Physiological and Growth Responses of C3 and C4 Plants to Reduced Temperature When Grown at Low CO2 of the Last Ice Age
Supported by the US Department of Energy (DE-FG02-95ER62124), the US National Science Foundation (0517668 and 0746822), and an American Fellowship to JK Ward from the American Association of University Women Educational Foundation.
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During the last ice age, CO2 concentration ([CO2]) was 180–200 μμmol/mol compared with the modern value of 380 μμmol/mol, and global 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 Medik. (C3) and Amaranthus retroflexus L. (C4) were grown at 200 μμmol/mol 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 5× the total mass of the C3 species versus 14× at the high temperature. This difference was due to a reduction in C4 growth at 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 had a growth advantage over C3 species during low [CO2] of the last ice age, although concurrent reductions in temperatures may have reduced this advantage.
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 μmol/mol during the last ice age, rose to 270 μmol/mol during the recent interglacial period (and just prior to the onset of the Industrial Revolution), and increased to 380 μmol/mol 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 °C 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-1,5-bisphosphate 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 °C) 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).
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 μmol/mol) the crossover temperature where C4 photosynthesis becomes favored over C3 photosynthesis occurs at 22–24 °C; whereas at ice age [CO2] (180 μmol/mol, 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 in some regions, 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 L. (C4; hereafter Amaranthus) and Abutilon theophrasti Medik. (C3; hereafter Abutilon) to reduced ice age temperatures (–8 °C) during growth at low [CO2] (200 μmol/mol) 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 (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.
On an absolute basis, the C3 (Abutilon) and C4 species (Amaranthus) exhibited large differences in total mass when grown at low [CO2] (200 μmol/mol) for 22 d. More specifically, the C4 species had five 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 for a total of 22 d, and therefore differences in final biomass mainly 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 producing 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 = 0.000 1, 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.
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 μmol/mol) for 22 d
|Abutilon theophrasti (C3)|
|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|
|Amaranthus retroflexus (C4)|
|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|
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 versus total mass at the high temperature (Table 2).
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 μmol/mol CO2 for 22 d at modern and glacial temperatures
|Abutilon theophrasti (C3)|| || || || |
|30/24 °C (modern)||0.26b||0.94||198a||0.98|
|22/16 °C (glacial)||0.41a||0.85||179b||0.98|
|Amaranthus retroflexus (C4)|| || || || |
|30/24 °C (modern)||0.74a||0.95||69b||0.93|
|22/16 °C (glacial)||0.79a||0.97||98a||0.97|
Overall, gs (stomatal conductance) was higher and SLM (specific leaf mass) was lower in the C3 species compared with 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 (non-significant 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 = 0.000 1), 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 μmol/mol CO2 and 1 400 μmol photons/m2 per s photosynthetic active radiation (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 (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 (Figure 1A) when grown at low [CO2]. Within species, reduced temperature did not have a significant effect on physiological responses (A, Pr, R (respiration); Figure 1A–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 (Figure 1A,B), and was not due to R that was similar between both species at both temperature treatments (Figure 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 (Figure 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).
This study involved growing C3 and C4 plants at low [CO2] (200 μmol/mol) with an 8 °C difference in temperature treatments (22/16 °C versus 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) exhibited a large advantage in absolute growth over the C3 species (Abutilon) at low [CO2]. The C4 species had five 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 μmol/mol), 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; Dippery et al. 1995; Sage 1995; Ward and Strain 1997; Ward et al. 1999; Ward et al. 2005; Sage and Kubien 2007), point to the dominant effect of low [CO2] in producing 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 C3Phaseolus vulgaris grown at 200 μmol/mol 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 years 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 that region.
In modern times (when [CO2] has ranged between 270 and 380 μmol/mol), C4 plants generally occupy regions with warm climates, and occur at low altitudes, and may dominant ecosystems during seasonal peaks in temperature (Kemp and Williams 1980; Rundel 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 decrease below 20 °C (Long 1983). Furthermore, Pittermann 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 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 μmol/mol CO2. It has been shown that A increases sharply between 20 °C 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 °C and 30 °C (by approximately 25%) at 360 μmol/mol CO2. However, in the same Sage (2002) study, Amaranthus showed very modest increases in photosynthetic rate across these same temperatures when measured at 180 μmol/mol CO2, and did not show any differences in photosynthetic rate at 100 μmol/mol 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 (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 used). 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 species (Berry and Björkman 1980; Pearcy and Ehleringer 1984; Sage 2002). In this study, the C3 plants grown at 200 μmol/mol 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 °C and 25 °C when grown at 200 μmol/mol 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].
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 high temperatures. This higher investment in root components may have been an early indication 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, 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.
Materials and Methods
Amaranthus retroflexus L. (C4 dicot) and Abutilon theophrasti Medik. (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 de-ionized water twice each day. Following emergence, pots were watered to saturation with half-strength 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 μmol/mol ([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 1 000 ± 50 μmol photons/m2 per s 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/m2 per s to prevent early initiation of flowering.
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, NE, USA). 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 relationships between root versus shoot mass and leaf area versus total mass by inclusion of data from both harvests (see statistical analyses).
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) 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 similarly to the method of Valentini et al. (1995) at growth CO2 and temperature conditions. 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 (PAM-2000, Walz, Germany) at each of 10 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, OH, USA) to match growth chamber humidity. Light levels for the response curves were attenuated with fine wire screens in 10 steps ranging from a maximum of 1 500 μmol photons/m2 per s 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 ΔCO2, ΔH2O, and Δ 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.
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 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).
(Handling editor: Scott Alan Heckathorn)
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.