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Global Change Biology (1999) 5, 857±867

Comparative responses of model C3 and C4 plants to

drought in low and elevated CO2
J O Y K . W A R D , * 1 D A V I D T . T I S S U E , ² R I C H A R D B . T H O M A S ³ and
BOYD R. STRAIN*
*Department of Botany, Duke University, Durham, NC 27708, USA, ²Texas Technical University, Lubbock, TX 79409-3131,
USA, ³West Virginia University, Morgantown, WV 26506, USA

Abstract
Interactive effects of CO2 and water availability have been predicted to alter the com-
petitive relationships between C3 and C4 species over geological and contemporary
time scales. We tested the effects of drought and CO2 partial pressures (pCO2) ranging
from values of the Pleistocene to those predicted for the future on the physiology and
growth of model C3 and C4 species. We grew co-occurring Abutilon theophrasti (C3)
and Amaranthus retro¯exus (C4) in monoculture at 18 (Pleistocene), 27 (preindustrial),
35 (current), and 70 (future) Pa CO2 under conditions of high light and nutrient avail-
ability. After 27 days of growth, water was withheld from randomly chosen plants of
each species until visible wilting occurred. Under well-watered conditions, low pCO2
that occurred during the Pleistocene was highly limiting to C3 photosynthesis and
growth, and C3 plants showed increased photosynthesis and growth with increasing
pCO2 between the Pleistocene and future CO2 values. Well-watered C4 plants exhib-
ited increased photosynthesis in response to increasing pCO2, but total mass and leaf
area were unaffected by pCO2. In response to drought, C3 plants dropped a large
amount of leaf area and maintained relatively high leaf water potential in remaining
leaves, whereas C4 plants retained greater leaf area, but at a lower leaf water potential.
Furthermore, drought-treated C3 plants grown at 18 Pa CO2 retained relatively greater
leaf area than C3 plants grown at higher pCO2 and exhibited a delay in the reduction
of stomatal conductance that may have occurred in response to severe carbon limita-
tions. The C4 plants grown at 70 Pa CO2 showed lower relative reductions in net
photosynthesis by the end of the drought compared to plants at lower pCO2, indicat-
ing that CO2 enrichment may alleviate drought effects in C4 plants. At the Pleistocene
pCO2, C3 and C4 plants showed similar relative recovery from drought for leaf area
and biomass production, whereas C4 plants showed higher recovery than C3 plants at
current and elevated pCO2. Based on these model systems, we conclude that C3 spe-
cies may not have been at a disadvantage relative to C4 species in response to low
CO2 and severe drought during the Pleistocene. Furthermore, C4 species may have an
advantage over C3 species in response to increasing atmospheric CO2 and more fre-
quent and severe droughts.
Keywords: Abutilon theophrasti, Amaranthus retro¯exus, C3 species, C4 species, carbon dioxide,
climate change, drought, Pleistocene
Received, resubmitted and accepted 19 December 1998

Correspondence: Dr Joy K. Ward, University of Utah, Depart-
ment of Biology, 257 South, 1400 East, Salt Lake City, UT 84112-
0840, USA, tel. + 1/801 5815927
1
Present address: University of Utah, Department of Biology,
257 South, 1400 East, Salt Lake City, UT 84112-0840, USA
Introduction
Before the Industrial Revolution (120 years ago), the
atmospheric CO2 partial pressure (pCO2) was » 27 Pa
(Barnola et al. 1987) and is projected to increase from the
current value of 36 Pa to 70 Pa CO2 before the end of the
21st century. Furthermore, global circulation models

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858 J . K . W A R D et al.
have predicted that some regions will have increases in
the frequency and severity of droughts, whereas other
regions will have higher precipitation as a result of
increased concentrations of greenhouse gases (Rind et al.
1990; Dirmeyer & Shukla 1996). Such climatic changes
may result in regional shifts in vegetation types and a
global redistribution of net primary production in the
future (Neilson & Marks 1994). On the other hand,
Vostok ice core data have indicated that pCO2 during the
late Pleistocene was as low as 18 Pa during glacial
maxima and ranged between 25 and 28 Pa CO2 during
interglacial periods (Barnola et al. 1987; Jouzel et al. 1993).
Furthermore, lower amounts of precipitation have been
predicted to occur during glacial maxima compared to
the present as indicated by higher amounts of dust
within portions of the ice core corresponding to glacial
periods (Yung et al. 1996).
Many studies have compared the responses of plants
with C3 and C4 photosynthetic pathways to atmospheric
pCO2 of the past (Polley et al. 1992; Polley et al. 1994;
Dippery et al. 1995; Sage 1995; Tissue et al. 1995) and
future (Coleman & Bazzaz 1992; Arp et al. 1993; Dippery
et al. 1995). Generally, C3 species show enhanced net
photosynthesis and growth with increasing pCO2
(Ceulemans & Mousseau 1994; Curtis 1996), whereas
C4 species are generally less affected by increasing pCO2
because of a lower CO2-saturation point for photosynth-
esis (Sage 1994). It has been predicted therefore that C3
species may have a competitive advantage over C4
species in the future as atmospheric pCO2 continues to
rise (Arp et al. 1993). On the other hand, at the low pCO2
of the Pleistocene, C4 species are expected to have an
advantage over C3 species as a result of the CO2-
concentrating mechanism in C4 plants that limits photo-
respiration and increases carboxylation ef®ciency. C3
plants grown at 20 Pa CO2 or lower show strong evidence
of stress such as large reductions in net photosynthesis
(Polley et al. 1993a, 1993b; Tissue et al. 1995; Beerling
1996) and biomass production that is reduced by as
much as 92% relative to that of plants grown at the
current pCO2 (Dippery et al. 1995). Low pCO2 has also
been shown to reduce or prevent reproduction in C3
species, whereas growth and reproduction of C4 species
are generally unaffected by low pCO2 (Allen et al. 1991;
Dippery et al. 1995; Ward & Strain 1997).
Interactive effects of CO2 and water availability may
alter the relative performance of C3 and C4 species. At
current pCO2, C4 species (particularly dicots) generally
require less water than C3 species because of higher CO2
uptake rates and greater stomatal resistance to water loss
(Stowe & Teeri 1978; Wentworth 1983; Ehleringer et al.
1997). Under conditions of elevated CO2 and drought, C3
species may be more competitive than C4 species as a
result of increased CO2 uptake rates coupled with greater
stomatal resistance that reduces water loss (Rogers et al.
1984; Tolley & Strain 1985; Marks & Strain 1989; Tyree &
Alexander 1993; Tschaplinski et al. 1995; HaÈttenschwiler
et al. 1997). It would be expected that reducing pCO2
below the current value in combination with the
imposition of drought will be more detrimental to C3
species than to C4 species, because reductions in
stomatal resistance would be necessary to regulate
carbon assimilation in C3 species, but would greatly
facilitate water loss (Baker et al. 1990; Polley et al. 1993a;
Polley et al. 1995; Ehleringer et al. 1997).
Here we report on the responses of C3 and C4 model
systems to the interactive effects of CO2 and water
availability to gain a more detailed understanding of the
competitive relationships between C3 and C4 species
during the Pleistocene and in response to future climate
change scenarios. The model system, involving co-
occurring annuals, has been used extensively for CO2
studies in the past (e.g. Bazzaz et al. 1989; Coleman &
Bazzaz 1992; Dippery et al. 1995; Tissue et al. 1995). We
hypothesized that low pCO2 and drought would affect C3
plants more negatively than C4 plants as a result of
greater stomatal opening, which would increase carbon
assimilation initially, but would also increase water loss in
C3 plants causing an eventual inhibition of photosynth-
esis. Furthermore, we postulated that C3 plants would be
less affected by drought in elevated CO2 because of
decreased water loss through transpiration and higher
carbon assimilation that would decrease the relative
advantage of C4 plants under drought conditions.
Materials and methods
Growth conditions
Seeds of Abutilon theophrasti (C3 dicot) and Amaranthus
retro¯exus (C4 dicot) were planted in 3.5-L plastic pots in
a 3 :3:1 (v/v) medium of turface, gravel, and sterilized
topsoil. Pots were distributed evenly and randomly
among four growth chambers at the Duke University
Phytotron (64 pots per species per chamber). Chambers
were maintained at pCO2 of 18 6 1 Pa (Pleistocene),
27 6 1 Pa (preindustrial), 35 6 3 Pa (current) and
70 6 4 Pa (predicted future). The CO2 partial pressures
were automatically monitored and controlled by infrared
gas analysis and by CO2 injection (Hellmers & Giles
1979). The 15 and 27 Pa CO2 chambers were scrubbed of
excess CO2 by passing chamber air over a mixture of
moist soda lime and vermiculite. Day/night tempera-
tures were 28/22 °C and the photoperiod was 14 h.
Photosynthetic photon ¯ux density (PPFD) during the
light period was maintained at 1000 6 50 mmol m±2s±1
with sodium vapor and metal halide lamps. Day/night
relative humidity was » 70/90%.
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RESPONSE OF C3 AND C4 PLANTS TO CO2 AND DROUGHT
Before seedling emergence, pots were watered to
saturation with de-ionized water twice daily. Following
emergence, seedlings were thinned to one per pot, and
all pots were watered to saturation with half-strength
Hoagland's solution (Downs & Hellmers 1978) each
morning and with de-ionized water each afternoon for
the next 27 days. Thereafter, water was withheld from
half of the plants of each species, chosen at random to
receive the drought treatment within each of the four
growth chambers. When drought-treated plants became
visibly wilted, they were re-watered in the same manner
as before the drought treatment.
Plant measurements
Total plant mass and total leaf area, which was measured
with an LI-3100 leaf area meter (Li-Cor, Inc., Lincoln,
NE), were determined for drought-treated and well-
watered (control) plants before the drought (27 days after
planting), at the end of drought (30±31 days after
planting), and 7 days after recovery from drought (37±
38 days after planting). Plant material was oven dried at
60 °C for 72 h and biomass was determined.
Leaf gas exchange parameters including net photo-
synthesis (A), stomatal conductance (gs), and transpira-
tion (E) were measured inside the growth chambers
under similar conditions as the growth environment with
a Li-Cor LI-6200 portable photosynthesis system. Leaf
water potential was measured with a Scholander
pressure chamber (Soil Moisture Equipment Corp., Santa
Barbara, CA, USA) and soil water potential was
measured with thermocouple soil psychrometers. Leaf
gas exchange measurements and leaf water potential
were measured on fully developed leaves from drought-
treated and well-watered plants before drought, each
day during drought, and at 2 and 7 days following
rewatering after wilting (recovery).
Statistical analysis
One-way analysis of variance (anova, Data Desk Version
5.0, Data Description Inc., Ithaca, NY) was used to test
the main effect of CO2 (as a ®xed factor) on growth (leaf
area, total mass), gas exchange (photosynthesis, stomatal
conductance, transpiration), and water status (leaf water
potential) of well-watered and drought-treated C3 and
C4 plants. Comparisons of the relative effects of CO2 and
drought on C3 and C4 species were conducted by one-
way anova with CO2 as the main effect on ratios from
random pairing of values from drought-treated and well-
watered plants at each time interval during the drought
and recovery period. Data were transformed when
necessary to meet the assumptions of anova. Multiple
comparisons of means were made by the Scheffe post hoc
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859
test. Relationships of photosynthesis to leaf water
potential and photosynthesis to transpiration (a measure
of instantaneous water use ef®ciency) were ®t with linear
regression equations. Analysis of covariance (ancova)
was used to determine whether there were signi®cant
differences between slopes from effects of CO2. All
treatment effects were considered signi®cant at P < 0.05.
Results
Well-watered plants
Well-watered Abutilon theophrasti (C3) showed evidence
of increasing leaf area with increasing pCO2 (P = 0.070,
Fig. 1a) and signi®cant increases in total dry mass
between 18 and 70 Pa CO2 (P = 0.046, Fig. 1b) at 38 days
of growth. Total leaf area (P = 0.30, Fig. 1a) and total dry
mass (P = 0.94, Fig. 1b) were unaffected by CO2 in well-
watered Amaranthus retro¯exus (C4) at 38 days of growth.
In response to increasing pCO2, photosynthesis increased
in both species (P = 0.0001 for Abutilon, P = 0.014 for
Amaranthus), and stomatal conductance (P = 0.0001 for
Abutilon, P = 0.0017 for Amaranthus) and transpiration
(P = 0.0001 for Abutilon, P = 0.0006 for Amaranthus) had
decreased signi®cantly at 28±31 days of growth (Fig.
2a±c).
Drought-treated plants
Leaf area and total biomass. Before drought, Abutilon plants
assigned to the drought treatment showed signi®cant
increases in leaf area and total mass between 18 and 70 Pa
CO2 (Table 1). Visible wilting occurred in Abutilon in all
CO2 treatments after 4 days without water when soil
water potentials reached ± 0.6 6 0.1 MPa (data not
shown). Following drought, plants grown at 70 Pa CO2
still had higher total mass than plants grown at 18 Pa
CO2, but CO2 no longer affected leaf area (Table 1). This
pattern of response continued throughout the recovery
period.
Before the imposition of drought, the ratios generated
from random pairing of leaf area values from plants
assigned to the drought treatment relative to those in the
well-watered (control) treatment were unaffected by CO2
treatment (and were not signi®cantly different from
unity) in Abutilon (P = 0.98, Fig. 3a). This was also the case
for all other measurements (for both C3 and C4 species)
involving relative responses of drought-treated to well-
watered plants before the drought treatment. A compar-
ison of drought-treated to well-watered Abutilon plants
of the same age showed that drought reduced leaf area
by 54±80% in all CO2 treatments, with plants in the 18 Pa
CO2 treatment being least affected by drought (P = 0.036,
Fig. 3a). The CO2 treatments had no effect on the ratios of
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860 J . K . W A R D et al.

Fig. 1 (a) Total leaf area and (b) total dry mass of well-watered
(never exposed to drought) Abutilon (C3) and Amaranthus (C4)
after 38 days of growth in four CO2 partial pressures (18, 27,
35 and 70 Pa). Values are means (n = 4) 6 SE. Different letters
within a species indicate statistically different responses to
CO2 at P < 0.05 (only for groups showing a signi®cant response
to CO2 from ANOVA).

leaf area of drought-treated plants relative to well-

watered plants following a 7-day recovery period
(P = 0.34), and drought-treated plants continued to have
relatively lower leaf area than well-watered plants. For
total dry mass of Abutilon, CO2 had no effect on the ratios
of drought-treated plants relative to well-watered plants
at the end of drought (P = 0.57) and at 7 days after
recovery from drought (P = 0.15, Fig. 3b).
Before drought, Amaranthus plants assigned to the
drought treatment only showed signi®cant increases in
leaf area between 27 and 35 Pa CO2, and there were no
signi®cant effects of CO2 on total mass (Table 1). Visible
wilting occurred in Amaranthus in all CO2 treatments
after 3 days without water when soil water potentials
reached ± 0.7 6 0.1 MPa (data not shown). At the end of
the drought, CO2 did not affect leaf area or total mass
(Table 1). At recovery, however, Amaranthus had lower
total mass at 18 Pa CO2 compared to 35 Pa CO2, and
similar leaf area in all CO2 treatments.
Drought reduced leaf area and dry mass of Amaranthus
in all CO2 treatments by about 46 and 44%, respectively
Fig. 2 (a) Photosynthesis, (b) stomatal conductance, and (c)
transpiration of well-watered Abutilon (C3) and Amaranthus
(C4) after 28±31 days of growth in four CO2 partial pressures
(18, 27, 35 and 70 Pa). Values are means (n = 16±20) 6 SE.
Different letters within a species indicate statistically different
responses to CO2 at P < 0.05 (only for groups showing a
signi®cant response to CO2 from ANOVA).
(P = 0.85 for leaf area, P = 0.85 for dry mass, Fig. 3c,d). At
7 days after recovery from drought, CO2 had no effect on
the leaf area of drought-treated plants relative to well-
watered plants (P = 0.65, Fig. 3c); however, there was a
signi®cant effect of CO2 on total dry mass (P = 0.040) that
indicated low recovery of plants grown at 18 Pa CO2
(Fig. 3d).
Leaf water potential and gas exchange of the C3 species. Leaf
water potential of Abutilon (C3) decreased as the drought

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RESPONSE OF C3 AND C4 PLANTS TO CO2 AND DROUGHT 861

Table 1 Leaf area and total mass of Abutilon (C3) and Amaranthus (C4) for plants assigned to the drought treatment with values for
the day before the onset of drought, the end of drought (visible wilting of leaves), and at 7 d after rewatering (recovery). Values are
means 6 SE (n = 4). Different letters within a species and time period indicate statistically different responses to CO2 at P < 0.05

Abutilon (C3) Amaranthus (C4)

Time of harvest CO2 (Pa) Leaf area (cm2) Total mass (g) Leaf area (cm2) Total mass (g)

b b
Before Drought 18 311 (64) 1.7 (0.3) 1100 (74) ab 8.3 (0.8) a
ab ab
27 525 (71) 3.2 (0.8) 776 (35) b 6.4 (0.5) a
a ab
35 653 (16) 3.8 (0.7) 1308 (110) a 10.0 (0.9) a
a a
70 673 (35) 4.8 (0.5) 925 (175) ab 7.2 (1.3) a

End of Drought 18 244 (16) a 1.8 (0.1) b

927 (94) a 9.9 (1.0) a

27 273 (109) a 4.2 (0.7) ab

743 (50) a 7.4 (0.9) a

35 302 (32) a 3.0 (0.8) ab

710 (105) a 6.8 (1.2) a

70 353 (97) a 5.4 (1.3) a

778 (59) a 9.1 (1.3) a

Recovery 18 702 (36) a 3.5 (0.4) c

1265 (93) a 14 (3) b

27 909 (72) a 7.4 (0.8) ab

2265 (517) a 23 (2) ab

35 669 (68) a 4.4 (0.4) cb

1818 (281) a 29 (3) a

70 1089 (231) a 10.4 (3.0) a

1800 (155) a 23 (3) ab

Fig. 3 (a) Leaf area and (b) total dry mass of Abutilon (C3) and (c) leaf area and (d) total dry mass of Amaranthus (C4) expressed as
ratios from random pairing of values from drought and well-watered treatments at different time intervals before and after the
drought period, and 7 days after re-watering at the end of the drought. The line at unity indicates no effect of drought on the
measured parameter. Values are means (n = 4) 6 SE. Different letters within a species and time period indicate statistically different
responses to CO2 at P < 0.05.

proceeded, but there was generally no absolute or plants) effect of CO2 on leaf water potential throughout
relative (drought-treated compared to well-watered the 4 days of exposure to drought (Table 2, Fig. 4a). At

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862 J . K . W A R D et al.

Table 2 Leaf water potential, photosynthesis, and stomatal conductance of Abutilon (C3) and Amaranthus (C4) for plants assigned to
the drought treatment with values for the day before the onset of drought, the end of drought (visible wilting of leaves), and at 7 d
after rewatering (recovery). Values are means 6 SE (n = 4). Different letters within a species and time period indicate statistically
different responses to CO2 at P < 0.05

Abutilon (C3) Amaranthus (C4)

CO2
Time of harvest (Pa) LWP (MPa) A (mmol m±2s±1) gs (mol m±2s±1) LWP (MPa) A (mmol m±2s±1) gs (mol m±2s±1)

a
Before Drought 18 ± 0.53 (0.13) 7.3 (0.8) b 1.24 (0.25) a
± 0.40 (0.01) a
22.8 (1.9) a
0.87 (0.11) a
a
27 ± 0.68 (0.06) 11.9 (1.4) ab 0.83 (0.11) a
± 0.42 (0.05) a
27.1 (1.8) a
0.92 (0.26) a
a
35 ± 0.68 (0.09) 14.9 (2.3) ab 1.20 (0.30) a
± 0.38 (0.05) a
27.6 (1.1) a
0.71 (0.06) a
a
70 ± 0.60 (0.10) 15.6 (2.4) a 0.55 (0.10) a
± 0.35 (0.03) a
26.1 (2.9) a
0.47 (0.12) a

a a a a
End of Drought 18 ± 1.88 (0.09) 0.10 (0.14) 0.030 (0.005) ± 2.45 (0.12) ± 0.46 (0.22) b 0.025 (0.001) a
a a a a
27 ± 2.10 (0.30) 0.13 (0.34) 0.023 (0.004) ± 2.18 (0.24) 0.98 (0.66) ab 0.030 (0.007) a
a
35 ± 2.00 (0.25) 1.8 (1.5) a 0.066 (0.037) a
± 1.73 (0.31) a
1.5 (1.3) ab 0.03 (0.01) a
a
70 ± 1.80 (0.10) 1.5 (1.0) a 0.016 (0.004) a
± 1.78 (0.13) a
7.4 (3.0) a 0.044 (0.009) a

a b a a a a
Recovery 18 ± 0.70 (0.04) 7.2 (1.3) 1.35 (0.43) ± 0.63 (0.03) 18.0 (3.1) 0.42 (0.13)
a
27 ± 0.60 (0.07) 10.0 (1.1) ab 1.06 (0.24) a
± 0.55 (0.06) a
19.3 (0.8) a
0.30 (0.09) a
a
35 ± 0.60 (0.07) 7.5 (1.6) b 0.72 (0.34) a
± 0.50 (0.07) a
18.8 (2.1) a
0.19 (0.01) a
a
70 ± 0.78 (0.06) 14.5 (2.0) a 0.67 (0.16) a
± 0.53 (0.03) a
25.6 (1.7) a
0.14 (0.01) a

2 days recovery from drought, leaf water potential of
plants grown at 18 Pa CO2 showed the lowest amount of
recovery (P = 0.021), but fully recovered after 7 days
(P = 0.19, Fig. 4a).
Before drought, Abutilon plants in the drought treat-
ment exhibited signi®cantly greater photosynthetic rates
at 70 Pa CO2 than at 18 Pa CO2, but showed similar rates
at the end of drought (Table 2). However, at recovery,
high CO2 increased the photosynthesis of drought-
treated plants (Table 2). Net photosynthesis was reduced
by about 93% in all CO2 treatments following 4 days
exposure to drought (Fig. 4b), and there was no relative
effect of CO2 on photosynthesis after 2 days (P = 0.92) or
7 days (P = 0.97) recovery from drought.
For Abutilon plants in the drought treatment, CO2 did
not affect stomatal conductance (gs) before drought, after
drought, or at recovery (Table 2). At the end of drought,
CO2 did not affect the relative responses of drought-
treated to well-watered plants for gs and E (P = 0.069 for
gs, P = 0.18 for E, data not shown for E) which were
reduced by » 94 and 87%, respectively (Fig. 4c). In
addition, CO2 had no relative effects on gs and E at
2 days (P = 0.80 for gs, P = 0.86 for E) or 7 days (P = 0.99 for
both E and gs) after rewatering.
Leaf water potential and gas exchange of the C4 species. In
Amaranthus, leaf water potential decreased as the
drought proceeded, but CO2 did not affect the absolute
values of leaf water potential before drought, at the end
of drought, or at recovery (Table 2). Furthermore, CO2
did not affect the response of drought-treated plants
relative to well-watered plants for leaf water potential
(Fig. 5a).
Before drought, the photosynthetic rates of
Amaranthus plants in the drought treatment were
unaffected by the CO2 treatments (Table 2); however,
by the end of the drought, plants grown at 70 Pa CO2
had signi®cantly higher photosynthetic rates than
plants grown at 18 Pa CO2. Relative comparisons
between drought-treated and well-watered plants
indicated that photosynthesis was reduced in all CO2
treatments as the drought progressed, but plants at
low CO2 (18 and 27 Pa) showed the greatest reduc-
tions in photosynthesis (99% reduction at 3 days),
whereas plants at 70 Pa CO2 were least affected (71%
reduction at 3 days, Fig. 5b). Furthermore, CO2 had no
relative effect on the recovery of photosynthesis from
drought at 2 days (P = 0.44) or 7 days (P = 0.75) after
rewatering (Fig. 5b). Stomatal conductance (gs) and
transpiration (E) of Amaranthus (C4) declined in all
CO2 treatments as the drought progressed, but the
absolute (Table 2) and relative (Fig. 5c) patterns did not
vary by pCO2 (data not shown for E). After 3 days of
exposure to drought, the response of drought-treated
plants relative to well-watered plants for gs and E was
reduced in all CO2 treatments by about 93 and 98%,
respectively. Furthermore, CO2 did not have a relative
effect on the recovery of gs and E at 2 days (P = 0.76
for gs, P = 0.83 for E) or 7 days (P = 0.49 for gs, P = 0.75
for E) after rewatering (Fig. 5c).
Relationships between leaf water potential and photosynth-
esis and between photosynthesis and transpiration in the

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RESPONSE OF C3 AND C4 PLANTS TO CO2 AND DROUGHT
Fig. 4 (a) Leaf water potential, (b) net photosynthesis, and
(c) stomatal conductance of Abutilon (C3) expressed as ratios
from random pairing of values from drought and well-watered
treatments at different time intervals: D0 is the day before
drought was induced, D1 is Day 1 of the drought treatment,
etc. and R2 and R7 are Days 2 and 7 of the recovery period
after re-watering. The line at unity indicates no effect of
drought on the measured parameter. Values are means
(n = 4) 6 SE. Different letters within a time period indicate sta-
tistically different responses to CO2 at P < 0.05.
C3 and C4 species. Net photosynthesis of both species
was reduced as leaf water potential declined in all
CO2 treatments (Fig. 6a,b). An analysis of covariance
demonstrated that there was no overall effect of CO2
on the relationship between photosynthesis and leaf
water potential in either Abutilon (P = 0.23) or Amar-
anthus (P = 0.24).
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Fig. 5 (a) Leaf water potential (b) net photosynthesis, and
(c) stomatal conductance of Amaranthus (C4) expressed as ra-
tios from random pairing of values from drought and well-wa-
tered treatments at different time intervals: D0 is the day
before drought was induced, D1 is day 1 of the drought treat-
ment, etc. and R2 and R7 are Days 2 and 7 of the recovery per-
iod after re-watering. The line at unity indicates no effect of
drought on the measured parameter. Values are means
(n = 4) 6 SE. Different letters within a time period indicate sta-
tistically different responses to CO2 at P < 0.05.
Both C3 and C4 species showed increasing transpira-
tion rate with increases in net photosynthesis (Fig. 7a,b).
The CO2 treatments resulted in signi®cantly different
slopes (P = 0.0078) in the C3 species (Fig. 7a). Relative to
35 Pa CO2, there was a 52% reduction at 18 Pa CO2 and a
15% increase at 70 Pa CO2 in the rate of increase of
photosynthesis with increasing transpiration rate for the
C3 species. In the C4 species, CO2 had no effect on the
L
864 J . K . W A R D et al.
Fig. 6 Net photosynthesis vs. leaf water potential of drought-
treated (a) Abutilon (C3) and (b) Amaranthus (C4) grown at 18,
27, 35 and 70 Pa CO2. Values include those from just before
drought (D0) to the end of drought (D3 or D4). Solid lines are
linear regression ®ts for each CO2 treatment.
relationship between transpiration and photosynthesis
(P = 0.65, Fig. 7b).
Discussion
Based on our theoretical understanding and past
studies of C3 and C4 physiology, we hypothesized
that low atmospheric pCO2 and reduced precipitation
of the late Pleistocene would favour C4 species over
C3 species, whereas increased pCO2 predicted for the
future would increase the competitiveness of C3
species relative to C4 species under drought condi-
tions. Therefore, we tested the interactive effects of
water availability and pCO2 of the past and future on
the physiology and growth of C3 and C4 plants to
gain further insights into the competitive relationships
between these functional types over geological and
contemporary time scales.
Abutilon (C3) grown under well-watered conditions
exhibited increased photosynthesis and growth in
response to increasing pCO2, as is commonly observed
Fig. 7 Net photosynthesis vs. transpiration of drought-treated
(a) Abutilon (C3) and (b) Amaranthus (C4) grown at 18, 27, 35
and 70 Pa CO2. Values include those from just before drought
(D0) to the end of drought (D3 or D4). Solid lines are linear
regression ®ts for each CO2 treatment.
in C3 species (Ehleringer & Monson 1993). Stomatal
conducatance was highest at the lowest growth pCO2
(18 Pa) and presumably resulted in an increased rate of
CO2 diffusion to the site of carboxylation. Increased
stomatal conductance has been proposed as a mechanism
for increased photosynthesis in C3 plants grown at low
pCO2, but it has not been observed in all plants (Sage &
Reid 1992). Despite increased stomatal conductance at
18 Pa pCO2, Abutilon exhibited a 41% reduction in net
photosynthesis in the 18 Pa CO2 treatment compared
with the 35 Pa CO2 treatment. Decreased photosynthesis
resulted in lower biomass production in the 18 Pa CO2
treatment than in the higher pCO2 treatments, suggesting
that, despite stomatal adjustment, low pCO2 of the past
was highly limiting to C3 photosynthesis and growth.
Abutilon exhibited signi®cantly reduced stomatal con-
ductance, but maintained the highest rate of net photo-
synthesis in the high CO2 (70 Pa) treatment. This high
rate of photosynthesis probably contributed to increased
growth at elevated CO2, which may have been further
enhanced by increased leaf area.
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RESPONSE OF C3 AND C4 PLANTS TO CO2 AND DROUGHT
Amaranthus (C4) grown under well-watered conditions
responded physiologically to pCO2 to a greater degree
than expected based on our theoretical understanding of
C4 biochemistry and the majority of previous CO2 studies
involving C4 species (Leegood & Osmond 1991;
Ehleringer & Monson 1993; Tissue et al. 1995). With
increasing pCO2, Amaranthus showed gradual increases
in net photosynthesis and decreases in stomatal conduc-
tance and transpiration that were similar to the responses
of the C3 species. These physiological responses, how-
ever, did not result in enhanced biomass production,
perhaps because of increased respiration or greater root
exudation (Norby 1994). Amaranthus has been shown to
reach CO2 saturation at a Ci of » 20 Pa CO2 (Tissue et al.
1995), indicating that Amaranthus grown at 18 Pa CO2 was
not CO2-saturated in our study. Similarly, Polley et al.
(1994) observed that Schizachyrium scoparium (C4) exhib-
ited decreased stomatal conductance and increased
photosynthetic rates when pCO2 was increased from 20
to 34 Pa, but plant growth remained unaffected. Garbutt
et al. (1990) reported decreased stomatal conductance and
increased photosynthetic rates in Amaranthus retro¯exus
(C4) and Setaria faberii (C4) in response to CO2 enrichment.
When grown in competition, elevated CO2 stimulated
biomass production of Amaranthus retro¯exus (C4) more
than that of Abutilon theophrasti (C3) (Bazzaz et al. 1989).
These results demonstrate that some C4 species are
responsive to changes in pCO2, despite theoretical
considerations to the contrary.
Both the C3 and C4 species showed drought-induced
reductions in total leaf area and biomass production, but
the speci®c responses to drought varied between species.
The C3 species responded to drought by quickly
senescing and dropping many older leaves, resulting in
large reductions in leaf area (54±80% reduction in
drought-treated relative to well-watered plants, 22±54%
reduction between the beginning and end of drought).
As a result, relatively higher leaf water potential was
maintained in new leaves of drought-treated C3 plants
compared to well-watered plants and between the
beginning and end of drought in comparison with the
C4 species. In contrast, the C4 species exhibited smaller
reductions in leaf area (46% reduction in drought-treated
relative to well-watered plants, 4±46% reduction between
the beginning and end of drought) compared to the C3
species. However, the C4 species showed a greater
reduction in leaf water potential than the C3 species in
drought-treated plants relative to well-watered plants
and between the beginning and end of drought. These
leaf loss patterns, as well as possible differences in C3
and C4 physiology, contributed to greater relative and
absolute recovery of leaf area and biomass production in
the > 18 Pa CO2 treatments in the C4 species than in the
C3 species.
# 1999 Blackwell Science Ltd., Global Change Biology, 5, 857±867
R
865
The effects of drought on total leaf area and biomass
production of the C3 and C4 species were differentially
affected by the pCO2 treatments. C3 plants grown at
18 Pa CO2 exhibited the smallest reductions in leaf area in
drought-treated plants relative to well-watered plants
and between the beginning and end of drought com-
pared with plants grown at higher pCO2. Retention of
relatively greater leaf area, even under drought condi-
tions, may be advantageous for C3 plants under
conditions of carbon limitation (Allen et al. 1991; Dippery
et al. 1995). In the C4 species, the CO2 treatment had little
effect on leaf area and biomass responses to drought;
however, when comparing drought-treated to well-
watered plants, C4 plants grown at 18 Pa CO2 had
signi®cantly lower recovery of leaf area and biomass
compared with plants grown at 35 Pa CO2. The C4 plants
grown at 18 Pa CO2 also showed the lowest absolute
recovery of leaf area and total mass between the
beginning and end of drought. Because the C3 species
lost the least amount of leaf mass during drought when
grown at 18 Pa CO2 and the C4 species showed the
lowest recovery from drought at 18 Pa CO2, relative
recovery of leaf area and biomass production from
drought was similar in the C3 and C4 species grown at
18 Pa CO2. These results suggest that competitive
relationships between co-occurring C3 and C4 plants
may not have been affected during shifts from wetter to
drier climates during the late Pleistocene. Furthermore,
at current and elevated pCO2, the C4 species showed
relatively greater recovery of leaf area and biomass
production compared to the C3 species, suggesting that
the C4 species would continue to be more competitive
than the C3 species in regions receiving more frequent
and severe droughts in the future.
The pCO2 treatments affected the initial responses of
the C3 species to drought. In drought-treated C3
plants grown at low CO2 (18 and 27 Pa), stomatal
conductance, photosynthesis, and transpiration were
not affected during early exposure to drought (D1)
relative to well-watered plants. As drought conditions
persisted, however, stomatal conductance decreased
rapidly, resulting in large reductions in photosynthesis
and transpiration. Thus, at low pCO2, stomatal control
may have been exerted primarily for regulation of
carbon assimilation during the initial stages of drought
and for regulation of water loss during the later stages
of drought. At pCO2 > 27 Pa, stomatal conductance,
photosynthesis, and transpiration of C3 plants were
immediately reduced at the initiation of drought (D1),
indicating that stomatal control was exerted to
regulate water loss throughout the entire drought
period. Despite these CO2-induced differences in the
initial response to drought, the C3 species showed
similar relative reductions in stomatal conductance,
L
866 J . K . W A R D et al.
photosynthesis, and transpiration in all pCO2 treat-
ments by the end of the drought (D4).
The C4 plants grown at 70 Pa CO2 exhibited lower
absolute and relative (drought-treated plants relative to
well-watered plants) reductions in photosynthesis by the
end of the drought compared to plants grown at 18 and
27 Pa CO2. This result is of interest because it is generally
assumed that C4 plants become CO2-saturated for
photosynthesis near the current atmospheric pCO2 (Sage
1994; Tissue et al. 1995). However, Patterson (1986)
observed that increases in pCO2 above the current value
reduced the effects of water stress on three C4 grass
species, and signi®cantly increased leaf area and total
mass. Therefore, elevated CO2 may not only reduce
drought effects in C3 annuals and trees (Tolley & Strain
1985; Tyree & Alexander 1993; Tschaplinski et al. 1995;
HaÈttenschwiler et al. 1997), but may also alleviate
drought effects in C4 species as well.
The relationship between net photosynthesis and leaf
water potential was not affected by pCO2 in either the C3
or C4 species. The C4 species exhibited higher photo-
synthetic rates across a wide range of leaf water
potentials than the C3 species. However, photosynthesis
of the C4 plants was still greatly reduced by the end of
the drought period, because these plants retained greater
leaf area during drought and thus had greater reductions
in leaf water potential. Instantaneous water use ef®ciency
(Fig. 7) was enhanced by CO2 enrichment in the C3
species but not in the C4 species (cf. Polley et al. 1992;
Polley et al. 1993a). These results suggest that C3 plants
may have had higher water requirements during the
Pleistocene compared to the present. Although C3 plants
grown at 70 Pa CO2 had higher water-use ef®ciency than
C3 plants grown at ambient CO2, their greater absolute
leaf area early in the drought treatment would have
increased whole plant water loss, resulting in similar
relative reductions in leaf water potential between CO2
treatments.
It is dif®cult to extrapolate our results to the effects of
long-term, gradual drought, because acclimation re-
sponses and growth modi®cations may substantially
modify responses to drought (e.g. Woodward & Bazzaz
1988; Chaves & Pereira 1992). Nonetheless, our study
provides information about the relative responses of C3
and C4 species to scenarios of severe and rapid drought
that may not allow for long-term acclimation responses
and may be more frequent in the future as a result of
global climate change. Furthermore, studies on the
responses of tree species to low pCO2 that occurred
during the Pleistocene are lacking, and therefore the C3
annual model system used in this study may provide
valuable insights into the possible responses of tree
seedlings to low pCO2 and reduced precipitation of the
late Pleistocene.
In summary, we found that the pCO2 that occurred
during the Pleistocene was highly limiting to C3
photosynthesis and growth under well-watered condi-
tions. Well-watered C4 plants grown in varying pCO2
exhibited physiological responses commonly found in C3
plants, but total mass and total leaf area were unaffected
by pCO2. The C3 plants grown at the Pleistocene pCO2
retained greater leaf area when subjected to drought than
plants at higher pCO2 and exhibited a delay in the
reduction of stomatal conductance compared to plants
grown at higher CO2 that may have been a response to
severe limitations in carbon availability. Furthermore, C3
and C4 plants showed similar abilities to recover from
drought with respect to leaf area and biomass production
at the Pleistocene pCO2, but C4 plants exhibited greater
recovery at current and elevated pCO2. The C4 plants
grown at 70 Pa CO2 also showed relatively lower
reductions in net photosynthesis in response to drought
than C4 plants at lower pCO2, indicating that elevated
CO2 may alleviate drought effects in C4 species.
Based on the observed responses of Abutilon theophrasti
and Amaranthus retro¯exus, we reject our hypotheses that
C3 species may be more severely affected by drought
than C4 species at low pCO2 and that C3 species may
have an advantage over C4 species during drought in
elevated pCO2. This study provides evidence that C3 and
C4 species may potentially respond to pCO2 and drought
in manners unpredicted by previous theoretical expecta-
tions.
Acknowledgements
We sincerely acknowledge Larry Giles, Beth Guy, Jeff Pippen
and Will Cook for their generous technical assistance. This
research was supported by the Department of Energy, CO2
Research Division, contract DE-FGO5±87ER60575, Electric
Power Research Institute contract RP3041±02, and NSF grant
BSR87±06429 for support of the Duke University Phytotron.
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