Title:
Physiological and Growth Responses of C3 and C4 Plants to Reduced Temperature
When Grown at Low CO2 of the Last Ice Age
Running Title:
Authors:
C3 and C4 Responses to Reduced Temperature at Low CO2
Joy K. Ward1*, David A. Myers2, and Richard B. Thomas2
1
Department of Ecology and Evolutionary Biology, University of Kansas,
Lawrence, KS 66045, USA;
2
Department of Biology, West Virginia University, Morgantown, WV 26506,
USA
*Author for Correspondence.
Tel: 785-864-5218
Fax: 785-864-5860
Email: [email protected]
Supported by the U.S. Department of Energy (DE-FG02-95ER62124), the U.S. National Science
Foundation (0517668 and 0746822), and an American Fellowship to J.K.W. from the American
Association of University Women Educational Foundation.
1
Abstract
During the last ice age, atmospheric [CO2] was 180-200 ppm compared with the modern value of
380 ppm, and temperatures were ~8 ˚C cooler. Relatively little is known about the responses of
C3 and C4 species to long-term exposure to glacial conditions. Here Abutilon theophrasti (C3)
and Amaranthus retroflexus (C4) were grown at 200 ppm CO2 with current (30/24 ˚C) and glacial
(22/16 ˚C) temperatures for 22 d. Overall, the C4 species exhibited a large growth advantage over
the C3 species at low [CO2]. However, this advantage was reduced at low temperature, where the
C4 species produced 5X the total mass of the C3 species versus 14X at the high temperature. This
difference was due to a reduction in C4 growth at the low temperature, since the C3 species
exhibited similar growth between temperatures. Physiological differences between temperatures
were not detected for either species, although photorespiration/net photosynthesis was reduced in
the C3 species grown at low temperature, suggesting evidence of improved carbon balance at this
treatment. This system suggests that C4 species exhibited a growth advantage over C3 species
during low [CO2] of the last ice age, although concurrent reductions in temperatures may have
reduced this advantage. (198 words)
Keywords: Abutilon theophrasti; Amaranthus retroflexus; C3 species; C4 species; climate
change; low CO2; low temperature; photorespiration; Pleistocene
2
Studying plant responses to global changes of the past provides valuable insights for
predicting future responses to a rapidly changing environment (Ward et al. 2005; Edwards et al.
2007; Jackson 2007). In addition, studies involving treatments that simulate past climates
provide a baseline for understanding the physiological functioning of plants prior to
anthropogenic influences (Polley et al. 1993a,b; Anderson et al. 2001; Sage and Coleman 2001;
Polley et al. 2002; Ward 2005). Atmospheric CO2 concentration ([CO2]) reached minimum
values of 180 ppm during the last ice age, rose to 270 ppm during the recent interglacial period
(and just prior to the onset of the Industrial Revolution), and increased to 380 ppm in the modern
atmosphere as a result of fossil fuel combustion and deforestation (EPICA 2004). Low [CO2]
that occurred during the last ice age is predicted to have produced carbon limitations within C3
plants, and may have reduced their distribution relative to C4 species (Polley et al. 1993a;
Dippery et al. 1995; Tissue et al. 1995; Ward and Strain 1999; Koch et al. 2004; Vidic and
Montanez 2004; Ward et al. 2005; Huang et al. 2006).
Over geologic time scales, periods of minimum [CO2] during glacial periods correlate
closely with reduced temperatures (~8 ºC reduction relative to modern times on a global average;
Petit et al. 1999; Sigman and Boyle 2000; but also see Cowling 1999). Knowledge of the effects
of reduced temperature on C3 and C4 plants grown at low [CO2] is critical for understanding
changes in plant competitive interactions and abundance during the last ice age. C3 plants are not
favored at low [CO2] as a result of increased oxygenase activity of rubisco (ribulose-1,5bisphosphate carboxylase-oxygenase) and reduced carboxylation activity in response to limiting
CO2 substrate (Pearcy et al. 1987; Tissue et al. 1995; Sage and Cowling 1999; Sage and Coleman
2001). C4 species, on the other hand, concentrate CO2 in bundle-sheath cells, and therefore are
much less negatively affected by reductions in atmospheric [CO2] (Dippery et al. 1995; Sage and
Coleman 2001; Ward 2005). With regard to temperature, C3 photosynthesis is positively affected
by reductions in temperature because oxygenase activity is reduced relative to carboxylation
activity, increasing quantum yield (ratio of CO2 molecules fixed to photons of light absorbed). In
contrast, the quantum yield of C4 photosynthesis is independent of temperature due to the
absence of temperature-dependent photorespiration (Ehleringer and Pearcy 1983). Furthermore,
low temperatures (especially below 20 ºC) often reduce the photosynthetic rates of C4 species
(Long 1983; Kubien et al. 2003), and low temperatures have been shown to make C4 plants more
susceptible to damage from photoinhibition (Fryer et al. 1995).
3
By combining the factors of CO2 and temperature, Ehleringer et al. (1997) modeled the
climate conditions under which C3 and C4 plants were favored based on quantum yield. The
authors found that at modern [CO2] (~380 ppm) the crossover temperature where C4
photosynthesis becomes favored over C3 photosynthesis occurs at 22-24 °C; whereas at ice age
[CO2] (180 ppm, 18,000 to 20,000 years ago), the crossover temperature occurs at approximately
15 °C. This quantum-yield model has been highly predictive of modern vegetation distributions
and clearly indicates that low [CO2] would have favored C4 species during the last ice age across
a wide range of regional temperatures (assuming sufficient moisture was present). The model
also predicts that reduced temperatures that occurred in conjunction with low [CO2] may have
increased the carbon gain of C3 species, although this likely did not compensate for the
pronounced negative effects of low [CO2].
In addition to physiological models, empirical studies examining the growth and
development of C3 and C4 species at reduced temperatures and low [CO2] are also needed to
better understand the functioning of ice age vegetation (Strain 1991; Sage and Coleman 2001;
Ward 2005). Thus, the objective of this study was to determine the physiological and growth
responses of co-occurring dicot annuals, Amaranthus retroflexus (C4; hereafter Amaranthus) and
Abutilon theophrasti (C3; hereafter Abutilon) to reduced ice age temperatures (-8 °C) during
growth at low [CO2] (200 ppm) that occurred during the last ice age. Past work has already
focused on the responses of these species to current and elevated [CO2] with temperature
interactions (see Ackerly et al. 1992; Coleman and Bazzaz 1992; Dippery et al. 1995, Tissue et
al. 1995; Ward et al. 1999; Sage and Kubien 2007), and therefore here the focus is on
temperature effects when all plants are grown at low [CO2]. We hypothesized that low
temperature would enhance the performance of the C3 species relative to the C4 species, but that
the low [CO2] growth conditions would produce higher performance in the C4 species overall.
Results
On an absolute basis, the C3 (Abutilon theophrasti) and C4 species (Amaranthus retroflexus)
exhibited large differences in total mass when grown at low [CO2] (200 ppm) for 22 d. More
specifically, the C4 species had 5 times the total mass of the C3 species when grown at the low
temperature (22 light/16 dark °C), and almost 14 times the total mass of the C3 species when
grown at the high temperature (30/24 °C; Table 1). In this case, all plants were grown from seed
4
for a total of 22 d, and therefore differences in final biomass reflect differences in relative growth
rate (change in biomass per unit biomass per unit time; hereafter RGR). Therefore, the RGR of
the C4 species greatly exceeded that of the C3 species at low [CO2]. In addition, Amaranthus has
inherently small seeds compared with Abutilon, and therefore initial seed size would not have
been a factor in providing a relative growth advantage of the C4 species over the C3 species when
grown at low [CO2]. Furthermore, the C3 and C4 species exhibited relative differences in their
responses to temperature for total mass and LA (leaf area) (significant species X temperature
interactions, P=0.0001, Table 1). The C3 species had similar total mass and LA at both the high
and low temperatures, whereas the C4 species showed a 65% reduction in total mass and a 55%
reduction in LA at the low temperature relative to the high temperature.
During growth at low [CO2], the C4 species allocated proportionally more biomass to
roots versus shoots in both temperatures compared with the C3 species (Table 2). In addition, the
C3 species allocated proportionally more biomass to roots (versus shoots) when grown at the low
temperature compared with the high temperature, whereas the C4 species was unresponsive to
temperature for allocation of root mass versus shoot mass (Table 2). Furthermore, the C3 species
had higher LA versus total mass at the high temperature compared with the low temperature. In
contrast, the C4 species had lower LA area versus total mass at the high temperature (Table 2).
Overall, gs (stomatal conductance) was higher and SLM (specific leaf mass) was lower in
the C3 species compared to the C4 species during growth at low [CO2] (Table 1). In response to
temperature treatments, the C3 and C4 species exhibited similar relative responses for gs (nonsignificant species X temperature interaction at P=0.64), whereby both species were
unresponsive to temperature. For SLM, the C3 and C4 species exhibited different relative
responses (significant species X temperature interaction at P=0.0001), with the C3 species
exhibiting similar SLM at both temperatures, and the C4 species showing a 20% reduction in
SLM between the high and low temperatures.
The Pr (photorespiration) of the C4 species was assumed to be negligible because there
were no statistical differences between values of A (net photosynthesis) measured at 2% versus
21% O2 at either temperature treatment (P=0.68 for high temperature, P=0.45 for low
temperature; data not shown) when measured at 180 ppm CO2 and 1400 µmol photons m-2 s-1
PAR. Therefore, Pr was only determined for the C3 species according to the method of Valentini
et al. (1995). The slopes of the relationship between photochemical efficiency of PSII
5
(photosystem II) and apparent quantum yield of CO2 assimilation (ΦCO2) for the C3 species were
9.42 (r2 = 0.83) and 10.18 (r2 = 0.93) for the high and low treatments, respectively (data not
shown).
Overall, the C3 species exhibited lower rates of A (by 38-45%) than the C4 species (Fig.
1a) when grown at low CO2. Within species, reduced temperature did not have a significant
effect on physiological responses (A, Pr, R (respiration); Fig. 1a,b,c), and there were no
significant species X temperature interactions for any of these physiological measurements
(P=0.46 for A, P=0.89 for R). Lower A in the C3 species versus the C4 species was a result of the
effects of Pr that reduced net carbon assimilation (Fig. 1a,b), and was not due to R that was
similar between both species at both temperature treatments (Fig. 1c).
Although a temperature effect was not detected for individual physiological
measurements in low [CO2]-grown plants, the ratio of Pr/A (measured simultaneously on the
same leaf area) for the C3 species decreased at the low temperature versus the high temperature
(Fig. 1b, insert). Furthermore, R/A was similar between temperature treatments within the C3
(warm = 0.15, cold = 0.15) and within the C4 species (warm = 0.08, cold = 0.10, data not
shown).
Discussion
This study involved growing C3 and C4 plants at low [CO2] (200 ppm) with an 8 °C difference in
temperature treatments (22/16 °C vs. 30/24 °C) in order to simulate the average global reduction
in temperature that occurred during the last ice age (Petit et al. 1999). Regardless of temperature,
the C4 species (Amaranthus retroflexus) exhibited a large advantage in absolute growth over the
C3 species (Abutilon theophrasti) at low [CO2]. The C4 species had 5 times the total mass of the
C3 species when grown at the low temperature and almost 14 times the total mass of the C3
species at the high temperature. In our past work, where the same C3 and C4 species were grown
at [CO2] near modern values (350 ppm), the C4 species only exhibited three times the biomass of
the C3 species at 28/22 °C (Dippery et al. 1995). Furthermore, in the present study, the C3 species
showed signs of carbon limitations with higher allocation of biomass to shoots versus roots (that
enhances total carbon assimilation) and lower photosynthetic rates relative to the C4 species.
Taken together, these results, along with those of other studies (Polley et al. 1993a,b; Sage 1995;
Dippery et al. 1995; Ward and Strain 1997; Ward et al. 1999; Ward et al. 2005; Sage and Kubien
6
2007), point to the dominant effect of low [CO2] in determining a growth advantage of C4 over
C3 species during the last ice age.
As hypothesized, the relative growth advantage of the C4 species was reduced at the low
temperature treatment in this study. This occurred because the C4 species exhibited a reduction in
growth at low temperature, whereas the C3 species was unaffected by temperature. This varies
from the results of Cowling and Sage (1998) with C3 Phaseolus vulgaris grown at 200 ppm CO2,
where plants showed a 70% increase in growth between 36 °C and 25 °C; it is important to note,
however, that the Phaseolus study involved a more extreme temperature range and a higher
maximum temperature compared with the present study.
It was not surprising that the C4 species exhibited a reduction in growth at the low
temperature treatment, although a 65% reduction was greater than anticipated based on
physiological predictions of C4 plants grown at low temperature (Sage and Kubien 2007). This
finding suggests that reductions in temperature during the last ice age, when combined with the
effects of low [CO2], may have greatly reduced the growth of some C4 species, and may have
limited their distribution. For example, C4 species were not detected in southern California, a
relatively warm region, between 12,000 and 28,000 yr BP, based on carbon isotope
measurements of mammal bone collagen that can be used to assess diet (Coltrain et al. 2004).
This may have been a result of reduced temperatures during the last ice age that limited the
competitive ability of C4 species in this region.
In modern times (when [CO2] has ranged between 270 and 380 ppm), C4 plants generally
occupy regions with warm climates, and occur at low altitudes, and may dominant ecosystems
during seasonal peaks in temperature (Rundel 1980; Kemp and Williams 1980; Pearcy et al.
1981), although exceptions have been described (Long 1999). This distribution pattern has been
primarily attributed to higher net photosynthesis and quantum yields of C4 species at warm
temperatures relative to C3 species. In addition, the light-saturated rate of A (net photosynthesis)
declines sharply in many C4 species as temperatures decreases below 20 °C (Long 1983).
Furthermore, Pitterman and Sage (2000) demonstrated that reduced rubisco activity may limit
photosynthesis in C4 plants grown at low temperatures (<17 °C), and Kubien et al. (2003)
showed that low rubisco content may limit C4 photosynthesis at a large range of sub-optimal
temperatures (using Flaveria bidentis with an antisense construct for the small subunit of
rubisco; see also Sage 2002). C4 plants may also be more susceptible to photoinhibition at low
7
temperatures because the reaction centers of photosystem II may be damaged and the efficiency
of energy transfer to these reaction centers may be diminished (Fryer et al. 1995).
Despite the findings of these past studies, the lower total mass of the C4 species grown at
the low temperature could not be attributed to any of the physiological measurements in the
present study. More specifically, the C4 species exhibited similar A, R, and gs in response to an 8
°C temperature difference when grown at 200 ppm CO2. It has been shown that A increases
sharply between 20 and 36 °C in the majority of C4 species when measured at current and
elevated [CO2] (Ghannoum et al. 2000). For example, Sage (2002) showed that the same C4
species, Amaranthus retroflexus, exhibited large increases in photosynthetic rate between 22 and
30 °C (by approximately 25%) at 360 ppm CO2. However, in the same study, Amaranthus
showed very modest increases in photosynthetic rate across these same temperatures when
measured at 180 ppm, and did not show any differences in photosynthetic rate at 100 ppm CO2.
Thus, as observed in the present study and in others, modern C4 plants may be relatively
temperature insensitive for photosynthesis when grown at low [CO2] representing the last ice
age. This response is driven by the inherent lack of temperature-sensitive oxygenase activity of
rubisco (shown to be the case here with no change in A between 2 and 21% O2) that differs from
C3 species, and is further driven by the insensitivity of PEP carboxylase (phosphoenolpyruvate
carboxylase) to temperature at low [CO2] conditions that is specific to C4 species (Sage and
Kubien 2007).
In the C4 species grown at low [CO2], partitioning of biomass between roots and shoots
was unaffected by temperature when differences in overall plant size were removed (with the
linear analysis employed). However, the C4 species grown at low temperature had greater LA per
total mass than plants grown at the high temperature. Despite this growth adjustment (with no
change in A per unit leaf area), the C4 species still produced lower biomass as a result of the
stressful effects of low temperature. This result illustrates the importance of considering
developmental and growth mechanisms, in addition to physiological responses, when evaluating
C3 and C4 responses to low [CO2] and temperature of the past.
When grown at low [CO2], the C3 species did not exhibit differences in physiology (A, Pr,
R, and gs) or in total mass, total leaf area, or SLM in response to the 8 °C difference in
temperature treatments. Past studies have indicated that the sensitivity of C3 photosynthesis to
temperature declines as plants become limited by CO2, much like the patterns exhibited by the C4
8
species (Berry and Björkman 1980; Pearcy and Ehleringer 1984; Sage 2002). In this study, the
C3 plants grown at 200 ppm CO2 were highly limited by CO2 availability, and for this reason,
they did not exhibit differences in A between temperature treatments. In addition, R of the C3
species was also unaffected by temperature in the present study. This result varies from the
findings of Cowling and Sage (1998) with Phaseolus vulgaris (C3) that exhibited a 68%
reduction in R between 36 and 25 °C when grown at 200 ppm CO2. Furthermore, several studies
have shown that C3 species are often conservative in the ratio of R versus A after long-term
exposure to increased temperature at current and elevated [CO2] (Dewar et al. 1999), and the
present study provides further evidence for this potential acclimation response at low [CO2] as
well.
Although it did not influence total biomass production, the C3 species did exhibit
differences in the partitioning of biomass in response to temperature, whereby plants grown at
the low temperature allocated significantly more biomass to roots versus shoots and had lower
LA versus total mass compared with plants grown at the high temperature. This higher
investment in root components may have been a response to the early stages of improved carbon
balance in the leaves of low temperature-grown plants at low [CO2]. This was evidenced by a
reduction in Pr versus A from simultaneous measurements on the same leaf area conducted at
low temperature. This result suggests that the carbon balance of the C3 species was beginning to
be positively affected by the ice age temperature treatment, but this response was not substantial
enough to increase total biomass production between temperature treatments.
In summary, this study with a C3 and C4 dicot suggests that low [CO2] that occurred
during glacial periods was likely a dominant factor that produced higher productivity of C4
species over C3 species. However, reduced temperatures in combination with low [CO2] may
have reduced the growth advantage of C4 species in some climates in the past. In addition, this
work provides the results of plant growth responses that support previous studies indicating
dominance of C4 species within ancient ecosystems during periods of low [CO2], particularly in
relatively warm regions (Street-Perrott et al. 1997, Cerling et al. 1997, Cerling et al. 1998).
Furthermore, the results of this study indicate that a combination of both leaf-level physiological
studies and growth studies are necessary for better understanding the mechanisms that
determined the distribution of C3 and C4 species within ancient ecosystems.
9
Materials and Methods
Growth conditions
Amaranthus (C4 dicot) and Abutilon (C3 dicot) that originated from old-field populations in
Illinois, USA were germinated and grown in monoculture in four growth chambers at the Duke
University Phytotron. Plants were grown in a 3:3:1 (v/v) medium of gravel, "Turface" and
sterilized topsoil in deep 3.5-L pots that did not restrict root development during the 22 d growth
period (Thomas and Strain 1991). Prior to emergence, pots were watered to saturation with deionized water twice each day. Following emergence, pots were watered to saturation with halfstrength Hoagland's solution (Downs and Hellmers 1978) each morning and with de-ionized
water each afternoon. Seedlings were thinned to one individual closest to the center of each pot
at 5 d after planting (both species emerged at 2 d after planting).
Two growth chambers were controlled at day/night temperatures with target values of
30/24 °C (Actual: 30.0 ± 0.9 SD/24.0 ± 0.3 °C and 30.0 ± 0.4/24.0 ± 0.2 °C), and two other
chambers were controlled at target values of 22/16 °C (Actual: 22.0 ± 0.4/16 ± 0.1 °C and 22.0 ±
0.5/16 ± 0.2 °C) to simulate current and glacial temperatures, respectively (Petit et al. 1999).
[CO2] within all growth chambers was maintained at 200 ± 10 ppm ([CO2] increased for 0.5 h
per day while plants were being watered and this time period was not included in the calculation)
by passing incoming air over a moist soda lime/vermiculite mixture to scrub CO2. Light/dark
periods were 14 h/10 h and the light level during the day was maintained at 1000 ± 50 µmol
photons m-2 s-1 PAR at plant height using metal halide and tungsten/halogen bulbs. Each dark
period was interrupted for 1 h with incandescent lighting at 50 µmol photons m-2s-1 to prevent
early initiation of flowering.
Growth measurements
Amaranthus and Abutilon from both temperature treatments were harvested at 15 d and 22 d after
emergence (n = 5-7 plants per chamber per harvest). At each harvest, total leaf area (LA) was
measured with a LI-3100 leaf area meter (Li-Cor, Lincoln, Nebraska). Plant material was
separated into roots, stems, and leaves and was oven dried (60 °C) for 48 h before mass was
measured. Specific leaf mass (SLM) was calculated as total leaf mass divided by the total leaf
area of individual plants. Partitioning of biomass was determined by comparing linear
10
relationships between root versus shoot mass and leaf area versus total mass by inclusion of data
from both harvests (see statistical analyses).
Physiological Measurements
Gas exchange parameters including stomatal conductance (gs), net photosynthesis (A), and dark
respiration (R) were measured at steady state conditions with an open system using a LI-6400
portable photosynthesis system (LI-Cor, Lincoln, Nebraska) at 20 d after planting. Conditions
within the leaf cuvette during measurements of gs and A were the same as those within growth
chambers during the light period. R was measured under dark conditions with all other
conditions in the leaf cuvette being similar to the light period (including temperature).
Pr of the C3 species (Abutilon) was measured according to the method of Valentini et al.
(1995) at growth CO2 and temperature conditions and with the following modifications. Gas
exchange and chlorophyll fluorescence were measured simultaneously from the most recently
mature leaves of Abutilon using the LI-6400 system and a pulse modulated fluorometer (PAM2000, Walz, Germany) at each of ten steps of a light response curve. Incoming air to the leaf
chamber was delivered from bottled 2% O2 in balanced nitrogen to provide non-photorespiratory
conditions. The incoming air was humidified by bubbling the air stream through distilled water
to saturation and then was scrubbed with “drierite” (W. A. Hammond Drierite Co., Xenia, Ohio)
to match growth chamber humidity. Light levels for the response curves were attenuated with
fine wire screens in ten steps ranging from a maximum of 1500 µmol photons m-2s-1 PAR to a
light level just below the light compensation point of each measured plant. For each light level,
gas exchange and fluorescence parameters were not recorded until ∆CO2, ∆H2O, and ∆ flow rate
(sample infrared gas analyzer – reference infrared gas analyzer) reached a total coefficient of
variation of less than 1% and stomatal conductance was stable. The ratio of Pr to A of the C3
species was calculated from simultaneous measurements of these two parameters on the same
leaf area.
Statistical analyses
Analyses of variance (ANOVAs) were conducted on data from individual plants at the second
harvest (22 d after emergence) for measurements of total mass, LA, gs, SLM, A, and R. Data
were tested for normality and loge transformed when necessary. The main effects of the analyses
11
included temperature, species, the interaction between these terms and the nested effect of
chamber(temperature). The chamber(temperature) variation was used as the error term for the
temperature effect, and other terms were tested over the residual variation. Pr was analyzed
without the species effect and species by temperature interaction because only C3 measurements
were taken. Treatment effects were considered significant at the P < 0.05 level.
The linear relationships of root versus shoot mass and leaf area versus total mass were
compared for the C3 and C4 species to determine if temperature regime affected biomass
partitioning at low CO2. Step-wise analysis of covariance (ANCOVA) was used to determine the
effects of species and temperature on these slopes (Samson and Werk 1986).
Acknowledgements
We thank Larry Giles, Beth Guy, Jeff Pippen, Will Cook, and Michael McGowan for
their generous technical assistance. We also thank Drs. Boyd Strain and James Ehleringer for
their insights into the design and results of this study.
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Figure legends
Figure 1. (a) Net photosynthetic rate (A, n=9-12), (b) photorespiration rate (Pr, n=5-6), and (c)
dark respiration rate (R, n=9-12) of Abutilon theophrasti (C3) and Amaranthus retroflexus (C4)
grown at 200 ppm CO2 for 20 d at either 30/24 °C (light/dark, modern) or 22/16 °C (glacial). The
insert in Figure 1b shows the ratio of photorespiration rate to net photosynthetic rate (Pr/A) of the
C3 species. Measurements were made at growth conditions and temperature during the light
period. This graph combines data from different chambers of the same treatment because no
significant chamber effect was detected. Different letters between temperatures and species
indicate significant differences at the P < 0.05 level according to ANOVA.
17
Tables
Table 1. The effects of modern and glacial temperatures on total mass (n=13-14), leaf area (LA,
n=13-14), stomatal conductance (gs, n=9-12) , and specific leaf mass (SLM, n=13-14) for
Abutilon theophrasti (C3) and Amaranthus retroflexus (C4) grown at low CO2 (200 ppm) for 22
d. Values are means ± 1 standard error. Data from different chambers within the same treatment
were combined because a chamber effect was not detected. Different superscript letters between
temperatures and species indicate significant differences at the P < 0.05 level according to
ANOVA.
Total mass (g)
LA (cm2)
gs (mol m-2 s-1)
SLM (g/m2)
30 / 24 °C (modern)
0.23 (0.04)c
49 (8)c
1.4 (0.1)a
32 (2)c
22 / 16 °C (glacial)
0.22 (0.03)c
39 (5)c
1.2 (0.1)a
34.9 (0.5)c
30 / 24 °C (modern)
3.1 (0.2)a
262 (13)a
0.52 (0.07)b
57 (2)a
22 / 16 °C (glacial)
1.1 (0.1)b
119 (11)b
0.43 (0.03)b
46 (1)b
Temperature treatment
Abutilon theophrasti (C3)
Amaranthus retroflexus (C4)
18
Table 2. Slopes and r2 values for the regressions of root mass (y) versus shoot mass (x) and leaf
area (y) versus total mass (x) for Abutilon theophrasti (C3) and Amaranthus retroflexus (C4)
grown at 200 ppm CO2 for 22 d at modern and glacial temperatures. Different letters within a
species and regression type indicate significant differences at the P < 0.05 level according to
ANCOVA (n=24).
Temperature treatment
Root mass (g) vs. shoot mass
Leaf area (cm2) vs. total mass
(g)
(g)
Slope
r2
Slope
r2
30 / 24 °C (modern)
0.26b
0.94
198a
0.98
22 / 16 °C (glacial)
0.41a
0.85
179b
0.98
30 / 24 °C (modern)
0.74a
0.95
69b
0.93
22 / 16 °C (glacial)
0.79a
0.97
98a
0.97
Abutilon theophrasti (C3)
Amaranthus retroflexus (C4)
19
Growth at 200 ppm
Modern 30 °C
Glacial 22 °C
Pr / A
30 °C
20
22 °C