) measured at
25 °C in leaves of the control plants (C), consisting of plants that did not suffer the 45 °C heat shock, and heated plants (H), plants after the 45 °C
heat-shock; (b) measured at 45° C in leaves of control plants (C). Error bars represent the SE of at least 4 replicates.
Table 3. Comparison of Rubisco activities (pmol CO2 g 1FW
min~l) of leaves of cork oak (Quercus suber L.) plants after 14
months either at ambient CO2 (350 ppm) or at elevated CO2
(700 ppm)
C = control plants (plants that did not suffer the 45 °C heat-shock);
H = heated plants (plants after the 45 °C heat-shock). Standard error
of at least 5 replicates. Comparison between treatment means was done
using Student's t test. Means significantly different are marked with
*(P<0.01).
0.0
0.8
0.7
o.e
Ambient CO2
Elevated CO2
kDa
58493726-
C
H
5.7±0.71*
1.9±0.32*
6.4 ±1.06*
2.0 ±0.46*
1C 2C 3C 4C 5C 6C
7H 8H9H10H11H 12H
I
Fig. 5. Immunodetection after SDS-PAGE, of the large subunit (LSU)
of Rubisco extracted from leaves of the controls (C), i.e. plants that
did not suffer the 45 °C heat shock, and heated plants (H), plants after
the 45 °C heat-shock, of cork oak (Quercus suber L.) as described by
Besford (1990) using extracts prepared as described by Wilkins el al.
(1994). Lanes 1C, 3C, 5C, 7H, 9H, and 11H were extracted from three
different plants grown at ambient CO2 (350 ppm). Lanes 2C, 4C, 6C,
8H, 10H, and 12H were extracted from three different plants grown at
elevated CO2 (700 ppm).
CO2. This is also consistent with the significant decreases
in the soluble protein, in the activity of Rubisco and in
the amount of the large subunit of Rubisco observed in
plants grown in the CO2-enriched atmosphere. These
results confirm earlier data obtained with tomato plants
(Besford, 1990), spruce trees (van Oosten et al, 1992)
and Prunus avium (Wilkins et al, 1994) indicating that
in some species a reduction in the steady-state amounts
of Rubisco occurs when they are grown in elevated CO2
(Bowes, 1991). One possible explanation for the downregulation of photosynthetic metabolism in response to
elevated CO2 is an inadequate sink strength as compared
to photoassimilate supply (Gunderson and Wullschleger,
o.s
Fig. 6. Photochemical efficiency of PSII (FJF^ in dark-adapted leaves
(30 min) of the control plants (C), consisting of plants that did not
suffer the 45 °C heat shock, and heated plants (H), plants after the
45 °C heat-shock, of cork oak (Quercus suber L.) plants grown for 14
months at ambient CO2 (solid bars) and elevated CO2 (open bars).
Error bars represent the SE of at least 5 replicates.
1994). In the present experiments, the increase in the pool
of total soluble sugars and starch in leaves of plants
grown at elevated CO2 suggests that the down-regulation
of photosynthesis in plants grown at elevated CO2 may
in fact be associated with a low demand for assimilates.
The feedback mechanism responsible for the decrease in
photosynthetic rates under elevated CO2 may include a
phosphate limitation resulting from inadequate triose
phosphate utilization, as described by Harley et al. (1992)
and/or the repression by sugars of transcriptional activity
of nuclear genes coding for chloroplast proteins as suggested by van Oosten and Besford (1994) and van Oosten
etal. (1994).
A loss of chlorophyll content in elevated CO2 plants
has been observed in several species exposed to elevated
CO2 (Besford, 1990) and this has been attributed to an
inhibition of the transcription of cab genes by the accumulation of soluble sugars (van Oosten et al., 1994). In this
experiment the decrease in chlorophyll concentration on
a leaf dry weight basis may be explained by the dilution
effect caused by the accumulation of starch, which resulted
in a decrease in SLA. Therefore, it is unlikely that the
lower chlorophyll concentration corresponded to a real
1760
Faria et al.
loss in chlorophyll from cells, as observed by Besford
(1990).
Stomata of plants grown under elevated CO2 became
less responsive to high temperatures (Fig. 3b). A similar
acclimation to elevated CO2 in the atmosphere was
observed in leaves of Quercus ilex trees growing in a
naturally CO2-enriched site as compared to ambient CO2grown trees (Chaves et al, 1995). The heat shock treatment caused a partial stomatal closure (Fig. 3b) which
may be a direct effect of the high temperature (Hall et al,
1976; Jarvis and Morison, 1981). However, although care
was taken to avoid plant water deficits (by maintaining
the plants well watered and a high relative humidity in
the chamber during the heat shock), the possibility can
not be completely disregarded of a confounding effect of
localized water deficit resulting from increased vapour
pressure difference between leaf and air at 45 °C as
compared to 25 °C (Schulze, 1993). However, changes in
bulk leaf water potential were not expected in those wellwatered plants of Q. suber because the same plants in the
greenhouse had moderate leaf water potentials at midday
(above -1.5 MPa) after more than 5h with a vapour
pressure difference below 1.7 ±0.07 kPa. The vapour pressure difference in the chamber during the heat shock
treatment was 1.9 + 0.1 kPa.
The maintenance of a positive net CO2 exchange rate
in plants grown at elevated CO2 after 4 h at 45 °C as
compared to values close to zero in plants grown at
ambient CO2, in spite of similar leaf stomatal conductance, indicates that stomatal closure was not the major
factor limiting photosynthesis at this high temperature.
The higher net carbon uptake rates in elevated CO2 plants
after heat-shock may result from a lower rate of photorespiration and/or a higher resistance of the photosynthetic
apparatus to heat stress. The latter is consistent with the
positive values of AmBX at 45 °C as compared to the
negative values observed in ambient CO2 plants as well
as with the faster recovery of A in elevated CO2 plants
after the heat shock treatment.
A greater tolerance to heat stress was observed in
elevated CO2-grown plants in spite of a decrease in
quantity and activity of Rubisco in plants from this
treatment. This suggests that Rubisco was not a major
limiting factor of photosynthesis in Q. suber under heat
stress conditions as might have been expected from studies
in other plant species under high temperature and irradiance (Stitt, 1991; Krapp et al, 1993; Stitt and Schulze,
1994). These authors have shown in transformed plants
with diminished amounts of Rubisco that one-sided limitation of photosynthesis by Rubisco can be avoided over
a wide range of growth conditions, but that in extreme
environmental conditions this capacity to adapt is
exhausted. The present data also suggest that Rubisco is
remarkably resistant to heat stress as shown by the
negligible losses in activity and no degradation of the LSU
observed in plants of both CO2 treatments after the
heat shock.
At the photochemical level, the negative effect of the
heat-shock treatment was slightly more pronounced in
ambient CO2 plants, which showed a decrease of 12% in
the maximal efficiency of energy conversion in PSII (given
by FJFm of dark-adapted leaves), as compared to a
decrease of 6% in elevated CO2 plants. This is consistent
with data reported by Long et al. (1992) showing a slight
increase in the maximum photochemical efficiency of PSII
measured at midday (with supra-optimal temperatures
for photosynthesis) in shoots of Scirpus olneyi growing
under elevated CO2 in open-top chambers as compared
with the controls grown at ambient CO2.
It can be speculated that the greater tolerance to high
temperature in plants grown at elevated CO2 may be
related to the stabilization of heat-susceptible enzymes
(Keeling et al, 1993) by the increased amounts of sugars
observed in these plants. Differences induced by growth
under elevated CO2 in the synthesis and/or degradation
of heat-shock proteins, acting as chaperones of key
enzymes of carbon metabolism or playing a role in the
stabilization of grana (Stapel et al, 1993), deserve consideration in future research.
In conclusion, it was apparent that growth in a high
CO2 atmosphere may protect Q. suber leaves from the
effects of high temperature. This was shown by the smaller
short-term (30 min) after-effect of heat shock in elevated
CO2 than in ambient CO2 plants, namely in the rates of
net photosynthesis (measured at 25 °C and 45 °C and
growth CO2 concentrations), AmK% (measured at 25 °C)
and efficiency of excitation energy capture by open photosystem II reaction centres (FJF^. This occurred in
spite of a 'down-regulation' of photosynthesis observed
in plants grown in a CO2-enriched atmosphere and may
reflect some protective mechanisms occurring at the membrane or enzymatic level, possibly linked to the increased
concentration of sugars observed in these leaves.
Acknowledgements
We thank COST 614 for providing the funds for the visit of T
Faria to HRI, UK, and Dr Richard C Leegood for critical
reading of the manuscript. T Faria and M Vaz received MSc
fellowships from JNICT, Lisbon, Portugal. This work was
financed by an EC project, contract Nr EV5V-CT92-0093.
References
Arnon DI. 1949. Copper enzymes in isolated chloroplasts:
polyphenol oxidase in Beta vulgaris. Plant Physiology 24,
1-15.
Besford RT. 1990. The greenhouse effect: acclimation of tomato
plants growing in high CO 2 , relative changes in Calvin cycle
enzymes. Journal of Plant Physiology 136, 458-63.
Bilger W, Schreiber U, Lange OL. 1984. Determination of leaf
Elevated CO2 and temperature in cork oak
resistance: comparative investigation of chlorophyll fluorescence changes and tissue necrosis methods. Oecologia 63,
256-62.
Bowes G. 1991. Growth at elevated CO 2 : photosynthetic
acclimation to elevated CO 2 . Plant, Cell and Environment
14, 795-806.
Bradford MM. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of proteins utilizing the
principle of protein-dye binding. Analytical Biochemistry
11, 248-54.
Chaves MM, Pereira JS, Cerasoli S, Clifton-Brown J, Miglietta
F, Raschi A. 1995. Leaf metabolism during summer drought
in Quercus ilex trees with lifetime exposure to elevated CO 2 .
Journal of Biogeography 22, 255-9.
Demmig-Adams B, Winter K, Krtger A, Czygan F-C. 1989.
Light response of CO 2 assimilation, dissipation of excess
excitation energy, and zeaxanthin content of sun and shade
leaves. Plant Physiology 90, 881-6.
Dijksrra P. 1989. Cause and effect of differences in specific leaf
area. In: Lambers H, Cambridge ML, Konings H, Pons TL,
eds. Causes and consequences of variation in growth rate and
productivity of higher plants. The Hague: SPB Academic
Publishing, 125^0.
Genty B, Briantais JM, Baker NR. 1989. The relationship
between quantum yield of photosynthetic electron transport
and quenching of chlorophyll fluorescence. Biochimica et
Biophysica Ada 990, 87-92.
Gunderson CA, Wullschleger SD. 1994. Photosynthetic acclimation in trees to rising CO 2 : a broader perspective.
Photosynthesis Research 39, 369-88.
Hall AE, Schuize ED, Lange OL. 1976. Current perspectives of
steady-state stomatal responses to the environment. In: Lange
OL, Kappen L, Schuize ED, eds. Water and plant life.
Ecological Studies 19. Berlin: Springer-Verlag, 169-88.
Harley PC, Thomas RB, Reynolds JF, Strain BR. 1992.
Modelling the photosynthesis of cotton grown in elevated
CO 2 . Plant, Cell and Environment 15, 271-82.
Janis PG, Morison JL. 1981. The control of transpiration and
photosynthesis by stomata. In: Jarvis PG, Mansfield TA, eds.
Stomatal physiology. Cambridge University Press, 247-79.
Keeling PL, Bacon PJ, Holt DC. 1993. Elevated temperature
reduces starch deposition in wheat endosperm by reducing
the activity of soluble starch synthase. Planta 191, 342-8.
Krapp A, Hoffman B, Schafer C, Stitt M. 1993. Regulation of
the expression of rbcS and other photosynthetic genes by
carbohydrates: a mechanism of the sink regulation of
photosynthesis. The Plant Journal 3, 817-28.
1761
Krause GH. 1994. The role of oxygen in photoinhibition of
photosynthesis. In: Foyer CH, Mullineaux PM, eds. Causes
of photooxidative stress and amelioration of defense systems in
plants. Florida: CRC Press. 43-76.
Lilley RM, Walker DA. 1974. Carbon dioxide assimilation by
leaves, isolated chloroplasts and ribulose bisphosphate carboxylase from spinach. Plant Physiology 55, 1087-92.
Long SP, Nie GY, Drake BG, Hendrey G, Lewin K. 1992. The
implications of concurrent increases in temperature and CO 2
concentration for terrestrial C3 photosynthesis. In: Murata
N, ed. Research in photosynthesis. The Netherlands: KJuwer
Academic Publishers, 811-17.
Schuize ED. 1993. Soil water deficits and atmospheric humidity
as environmental signals. In: Smith JAC, Grffiths H, eds.
Plant responses to water deficits-from cell to community. SEB
Environmental Plant Biology Series. Oxford: Bios Scientific
Publishers, 129-45.
Stapel D, Kruse E, Kloppstedek K. 1993. The protective effect
of heat shock proteins against photoinhibition under heat
shock in barley (Hordeum vulgare). Journal of Photochemistry
and Photobiology, Series B 21, 211-17.
Stitt M. 1991. Rising CO 2 levels and their potential significance
for carbon flow in photosynthetic cells. Plant, Cell and
Environment 14, 741-62.
Stitt M, Lilley RMcC, Gerhardt R, Heldt HW. 1989.
Determination of metabolite levels in specific cells and
subcellular compartments of plant leaves. Methods in
Enzymology 174, 518-52.
Stitt M, Schuize D. 1994. Does Rubisco control the rate of
photosynthesis and plant growth? An exercise in molecular
ecophysiology. Plant, Cell and Environment 17, 465-87.
van Oosten JJ, Affif D, Dizengremel P. 1992. Long-term effects
of a CO2-enriched atmosphere on enzymes of the primary
carbon metabolism of spruce trees. Plant Physiology and
Biochemistry 30, 541-7.
van Oosten JJ, Besford RT. 1994. Sugar feeding mimics effect
of acclimation to high CO 2 -rapid down-regulation of Rubisco
small subunit transcripts but not of the large subunit
transcripts. Journal of Plant Physiology 143, 306-12.
van Oosten JJ, Wilkins D, Besford RT. 1994. Regulation of the
expression of photosynthetic nuclear genes by CO 2 is
mimicked by regulation by carbohydrates: a mechanism for
the acclimation of photosynthesis to high CO2? Plant, Cell
and Environment 17, 913-23.
Willkins D, van Oosten JJ, Besford RT. 1994. Effects of elevated
CO 2 on growth and chloroplast proteins in Prunus avium.
Tree Physiology 14, 769-79.