Plant, Cell and Environment (2000) 23, 649–656
Growth in elevated CO2 protects photosynthesis against
high-temperature damage
DANIEL R. TAUB,1 JEFFREY R. SEEMANN2 & JAMES S. COLEMAN1
1
Division of Earth and Ecosystem Sciences, Desert Research Institute, 2215 Raggio Parkway, Reno, NV 89512, USA, and
Department of Biochemistry, University of Nevada, Reno, NV 89557, USA
2
ABSTRACT
INTRODUCTION
We present evidence that plant growth at elevated atmospheric CO2 increases the high-temperature tolerance of
photosynthesis in a wide variety of plant species under
both greenhouse and field conditions. We grew plants
at ambient CO2 (~ 360 mmol mol-1) and elevated CO2
(550–1000 mmol mol-1) in three separate growth facilities,
including the Nevada Desert Free-Air Carbon Dioxide
Enrichment (FACE) facility. Excised leaves from both the
ambient and elevated CO2 treatments were exposed to temperatures ranging from 28 to 48 °C. In more than half the
species examined (4 of 7, 3 of 5, and 3 of 5 species in the
three facilities), leaves from elevated CO2-grown plants
maintained PSII efficiency (Fv/Fm) to significantly higher
temperatures than ambient-grown leaves. This enhanced
PSII thermotolerance was found in both woody and herbaceous species and in both monocots and dicots. Detailed
experiments conducted with Cucumis sativus showed that
the greater Fv/Fm in elevated versus ambient CO2-grown
leaves following heat stress was due to both a higher Fm
and a lower Fo, and that Fv/Fm differences between elevated and ambient CO2-grown leaves persisted for at least
20 h following heat shock. Cucumis sativus leaves from
elevated CO2-grown plants had a critical temperature for
the rapid rise in Fo that averaged 2·9 °C higher than leaves
from ambient CO2-grown plants, and maintained a higher
maximal rate of net CO2 assimilation following heat shock.
Given that photosynthesis is considered to be the physiological process most sensitive to high-temperature damage
and that rising atmospheric CO2 content will drive temperature increases in many already stressful environments,
this CO2-induced increase in plant high-temperature
tolerance may have a substantial impact on both the productivity and distribution of many plant species in the 21st
century.
High temperature is a common stress for plants, restricting
growth and productivity (Boyer 1982) and influencing the
distribution of species (Grace 1987). Models of global
climate predict that global mean surface air temperatures
will rise by 1·5–4·5 °C by the middle of the 21st century due
to increased atmospheric concentrations of CO2 and other
trace gases (Manabe 1998; Ramnathan 1998). Extreme
high-temperature events are also anticipated to increase
greatly in frequency (Wagner 1996). Plants are thus likely
to experience increasing high-temperature stress in their
natural communities, especially as the rate of climate
change may exceed the rates at which plant species can
migrate (Davis 1986).
One of the primary results of high-temperature stress is
damage to photosynthetic electron transport. Alexandrov
(1977), reviewing several decades of research, found that
photosynthesis was among the plant functions most sensitive to high-temperature damage. A review by Berry &
Bjorkman (1980), as well as a variety of subsequent studies
(e.g. Havaux 1993a; Heckathorn et al. 1998), have found
electron transport through photosystem II (PSII) to be
the component of photosynthesis most susceptible to
irreversible high-temperature damage.
In addition to affecting climate, elevated atmospheric
levels of carbon dioxide directly influence a variety of plant
processes, particularly growth (Curtis & Wang 1998;
Poorter, Roumet & Campbell 1996) and photosynthesis
(Curtis 1996; Moore et al. 1999). Two recent studies have
suggested that elevated CO2 can also increase the capacity
of PSII to tolerate high-temperature events. Huxman et al.
(1998) found that the maximum photochemical efficiency
of PSII (Fv/Fm) declined substantially in response to a fourday high-temperature event in plants of Yucca whipplei
growing at 360 mmol CO2 mol-1. By contrast, the Fv/Fm of
plants growing at elevated CO2 (700 mmol mol-1) was
largely unaffected throughout the high-temperature treatment. In two other Yucca species, there was no effect of
elevated CO2 on thermotolerance, as measured by Fv/Fm.
In a second study, Faria et al. (1996) found that the capacity of Quercus suber seedlings to withstand a 4 h, 45 °C heat
stress was greater in plants grown at elevated CO2
(700 mmol mol-1) than ambient CO2 (350 mmol mol-1). This
effect was apparent in both Fv/Fm and the maximal rate of
net CO2 assimilation by leaves.
Key-words: chlorophyll fluorescence; elevated CO2; free-air
carbon dioxide enrichment; heat shock; photosystem II;
thermotolerance.
Correspondence: Daniel Taub. Fax: +1 775 673 7485; e-mail:
[email protected]
© 2000 Blackwell Science Ltd
649
650 D. R. Taub et al.
In both the experiments of Huxman et al. (1998) and
Faria et al. (1996), heat stress was applied in the light. Their
results therefore do not unambiguously demonstrate
whether growth at elevated CO2 was protective against the
direct effects of high temperature per se, or instead mitigated against high-temperature-induced photo-inhibition
(Bongi & Long 1987; Koniger, Harris & Pearcy 1998). To
more directly examine the effect of growth CO2 levels on
PSII high-temperature tolerance, we conducted several
experiments examining the responses of a variety of plants
grown at different CO2 levels to heat stress in the dark. To
assess the generality of our findings, these experiments
included studies on a variety of herbaceous and woody
plant species grown in three separate elevated-CO2 growth
facilities, including plants grown in elevated CO2 under
otherwise natural conditions at the Nevada Desert FreeAir Carbon Dioxide Enrichment (FACE) facility.
MATERIALS AND METHODS
Growth conditions
Plants were grown in three separate CO2-controlled plant
growth facilities: naturally lit growth chambers at the
Desert Research Institute, Reno, Nevada; the Nevada
Desert FACE Facility, Nye County, Nevada; and a
glasshouse at the University of Nevada, Reno. For the
naturally lit growth chambers, CO2 levels were
350 mmol CO2 mol-1 (ambient) and 750 mmol CO2 mol-1
(elevated), and two chambers of each CO2 level were used.
Day/night growth temperatures were 28/18 °C; maximum
photosynthetic photon flux density was approximately
1600 mmol photons m-2 s-1. Plants were grown from seed
in a commercial potting mixture (Supersoil, Rod McLellan
Co., San Mateo, California, USA) and fertilized daily with
quarter-strength Plantex 15-15-18 fertilizer (Plantco,
Ontario, Canada). Species examined were Cucumis sativus
L. c.v. Poinsett 76, Glycine max (L.) Merr. c.v. Williams,
Gossypium hirsutum L. c.v. DPL-77, Hordeum vulgare L.
c.v. Poco, Nicotiana sylvestris Speg. & Comes, Phaseolus
vulgaris L. c.v. Little Linden, and Triticum aestivum L. c.v.
Yamhill. Depending on the species, plants were between
26 and 38 days old at the time of the high-temperature
experiments.
Plants at the FACE site are naturally occurring vegetation surrounded by 25 m diameter rings fumigated with
ambient (~ 360 mmol CO2 mol-1) or CO2-enriched air
(550 mmol CO2 mol-1; n = 3 rings per treatment). Species
examined were Achnatherum hymenoides (Roemer & J.A.
Schultes) Barkworth, Ambrosia dumosa (Gray) Payne,
Baileya multiradiata Harvey & Gray ex Gray, Larrea
tridentata (Sesse & Moc. ex DC.) Coville, and Lycium
pallidum Miers. Leaves were tested for thermotolerance of
PSII on 19–20 May 1999, and for L. tridentata, on 3 March
1999 as well. Mean daily minimum/maximum temperatures
were – 6·9/21·0 °C for the two weeks prior to 3 March and
5·5/29·3 °C for the two weeks prior to 20 May. Technical
details of the FACE facility can be found in Jordan et al.
(1999).
Glasshouse CO2 levels were 1000 mmol CO2 mol-1 (elevated) and 360 mmol CO2 mol-1 (ambient). Day/night temperatures were 27/16 °C; maximum photosynthetic photon
flux density was approximately 2000 mmol photons m-2 s-1.
Plants were grown from seed in supersoil and fertilized
daily with Peters Professional 15-16-17 fertilizer (Scotts,
Allentown, Pennsylvania, USA). Species examined were
Beta vulgaris L. c.v. Early Wonder, Brassica oleracea var.
Botrytis L. c.v. Amazing, Cucumis sativus L. c.v. Poinsett 76,
Cucurbita pepo L. c.v. Baby Bear and Lycopersicon
pimpinellifolium (Jusl.) P. Mill. Depending on the species,
plants were between 26 and 37 days old at the time of the
high-temperature experiments.
Determination of thermotolerance of PSII
efficiency (Fv/Fm)
Heat treatments were performed on 20 mm diameter leaf
discs (dicots) or leaf segments (grasses); for microphyllous
desert species at the NDFF entire leaves were used; the
species examined are listed in Table 1. Discs or segments
cut from a single leaf were treated across the entire range
of temperatures used for temperature curves where the size
of the leaf allowed. For microphyllous desert shrub species,
leaves from a single terminal branch were treated across
the range of temperatures. Leaves, leaf discs or leaf segments were floated on distilled water in test tubes set in
temperature-controlled water baths; the water in the baths
and test tubes was at the set-point when the leaves were
added. Heat treatments were performed in the dark. Duration of heat treatments varied from 1 to 4 h, depending
on the experiment. Experiments with an extended period
of room-temperature treatment following the heat stress
were performed with leaf discs of Cucumis sativus
and Nicotiana sylvestris. For these experiments, discs
were kept in the dark in moist paper towels between
measurements.
Maximal PSII efficiency of light capture was determined
as Fv/Fm, where Fv = (Fm - Fo)/Fm and Fm and Fo are the
maximal and initial fluorescence yield, respectively, of a
dark-adapted leaf. For plants grown in the naturally lit
chambers and glasshouse, this was measured with a
PAM 101 fluorometer (Walz) with a saturating light
pulse provided by a xenon lamp (ILC Technology Model
R300-4). For plants at the Nevada Desert FACE facility,
Fv/Fm was determined with an FMS 2 fluorimeter system
(Hansatech Instruments, Kings Lynn, Norfolk, UK), using
the unit’s internal light sources.
Determination of the critical temperature for
rapid rise in Fo
We utilized the Fo–temperature curve technique of
Schreiber & Berry (1977) as an additional assay of PSII
thermotolerance. This technique has been widely used to
determine the temperature at which irreversible damage to
PSII occurs (e.g. Bilger, Schreiber & Lange 1984; Havaux
1993b; Koniger et al. 1998; Rekika, Monneveux & Havaux
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 649–656
Elevated CO2 and high-temperature tolerance 651
Table 1. Comparison of PSII thermotolerance for plant species grown in elevated and ambient CO2
Species
Plant type
n
T50 in elevated
CO2 (°C)
T50 in ambient
CO2 (°C)
Plants grown in naturally lit growth chambers at 750 (elevated) and 350 (ambient) ppm CO2
Cucumis sativus
Agricultural dicot
4
40·47 ± 0·21
37·91
Glycine max
Agricultural dicot
4
42·14 ± 0·21
40·80
Gossypium hirsutum
Agricultural dicot
4
40·73 ± 0·28
40·39
Hordeum vulgare
Agricultural monocot
4
37·40 ± 0·13
37·30
Nicotiana sylvestris
Agricultural dicot
4
41·56 ± 0·36
40·43
Phaseolus vulgaris
Agricultural dicot
4
38·16 ± 0·25
37·99
Triticum aestivum
Agricultural monocot
4
38·22 ± 0·26
37·39
Difference in T50
(elevated – ambient) (°C)
± 0·60
± 0·41
± 0·26
± 0·37
± 0·64
± 0·35
± 0·11
2·56**
1·34*
0·34 (ns)
0·10 (ns)
1·13*
0·17 (ns)
0·83*
Plants growing naturally at Nevada Desert Face facility at 550 (elevated) and 360 (ambient) ppm CO2
Ambrosia dumosa
Deciduous shrub
5
44·76 ± 0·39
43·63 ± 0·29
Achnatherum hymenoides
Perennial grass
4
44·47 ± 0·19
44·02 ± 0·15
Baileya multiradiata
Perennial forb
5
43·23 ± 0·62
44·50 ± 0·71
Lycium pallidum
Deciduous shrub
5
44·27 ± 0·18
42·63 ± 0·19
Larrea tridentata (May 20)
Evergreen shrub
4
46·18 ± 0·20
45·69 ± 0·26
Larrea tridentata (March 3)
Evergreen shrub
6
45·22 ± 0·33
44·62 ± 0·07
1·13*
0·45#
-1·35 (ns)
1·65***
0·50#
0·60*
Plants grown in glasshouse at 1000 (elevated) and 370 (ambient) ppm CO2
Beta vulgaris
Agricultural dicot
5
38·61 ± 0·24
Brassica oleracea
Agricultural dicot
5
38·31 ± 0·25
Cucumis sativus
Agricultural dicot
4
43·00 ± 0·26
Cucurbita pepo
Agricultural dicot
6
41·85 ± 0·32
Lycopersicon pimpinellifolium
Agricultural dicot
4
39·91 ± 0·22
38·03
37·84
41·38
40·88
39·05
± 0·20
± 0·34
± 0·33
± 0·21
± 0·36
0·59#
0·47 (ns)
1·63**
0·97*
0·86*
Values shown are the temperatures (mean ± standard error) that caused a 50% decrease in the maximal efficiency of PSII (Fv/Fm) relative
to non-heat-stressed controls (T50). n, number of leaves for each treatment, each from a separate plant. Significance levels: ns, P > 0·10;
# 0.10 > P > 0·05; * 0.05 > P > 0·01; ** 0.01 > P > 0·001; *** 0.001 > P
1997). The critical temperature (Tcrit) for PSII damage
obtained by this technique has been shown in a variety of
species to correspond closely to the temperature at which
the capacity for photosynthetic fixation as measured by gas
exchange becomes unstable, declining with continued
exposure to a constant temperature (Seemann, Berry &
Downton 1984). This assay was used for plants of Cucumis
sativus grown in naturally lit growth chambers. A 20 mm
diameter leaf disc (dark-adapted for 45 min prior to
measurement) was placed in a specially constructed waterjacketed brass chamber with a glass window through which
chlorophyll fluorescence could be monitored. Leaves were
heated at 1 °C min-1 by increasing the temperature of the
water flowing through the chamber jacket, while initial
chlorophyll fluorescence (Fo) was continuously monitored
with the measuring beam of a PAM 101 fluorometer (Walz,
Effeltrich, Germany). Leaf temperature was monitored
with a type t thermocouple appressed to the underside of
the leaf and a LI-1000 data logger (Li-Cor, Lincoln,
Nebraska, USA). Tcrit was determined as the temperature
at the point of intersection of lines fitted to the linear portions of the fluorescence curve, as in Fig. 4.
Determination of the maximal rate of net CO2
assimilation (Amax)
Leaves of Cucumis sativus from plants grown in the naturally lit growth chambers were separated at the mid-vein
and the portions on either side were floated in the dark on
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 649–656
beakers of distilled water in water baths at 28 °C and 40 °C.
Immediately following heat treatment for 1 h, Amax was
measured at a photosynthetic photon flux density of
1600 mmol photons m-2 s-1, 1900 mmol CO2 mol-1 and leaf
temperature of 28 °C using an LI-6400 photosynthesis
system (Li-Cor, Lincoln, Nebraska, USA). To prevent
desiccation of leaves during measurement, the air stream
entering the leaf chamber was humidified, and the measurements made rapidly. The values obtained by this
method should be regarded as an approximate Amax; values
obtained by gas exchange on intact leaves were approximately 3 mmol CO2 m-2 s-1 higher than those reported here.
RESULTS
There was a striking difference in the response to the heat
shock between Cucumis sativus leaves grown at ambient
and elevated CO2 (Fig. 1). PSII efficiency (Fv/Fm) was substantially reduced in ambient leaves treated at 36 °C relative to 28 °C controls, and further declined with each
increment in temperature up to 42 °C. By contrast, Fv/Fm
in leaves grown in elevated CO2 did not decline substantially even at 38 °C, and was greater in elevated than
ambient CO2 leaves following treatment at all temperatures from 36 to 40 °C. To facilitate interspecies comparison, we estimated the temperature at which Fv/Fm declined
to 50% of its control (28 °C) value (T50) by interpolation
between observed data points. For Cucumis sativus, T50
values of the leaves of plant grown at ambient CO2 aver-
652 D. R. Taub et al.
Fm than ambient-grown leaves following heat treatment
(Fig. 2b,c).
Differences between elevated and ambient leaves in
terms of Fv/Fm following heat shock also persisted during
recovery from heat shock. Figure 3(a) shows a significantly
higher Fv/Fm in elevated than ambient leaves of Cucumis
sativus measured immediately following heat treatment.
Figure 3(b) shows Fv/Fm for the same leaves following a
20 h recovery period. Some recovery of PSII function is
evident for both ambient and elevated leaves, but the differences between them persist, and are of approximately
the same magnitude as immediately following heat treat-
Figure 1. Maximal efficiency of PSII electron transport (Fv/Fm)
in leaves of Cucumis sativus following 4 h treatment in the dark
at the indicated temperature. Leaves were from plants grown at
350 and 750 mmol mol-1 CO2. Each point shows the mean and
standard error of four leaves (from four separate plants).
aged 37·9 °C, while those from plants grown at elevated
CO2 averaged 40·5 °C, an increase in temperature tolerance
of 2·6 °C.
The results of similar experiments for a variety of plant
species are shown in Table 1. As indicated, these experiments were conducted on plants grown in several different
facilities, and the levels of CO2 used in the elevated CO2
treatments varied from 550 to 1000 mmol CO2 mol-1.
Nonetheless, an elevated CO2-associated enhancement of
PSII thermostability was observed in more than half the
species tested in each facility, and at each level of elevated
CO2. The average increase in thermostability in the elevated CO2 treatments was 0·92, 0·50 and 0·90 °C across all
species for the naturally lit growth chambers, the FACE site
and the greenhouse, respectively. Averaged across only
those species which showed a significant increase in thermotolerance, the mean thermotolerance increase was 1·5,
1·1 and 1·2 °C for the naturally lit growth chambers, the
FACE site and the greenhouse, respectively.
This effect was also consistently found using a wide
variety of heat-treatment protocols. We heat-treated leaves
floating on water with either the abaxial or the adaxial side
down, and also within vials so that the discs remained dry,
with results similar to those shown in Table 1. We also performed the experiments both with leaves taken directly
from their well-lit growing conditions, and with leaves darkadapted for up to 1 h before heating (data not shown), with
results similar to those shown in Fig. 1 and in Table 1.
The difference between elevated and ambient-grown
leaves in quantum yield of PSII was apparent within 10 min
of the onset of thermal stress (Fig. 2a), suggesting that the
factors protecting PSII in elevated CO2-grown leaves are
either present prior to the initiation of heat stress, or are
very rapidly induced. The difference between elevated and
ambient leaves was not simply a matter of heat-induced
fluorescence quenching in ambient-grown leaves, since
elevated CO2-grown leaves had both lower Fo and higher
Figure 2. Time course of changes in maximal efficiency of PSII
(Fv/Fm) and initial (Fo) and maximal chlorophyll fluorescence
(Fm) in leaves of Cucumis sativus heated at 40 °C. Leaves are
from plants grown at 350 or 750 mmol mol-1 CO2. Points
represent the mean and standard error of four leaves (from four
separate plants).
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 649–656
Elevated CO2 and high-temperature tolerance 653
Figure 3. Maximal efficiency of PSII (Fv/Fm) in leaf discs of
Cucumis sativus treated at 28 or 40 °C for 4 h. (a) Immediately
following heat treatment. (b) The same discs following 20 h
recovery at room temperature. Leaves are from plants grown at
350 or 750 mmol mol-1 CO2. Bars represent mean and standard
error of four leaves (from four separate plants).
ment. We have obtained similar results with leaves of Nicotiana sylvestris following a 24 h post-heat stress recovery
period (data not shown).
Critical temperatures for thermal damage to PSII
obtained from Fo–temperature curves averaged nearly
3 °C higher for Cucumis sativus leaves from plants grown
in elevated CO2 than in leaves from plants grown at
ambient CO2 (Fig. 4). This result corresponds well with the
thermotolerance difference observed between elevated and
ambient CO2-grown Cucumis sativus plants as assessed by
Figure 5. Maximal net photosynthetic rates (Amax) for leaves of
Cucumis sativus following a 1 h treatment at 28 or 40 °C. Leaves
were from plants grown at 750 or 350 mmol mol-1 CO2. Amax was
measured at 28 °C, 1900 ppm CO2 and a photosynthetic photon
flux density of 1600 mE m-2 s-1. Each bar shows the mean and
standard error of four leaves (from four separate plants). Means
labelled with different letters differ significantly at a = 0·05.
the treatment temperature resulting in a 50% decline in
Fv/Fm (Table 1).
The enhancement of photosynthetic thermotolerance by
elevated CO2 was also seen in the response of leaf CO2
uptake (Fig. 5). Following a non-damaging control temperature treatment (28 °C), ambient CO2-grown Cucumis
sativus leaves had significantly higher CO2 assimilation
rates than elevated CO2-grown leaves (Fig. 5), most likely
reflecting down-regulation of Calvin cycle enzymes in elevated CO2 plants, as is observed in many plant species
(Moore et al. 1999). Heat-stress treatment of ambient
CO2 leaves (40 °C for 1 h) resulted in a 90% decline in net
assimilation relative to 28 °C controls, while elevated CO2
leaves suffered a much less drastic decline of 43%.
DISCUSSION
Figure 4. Representative fluorescence traces for the
determination of the critical temperature (Tcrit) for PSII damage.
A leaf disc is heated at ~ 1 °C min-1 while initial fluorescence
(Fo) is measured with a weak measuring beam. Leaf temperature
is measured with a thermocouple appressed to the leaf surface.
The threshold temperature is determined by the temperature at
the point of intersection of lines fitted to the linear portions of
the curves, as shown. Representative curves are shown for leaves
of Cucumis sativus grown at 350 and 750 mmol mol-1 CO2. No
significance should be inferred for the relative height of the two
curves, which is arbitrary. The insert shows mean values for Tcrit
for leaves of Cucumis sativus grown at 350 and 750 mmol mol-1
CO2.
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 649–656
Several important conclusions can be drawn as a result of
these experiments. First, the elevated CO2-associated
enhancement of PSII high-temperature tolerance seen in
various species appears to be real and not an experimental
artefact. This conclusion is based on both the fact that CO2enhanced thermotolerance has been seen in plants grown
in elevated CO2 in three separate facilities (Table 1; five
facilities if one includes the results of Faria et al. 1996 and
Huxman et al. 1998), and that similar results were found
using a variety of experimental protocols for exposure
of leaves and plants to high temperature. This CO2
enhancement of high-temperature tolerance was also discernable using several different measures: quantum efficiency of PSII, the critical temperature for fluorescence rise,
and gas-exchange measurement of photosynthetic CO2
assimilation.
654 D. R. Taub et al.
The thermotolerance-enhancing effect of elevated CO2
also appears to be widespread and common among plant
species, although apparently not ubiquitous. We have found
the effect in both a monocot (Triticum aestivum, Table 1;
also Yucca whipplei in Huxman et al. 1998) and several dicot
species (Table 1; also Faria et al. 1996), including annual agricultural species (Cucumis sativus, Cucurbita pepo, Glycine
max, Nicotiana sylvestris, Triticum aestivum), deciduous
shrubs (Ambrosia dumosa, Lycium pallidum) and an evergreen shrub (Larrea tridentata); the similar effect found by
Faria et al. (1996) was for a tree species (Quercus suber). It
also appears that this CO2 enhancement of PSII thermotolerance is relevant to plants growing in their natural environment, as this effect was found for several species at the
Nevada Desert FACE facility (NDFF) in southern Nevada
(Table 1). That a CO2 thermotolerance effect has been seen
for several species at the NDFF, under essentially natural
conditions, against the full background of ecological
processes and environmental and genetic variation, strongly
suggests that this effect is truly relevant to the elevated CO2
conditions that will occur in natural ecosystems in the near
future. In short, the enhancement of thermotolerance in the
machinery of PSII is a phenomenon that is genuine,
common among plant species, and relevant to natural circumstances of plant growth.
Although the levels used for the elevated CO2 treatments differed greatly at our three experimental facilities
(550, 750 and 1000 mmol CO2 mol-1), we did not see any
clear differences among the sites in the magnitude of
thermotolerance enhancement. Considering the different
species studied, and the differences in growth conditions
(e.g. light levels, temperature, nutrient availability) at the
three facilities, it is not possible to draw conclusions about
the dose-dependence of CO2 effects on thermotolerance.
Experiments with individual species grown across a CO2
gradient under otherwise uniform conditions will be
needed to address this question.
In contrast to our findings, two previous studies have
found slightly lower values of Fv/Fm in leaves of several
Eucalyptus species exposed to high temperatures in elevated versus ambient CO2 (Roden & Ball 1996a,b). In both
these studies, exposure to high temperatures was over a
period of 8 weeks, suggesting that the effect of elevated
CO2 on long-term acclimatization of PSII to chronic exposure to high temperatures might differ from its effect on
tolerance of acute heat shock.
Virtually nothing is known of the mechanism(s) responsible for increased PSII thermotolerance in plants grown at
elevated CO2. We propose that the factors responsible for
this phenomenon are likely to be a subset of those that have
been shown to protect PSII in plants acclimatized to hightemperature conditions, or to be associated with inter- or
intra-specific variation in PSII thermostability. Several such
factors have been identified, including production of a
chloroplast-localized small heat shock protein (ChSmHSP;
Heckathorn et al. 1998; Stapel, Kruse & Kloppstech 1993);
increases in thylakoid membrane lipid fatty acid saturation (Hugly et al. 1989; Pearcy 1978; Thomas et al. 1986);
increased solute concentrations in the chloroplast stroma
(Santarius 1973; Seemann, Downton & Berry 1986;
Williams, Brain & Dominy 1992); increased levels of the
carotenoid pigment zeaxanthin (Gruszecki & Strzalka
1991; Havaux 1998; Havaux & Gruszecki 1993; Tardy &
Havaux 1997); and emission of the hydrocarbon isoprene
(Sharkey & Singsaas 1995; Singsaas et al. 1997) (although
see Logan & Monson, 1999).
There is little evidence as to whether any of these factors
are influenced by growth CO2 levels. No published study
has investigated the expression of any plant heat shock
protein under elevated CO2. The only study we are aware
of that examined the effect of growth at elevated CO2 on
lipid composition found increased saturation of some
classes of thylakoid lipids, but decreased saturation of
others (Williams et al. 1998). To our knowledge, the only
studies to have reported zeaxanthin contents in plants
grown at elevated CO2 are those of Roden and colleagues
using several Eucalyptus species (Roden & Ball 1996a,b;
Roden, Egerton & Ball 1999). These studies did not find
statistically significant differences between elevated and
ambient CO2 plants for either the total quantity of xanthophyll cycle pigments (violaxanthin (V) + anteraxanthin
(A) + zeaxanthin (Z)) or for the proportion of deepoxidated pigments (A + Z/V + A + Z) in any of the
species.
Both increases and decreases in isoprene emission have
been reported for plants grown under elevated CO2
(Sharkey, Loreto & Delwiche 1991;Tognetti et al. 1998). Isoprene, however, is not a likely source of the CO2 enhancement of thermotolerance we observed in our experiments.
Isoprene, as a volatile compound, has no pools or storage
in the leaf, and its concentration in leaves closely tracks
current synthesis (Monson et al. 1991; Sharkey & Singsaas
1995). All studies agree that isoprene production, concentration and emission are light-dependent (Loreto &
Sharkey 1990; Monson et al. 1991; Sharkey & Loreto 1993;
Wildermuth & Fall 1996), ceasing rapidly with the onset of
darkness (Monson et al. 1991). As the experimental data we
have presented were all obtained on leaves heat-stressed in
the dark, it is very unlikely that these leaves would have
contained isoprene during the heat treatment. In addition,
isoprene is produced in substantial quantities almost exclusively by woody plants (Harley, Monson & Lerdau 1999),
and we have observed CO2 enhancement of thermotolerance in several herbaceous species (Table 1). In particular,
two of the species in which we have found this effect
(Glycine max and Triticum aestivum) have been confirmed
to emit only extremely small quantities of isoprene (Evans
et al. 1982) produced non-enzymatically (Manuel Lerdau,
personal communication). These quantities are several
orders of magnitude below those required for thermal
protection of PSII (Singsaas et al. 1997).
A connection between elevated CO2 and increased levels
of chloroplast solutes is better documented than for any of
the other proposed mechanisms. Growth at elevated CO2
effects profound alterations in cellular and subcellular
concentrations of many soluble compounds (Poorter et al.
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 649–656
Elevated CO2 and high-temperature tolerance 655
1997), particularly sugars, sugar alcohols and other photosynthetic metabolites (Cheng, Moore & Seemann 1998;
Moore, Palmquist & Seemann 1997; Sicher & Kremer
1996), and Ferris & Taylor (1994) found that total osmotic
concentration increased in the leaves of several species
grown in elevated CO2. However, there is no direct evidence of an association between increases in particular
solutes, or in total solute concentration, and the enhancement of thermotolerance in plants grown in elevated CO2.
There is clearly insufficient evidence at the moment to
link any of these putative mechanisms to the phenomenon
of enhancement of PSII thermotolerance at elevated CO2,
although each deserves careful examination to determine
its possible role. There is also no evidence to suggest
reasons for the differences observed among species in the
response of PSII thermotolerance to elevated CO2. Such
differences among species might prove important in determining the response of plant communities to an increased
prevalence of high-temperature events in the elevated CO2
world of the near future.
ACKNOWLEDGMENTS
Craig Biggart, Terri Charlet, Travis Huxman, Dean Jordan,
Annette Risley, David Schorran, Liz Sotoodeh, Dianne
Stortz-Lintz and Stephen Zitzer provided valuable assistance in various aspects of this work. Grant Cramer,
Charley Knight, Manuel Lerdau, Michael Loik, Heiko
Lokstein, Tom Owens, David Wolfe and a host of people at
the 1999 Annual Meeting of the Ecological Society of
America provided helpful discussion of the work in
progress. This work was supported by National Science
Foundation grant IBN-9357302 to J.S.C.
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Received 23 September 1999; received in revised form 20 January
2000; accepted for publication 20 January 2000
© 2000 Blackwell Science Ltd, Plant, Cell and Environment, 23, 649–656