*Present address: Department of Biology, University of Utah, Salt Lake City, UT 84112, USA
Photosynthetic performance at low temperature of Bouteloua gracilis Lag., a high-altitude C4 grass from the Rocky Mountains, USA
Article first published online: 25 DEC 2001
© 2000 Blackwell Science Ltd
Plant, Cell & Environment
Volume 23, Issue 8, pages 811–823, August 2000
How to Cite
Pittermann, J. and Sage, R. F. (2000), Photosynthetic performance at low temperature of Bouteloua gracilis Lag., a high-altitude C4 grass from the Rocky Mountains, USA. Plant, Cell & Environment, 23: 811–823. doi: 10.1046/j.1365-3040.2000.00599.x
- Issue published online: 25 DEC 2001
- Article first published online: 25 DEC 2001
- C4 photosynthesis;
- gas exchange;
- Rubisco activity;
- sub-alpine plants;
- temperature responses
The mechanisms controlling the photosynthetic performance of C4 plants at low temperature were investigated using ecotypes of Bouteloua gracilis Lag. from high (3000 m) and low (1500 m) elevation sites in the Rocky Mountains of Colorado. Plants were grown in controlled-environment cabinets at a photon flux density of 700 μmol m−2 s−1 and day/night temperatures of 26/16 °C or 14/7 °C. The thermal response of the net CO2 assimilation rate (A) was evaluated using leaf gas-exchange analysis and activity assays of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPCase) and pyruvate,orthophosphate dikinase (PPDK). In both ecotypes, a reduction in measurement temperature caused the CO2-saturated rate of photosynthesis to decline to a greater degree than the initial slope of A versus the intercellular CO2 response, thereby reducing the photosynthetic CO2 saturation point. As a consequence, A in normal air was CO2-saturated at sub-optimal temperatures. Ecotypic variation was low when grown at 26/16 °C, with the major difference between the ecotypes being that the low-elevation plants had higher A; however, the ecotypes responded differently when grown at cool temperature. At temperatures below the thermal optimum, A in high-elevation plants grown at 14/7 °C was enhanced relative to plants grown at 26/16 °C, while A in low-elevation plants grown at 14/7 °C was reduced compared to 26/16 °C-grown plants. Photoinhibition at low growth temperature was minor in both ecotypes as indicated by small reductions in dark-adapted Fv/Fm. In both ecotypes, the activity of Rubisco was equivalent to A below 17 °C but well in excess of A above 25 °C. Activities of PEPCase and PPDK responded to temperature in a similar proportion relative to Rubisco, and showed no evidence for dissociation that would cause them to become principal limitations at low temperature. Because of the similar temperature response of Rubisco and A, we propose that Rubisco is a major limitation on C4 photosynthesis in B. gracilis below 17 °C. Based on these results and for theoretical reasons associated with how C4 plants use Rubisco, we further suggest that Rubisco capacity may be a widespread limitation upon C4 photosynthesis at low temperature.
C4 plants are common at low latitude and altitude, but are rare to absent at latitudes and altitudes having cool temperate or boreal climates ( Long 1983; Collatz et al. 1998 ). Where growing season temperatures average less than 13 °C, C4 plants are rare; where they average above 22 °C, C4 plants are typically dominant members of herbaceous plant communities ( Long 1983; Sage et al. 1999 ). In temperate grasslands where C3 and C4 grass species co-exist, the C3 to C4 biomass composition of the vegetation is correlated with seasonal temperature. C3 plants have superior growth during cool springs, whereas C4 grasses dominate during mid-summer ( Kemp & Williams 1980; Monson et al. 1983 ). Despite this understanding of the geographic and seasonal distribution of C4 plants, the underlying mechanisms controlling these trends are not clearly understood. In particular, is the lack of C4 plants in cooler climates the result of an inherent limitation in the C4 pathway, or is it a merely a reflection of recent C4 origin in tropical climates? Numerous hypotheses have been proposed to explain the absence of C4 species in low-temperature habitats (for reviews see Long 1983; Ehleringer & Monson 1993; Leegood & Edwards 1996). The leading explanations can be grouped into three general categories.
- 1There is a loss of adaptive value of the C4 syndrome at low temperature simply because it is less efficient than C3 photosynthesis; no injury or enzyme dissociation explains C4 failure.
- 2There is a failure of the C4 photosynthetic apparatus because of chilling injury to one or more of the enzymatic steps of the C4 cycle.
- 3C4 plants are mal-adapted to cold environments as a result of their subscript tropical origin rather than any inherent flaw in the C4 pathway at low temperature. Failure in the cold results from chilling injury throughout the plant, rather than in just the photosynthetic apparatus.
Consistent with the first hypothesis, most C4 species show inferior photosynthesis and growth relative to ecologically similar C3 species below 20 °C ( Berry & Raison 1981; Pearcy et al. 1981 ; Christie & Detling 1982). Quantum yields of C4 species are inferior below about 25 °C, and modelled differences in C3 and C4 quantum yield predict the distribution of a photosynthetic pathway within a grass flora along latitude and altitude gradients ( Ehleringer 1978; Cerling et al. 1997 ). Substantial research has addressed the second hypothesis, showing that a number of enzymes in the mesophyll reactions of the C4 cycle are chilling-sensitive ( Leegood & Edwards 1996; Matsuba et al. 1997 ). Both pyruvate, orthophosphate dikinase (PPDK, EC 18.104.22.168) and phosphoenolpyruvate carboxylase (PEPCase, EC 22.214.171.124) can dissociate in vitro below 8–12 °C. However, species and ecotypic variation is noted in the cold lability of C4 enzymes, indicating that they are not inherently prone to failure in chilling situations ( Sugiyama et al. 1979 ; Krall & Edwards 1993; Simon & Hatch 1994; Matsuba et al. 1997 ). Instead, they may be optimized for the thermal environment the plants are adapted to, with reduced stability at temperatures outside the optimum range. A key question that remains is whether C4 plants adapted to low temperature exhibit enzyme lability at chilling temperatures (< 12 °C). Despite increasing in popularity in recent years, the hypothesis that C4 plants are mal-adapted for cold climates has not received much experimental attention ( Ehleringer & Monson 1993; Long 1999). C4 taxa are largely tropical in origin, and probably evolved less than 20–30 million years ago ( Kellogg 1999). According to this hypothesis, the apparent failure of C4 species in low-temperature settings may result from insufficient time for tropical C4 taxa to radiate into cooler habitats, rather than any barrier associated with C4 physiology.
At present, none of these hypotheses are widely accepted and it remains unknown whether there is any inherent limitation in the C4 syndrome at low temperature. One approach that can minimize difficulties associated with cold injury is to study temperature responses of C4 plants from the coldest habitats where C4 species occur. Numerous C4 species in the genus Bouteloua, Miscanthus, Muhlenbergia, Spartina and Setaria occur at high latitude or altitude ( Sage et al. 1999 ). One species, Bouteloua gracilis (blue grama) is an NAD-ME-type C4 grass that grows from sub-boreal zones of western Canada to Chihuahuan desert communities of northern Mexico, and from an elevation below 1000 m to over 3000 m ( Hitchcock & Chase 1971; Gutierrez et al. 1974 ). Because of its widespread distribution, it encounters as much thermal variation during the growing season as any C4 species, and at the upper end of its distribution it will experience repeated chilling. Presumably, B. gracilis is tolerant of chilling, so that it should be possible to evaluate C4 photosynthetic performance at low temperature with minimal interference from chilling injury.
In the present study, we evaluate mechanisms controlling the temperature response of photosynthesis in low- and high-elevation ecotypes of Bouteloua gracilis using an experimental approach that combines gas-exchange analysis and assays of the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 126.96.36.199) PPDK and PEPCase. The study was designed to evaluate short-term limitations on photosynthesis as a function of temperature, as well as assess how these limitations vary between the ecotypes and during thermal acclimatization. To provide a more comprehensive analysis of possible limitations, we interpret the results in the context of the recent model of C4 photosynthesis by von Caemmerer & Furbank (1999).
METHODS AND MATERIALS
High- and low-elevation ecotypes of Bouteloua gracilis Lag. were collected in August 1996 near Grant, Colorado, USA (39°5′N, 105°30′W; 3000 m) and Dawson Butte, Colorado (39°10′N, 104°30′W; 1500 m), respectively. Mean minimum temperatures during the growing season are 0 °C (June) to 4 °C (July) at the 3000 m location, and 10° to 14 °C near Dawson Butte ( Bowman & Turner 1993; National Weather Service, 1999). Average maximum temperatures for July are 20–23 °C at 3000 m in the central Rockies, and 29–31 °C on the Prairie near Dawson Butte. Because of the high solar radiation level in the Rocky Mountains, maximum leaf temperatures may be 3–10 °C greater than air temperature at these sites. At the high-altitude site, night-time radiation loss may reduce leaf temperature to below air temperature by a few degrees, such that leaves may become frosted on cooler nights during the growing season ( Jordan & Smith 1995).
Transplants were grown in 4 L pots of soil (60% Pro-mix, Premier Brands, Redhill, Pennsylvania, USA; 20% perlite, 20% sand) in Conviron E-15 growth cabinets. Plants were grown at 26 °C day/16 °C night temperatures, with 16 h photoperiods and a daytime photon flux density of 700 μmol m−2 s−1. Plants were watered daily and fertilized twice-weekly with a half-strength Hoagland's solution.
To evaluate the effects of prolonged chilling on the photosynthetic performance of the two B. gracilis ecotypes, tillers established at 26/16 °C were transferred to 14/7 °C at a light intensity of 700 μmol m−2 s−1. These plants were grown for a minimum of 17 d prior to gas exchange to allow acclimatization to the cooler conditions.
Measurement of photosynthetic CO2 assimilation and chlorophyll fluorescence
Photosynthetic and stomatal responses to intracellular CO2 partial pressure (Ci), temperature, and light were measured using a null balance gas-exchange system modified for humidity control ( Sharkey 1985; Field et al. 1989 ). Gas concentration in the leaf chamber was established using three mass flow controllers (Model 840, Sierra Instruments, Monterey, California, USA) that regulated the mixing ratio of pure N2, O2 and 3% CO2 in air. Additionally, 3% CO2 in air was added to the leaf chamber via a fourth flow controller to offset photosynthetic depletion of CO2 from the air stream. Water vapour and CO2 content of the air stream before and after the leaf chamber were measured using an infra-red gas analyser (Li-Cor 6262, Lincoln, Nebraska, USA). Thermal control was provided by Peltier thermoelectric devices (Melcor, Trenton, New Jersey, USA) and a refrigerated water bath. During each gas-exchange experiment, light levels were adjusted to just saturate photosynthesis. Because the light saturation point declined with temperature (data not shown), light levels at the lower temperatures were typically set near 1000 μmol m−2 s−1, while at the warmer temperatures they were set 50–100% higher. The vapour pressure differences between leaf and air (VPD) were 8 to 12 mbar below 25 °C, but exceeded 20 mbar above 30 °C. Gas-exchange parameters were calculated according to von Caemmerer & Farquhar (1981).
To evaluate effects of long-term chilling on the photochemistry of B. gracilis, the dark-adapted Fv/Fm response was measured before and after 28 d of growth at 14/7 °C. Nine leaves from three plants per treatment were dark-treated for 30 min before Fv/Fm was measured using a pulse-modulated fluorimeter (Optisciences OS-500, Haverhill, Massachusetts, USA).
Enzyme and chlorophyll assays
Enzyme activity was determined on leaves from plants used for gas-exchange analysis. Leaf samples were collected from 4–6 plants exposed to atmospheric levels of CO2 and a light intensity above 1000 μmol photons m−2 s−1. After gas-exchange measurements, 3–5 cm2 of leaf area from 4–6 leaf blades were quickly harvested and frozen in liquid N2, after which they were stored at –80 °C until assay.
The activity of PEPCase (modified from Ashton et al. 1990 ) and both activity and content of Rubisco ( Sage & Seemann 1993) were determined using aliquots from a common leaf extract. A leaf sample (2–3 cm2) was extracted in 3 mL of 100 m M HEPES buffer containing 2 m M EDTA, 5 m M MgCl2, 5 m M DTT, 0·14% (w/v) BSA, 1% PVPP, 4 m M amino-n-caproic acid, 0·8 m M benzamide and 2 m M NaH2PO4 at 0 °C. For the Rubisco activity assay, an aliquot of the extract was incubated at 0 °C for 10 min in the presence of 10 m M NaHCO3 and 20 m M MgCl2 in order to fully carbamylate Rubisco. Rubisco activity was assayed using the carbamylated extract by determining the rate at which 14CO2 was incorporated into acid-stable extracts. Assays were conducted in triplicate for 30–120 s at 1, 7, 13, 17, 23, 30 and 36 °C in an assay buffer containing 100 m M Bicine at pH 8·2, 1 m M EDTA, 20 m M MgCl2, 1 m M DTT, 0·1–0·3 units ml−1 ribulose-5-P kinase, 1 unit ml−1 phospho-ribulo-isomerase, 2 m M ATP, 2 m M ribose-5-P and 15 m M [14C]NaHCO3 (ICN Pharmaceuticals, Costa Mesa, California, USA). Acid-stable radioactivity was determined by liquid scintillation spectroscopy.
Rubisco content was determined by measuring the amount of 14C-carboxy-arabinitol bisphosphate (CABP) that bound to Rubisco catalytic sites, assuming a binding ratio of 6·5 CABP molecules per Rubisco molecule ( Butz & Sharkey 1989). Aliquots of the carbamylated Rubisco extract were incubated for 30 min in the presence of 40 μM CABP at room temperature, followed by a 3 h incubation at 37 °C in the presence of rabbit anti-Rubisco serum. The resulting immunoprecipitated Rubisco–CABP complexes were then filtered with a Gelman Supor-200 membrane filter (Ann Arbor, Michigan, USA) and thoroughly rinsed with a 10 m M sodium phosphate buffer (pH 7·6, with 10 m M MgCl2 and 150 m M NaCl). The radioactivity of the 14CABP–Rubisco complex that was bound to the filters was counted by liquid scintillation spectroscopy.
PEPCase was assayed for 30–60 s by injecting 20 μL of unmodified leaf extract into 480 μL of 50 m M Bicine buffer (pH 8·2) containing 1 m M EDTA, 5 m M MgCl2, 5 m M DTT, 5 m M PEP, 0·2 m M NADH, 2 units ml−1 malate dehydrogenase, 5 m M glucose-6-phosphate and 5·4 m M [14C]NaHCO3. Acid-stable radioactivity was quantified using liquid scintillation spectroscopy.
The activity of PPDK was assayed spectrophotometrically by coupling the production of PEP to NADH oxidation via PEPCase and malate dehydrogenase ( Ashton et al. 1990 ). Leaves (1·5–2·5 cm2) were rapidly (< 90 s) extracted in 2·5 mL of 50 m M HEPES pH 8·2 buffer containing 1% (w/v) PVPP, 0·14% (w/v) BSA, 5 m M amino-n-caproic acid, 1 m M benzamide, 5 m M DTT, 2 m M NaH2PO2, 14% (v/v) glycerol and 1 m M EDTA. The extract was centrifuged for 10 s at 8000 g, and the resulting supernatant incubated for 30 min at 25 °C. During this incubation period, the in vitro activity of PPDK rose by about 10–30% before stabilizing at a maximum value. The assay was initiated by adding 20 μL of supernatant to 480 μL of an assay buffer (50 m M HEPES, pH 8·2, 7 m M DTT, 2 m M glucose-6-phosphate, 5 units ml−1 malate dehydrogenase, 2 m M pyruvic acid, 2 mm ATP, 1 m M NaH2PO4, 8 m M MgCl2, 5 mm (NH4)2SO4, 10 m M NaHCO3, 0·2 m M NADH, 0·5 units PEPCase, 0·5 units myokinase and 0·5 units pyrophosphatase. PPDK activities were recorded at 7, 10, 13, 17, 23, 30 and 36 °C over 7–10 min using a Hewlett-Packard 8452A diode array spectrophotometer to measure change in absorbance at 340 nm.
Chlorophyll, carbon and nitrogen content
Chlorophyll content was determined on aliquots of the leaf extract used for Rubisco assay. Following tissue homogenization, 200 μL aliquots were added to 800 μL of N,N-dimethyl formamide, vortexed and allowed to incubate for 15 min in darkness. After a second vortexing, the extract was centrifuged at 12 000 g for 2 min and the chlorophyll content determined spectrophotometrically at 647 and 664 nm ( Porra et al. 1989 ). Nitrogen content of B. gracilis leaves from the various growth treatments was assessed using a LECO carbon: nitrogen analyser (LECO Corp., St Joseph, Michigan, USA).
CO2 assimilation rate in Bouteloua gracilis
In both ecotypes, the rate of net CO2 assimilation (A) in plants grown at 26/16 °C rose from 7 °C to an optimum near 36 °C ( Fig. 1). At all measurement temperatures, the low-elevation grasses had photosynthetic rates that were 40% (low temperature) to 15% (high temperature) greater than in the high-elevation plants. Little ecotypic differentiation was observed in the response of stomatal conductance to temperature ( Fig. 2a). Stomatal conductance in both ecotypes increased from 0·1 mol m−2 s−1 at 10 °C to 0·3 mol m−2 s−1 at the optimum temperature for photosynthesis, but declined above 36 °C where VPDs exceeded 20 mbar. The Ci/Ca response (where Ca is the ambient CO2 partial pressure) was similar in both ecotypes in that it decreased from near 0·7 at the lower temperatures to near 0·3 at 40 °C; however, the high-elevation ecotype had greater Ci/Ca below 20 °C ( Fig. 2b).
The response of A to Ci was compared in the ecotypes at 33, 23 and 13 °C ( Fig. 3). As temperature declined, CO2-saturated A declined to a greater degree than the initial slope of the A versus Ci response. In both ecotypes at 23 and 33 °C, there was no effect on the initial slope. At 13 °C, the initial slope declined in the low-elevation ecotype, but remained unaffected in the high-elevation ecotype. As a consequence of the different responses of the CO2-saturated plateau and the initial slope, the CO2 saturation point declined from 100 to 150 μbar at 33 °C to < 60 μbar at 13 °C. The net CO2 assimilation rate was similar between the ecotypes in the initial slope region of the CO2 response, but the high-elevation ecotype exhibited a sharper transition to the CO2-saturated plateau. The operating Ci (Ci at a Ca of 360 μbar) corresponded to the CO2-saturated region of the A/Ci curve, being > 200 μbar in both ecotypes at 13, 23 and 33 °C (see arrows, Fig. 3). No major change was observed in the operating Ci with temperature change in these experiments, which contrasts with the response noted in Fig. 2(b). We attribute this discrepancy to maintenance of VPD near 10 mbar in the A/Ci experiments.
Differences between low- and high-elevation ecotypes of B. gracilis
Net CO2 assimilation rate, Rubisco activity, Rubisco content and the molar activity of Rubisco were signifi-cantly higher in the low-elevation varieties than the high-elevation plants ( Table 1). There were no significant differences in PEPCase activity or chlorophyll and nitrogen content between the ecotypes ( Table 1).
|Low elevation (1500 m)||High elevation (3000 m)|
|Assimilation rate (μmol m–2 s–1)||30·1 ± 0·6a||22·5 ± 0·9b|
|Rubisco content (g m–2)||0·66 ± 0·05a||0·58 ± 0·04b|
|Rubisco activity (μmol m–2 s–1)||41·4 ± 1·9a||27·3 ± 4·1b|
|Rubisco molar activity (mol mol s–1)||28 ± 2a||23 ± 2b|
|PEPCase activity (μmol m–2 s–1)||126 ± 12||115 ± 15|
|Chlorophyll content (μmol m–2)||344 ± 14||318 ± 12|
|Leaf nitrogen content (mmol m−2)||86 ± 4||93 ± 4|
The thermal response of Rubisco, PEPCase and PPDK activities in B. gracilis
The temperature response of Rubisco activity was measured in leaf tissue used for the gas-exchange measure-ments presented in Fig. 1. Rubisco activity from low-elevation grasses was 30–50% greater than from the high-elevation plants ( Fig. 4). In both types, Rubisco activity increased with increasing temperature in an exponential manner, showing little sign of levelling over the range studied ( Fig. 4). Below 17 °C, Rubisco activity was identical in response and magnitude to A in both ecotypes. As temperature rose above 20 °C, Rubisco activity increasingly rose above the corresponding value of A at a given temperature, such that it was 2–3 times greater at the thermal optimum of A at 36 °C. The thermal response of PEPCase activity rose in a similar manner to Rubisco activity, but in both ecotypes it was about three times greater than Rubisco activity at all measurement temperatures ( Fig. 4).
The activity of PPDK increased substantially in response to higher temperature, reaching 31 μmol m−2 s−1 at 36 °C in the low-elevation ecotype and 22 μmol m−2 s−1 in the high-elevation grasses ( Fig. 5a). The activity of PPDK in B. gracilis was lower than that required to sustain observed CO2 assimilation rates. We believe this was because of incomplete extraction or the presence of inhibitory compounds such as phenolics in the extract. Despite this limitation, the total rate of PPDK activity was roughly proportional to Rubisco activity as temperature declined ( Fig. 5b).
In both ecotypes at 7–17 °C, the Q10 of photosynthesis was near 2·6 and its apparent activation energy (Ea) was 62–68 kJ mol−1 ( Table 2). Between 7 and 17 °C, the Ea and Q10 values of A were similar to the Ea and Q10 values for PPDK in both ecotypes and for Rubisco from the low-elevation plants; however, they were less than the corresponding values observed for PEPCase from both ecotypes, and for Rubisco from the high-elevation ecotype. Above 17 °C, the Q10 and Ea values for photosynthesis were similar in the two ecotypes, but were much less than values observed at 7–17 °C. In each ecotype above 17 °C, the Ea and Q10 values of A were about half those of Rubisco and PPDK, and slightly less than those of PEPCase. Arrhenius plots for the enzyme responses to temperature showed slope changes near 17 °C for PEPCase in both ecotypes, and near 17 °C for Rubisco from the high-elevation ecotype ( Fig. 6).
|CO2 assimilation rate||Rubisco||PEPCase||PPDK|
|7–17 °C||17–34 °C||7–17 °C||17–36 °C||7–17 °C||17–36 °C||7–30 °C|
|Apparent Ea||61·6 ± 1·5||27·5 ± 1·7||63·4 ± 6·9||55·5 ± 2·9||80·3 ± 11·1||43·1 ± 6·4||62·7 ± 4·7|
|Apparent Ea||67·6 ± 13·3||32·5 ± 2·9||87·0 ± 16·7||62·0 ± 8·5||82·6 ± 6·4||46·0 ± 8·5||53·7 ± 4·8|
The effects of prolonged chilling on the photosynthetic and fluorescence responses in the B. gracilis ecotypes
Relative to plants grown at 26/16 °C, photosynthesis was inhibited below a measurement temperature of 25 °C in low-elevation plants grown at 14/7 °C ( Fig. 7a). Plants of the high-elevation ecotype had slightly higher A at measurement temperatures below 30 °C when grown at 14/7 °C than when grown at 26/16 °C ( Fig. 7b). Additionally, high-elevation plants grown at the lower temperature had a wide thermal optimum of photosynthesis centred on 30 °C instead of the 37 °C optimum exhibited by the warm-grown plants. As a result of these shifts, A below 25 °C was similar between the ecotypes following growth at 14/7 °C. In both ecotypes, the 14/7 °C-acclimatized plants demonstrated no obvious qualitative difference in their A/Ci response compared to the 26/16 °C-grown grasses (data not shown). Nitrogen content was also unaffected, being near 90 mmol m−2 in both ecotypes grown at 14/7 °C.
A slight difference in Fv/Fm values was observed between the two ecotypes following 28 d of growth at 14/7 °C. Prior to chilling, both ecotypes exhibited similar dark-adapted Fv/Fm values (0·76 ± 0·01 in the high-elevation ecotype, 0·74 ± 0·01 in the low-elevation ecotype, means ± SE). After 28 d growth at 14/7 °C, Fv/Fm in the high-elevation ecotype was 0·70 ± 0·01, slightly greater than the value of 0·65 ± 0·04 seen in the low-elevation ecotype (differences significant at P < 0·05).
Photosynthetic limitations in Bouteloua gracilis as a function of temperature
Our data indicate that Rubisco is the predominant control over the temperature response of photosynthesis in B. gracilis below 17 °C. Below this temperature, A was CO2-saturated, as it is theoretically predicted to be if Rubisco capacity is limiting ( von Caemmerer & Furbank 1999). Moreover, despite ecotypic differences in biochemical and photosynthetic parameters, the activity of Rubisco was equivalent to A in both low- and high-elevation ecotypes of B. gracilis below 17 °C. In C4 plants, a near 1:1 association between A and in vitro Rubisco activity should occur if Rubisco capacity is limiting, because the high CO2 level in the bundle sheath allows Rubisco to operate near its Vmax ( von Caemmerer & Furbank 1999).
In theory, the CO2-saturated rate of A could also be determined by a ribulose-1,5-bisphosphate (RuBP) regeneration limitation, or phosphoenol pyruvate (PEP) regeneration limitations caused by low PPDK activity ( von Caemmerer & Furbank 1999). While we cannot comment directly on the controlling role of RuBP regeneration, we do not think it is high below 17 °C because of the close association between Rubisco activity and A. If RuBP regeneration became the predominant control over A at low temperature, we would expect A to fall below Rubisco activity. A similar result would also be expected if PPDK capacity limited the thermal response of A at low temperature. Low-temperature inhibition of photosynthesis in C4 plants has often been attributed to the cold lability of PPDK ( Long 1983; Edwards et al. 1985 ; Simon & Hatch 1994), but we saw little evidence for this here. The ratio of PPDK activity relative to Rubisco activity did not decrease appreciably at cooler temperatures in B. gracilis, and no obvious slope change occurred in the Arrhenius plot of PPDK. A change in the slope of the Arrhenius plot of PPDK indicates dissociation, and previous studies have reported slope changes below 12 °C ( Shirahashi et al. 1978 ; Edwards et al. 1985 ). Dissociation of PPDK in the cold may not be a universal phenomenon in C4 plants because cold-adaptive genotypes possess more stable forms of the enzyme at low temperature ( Usuda et al. 1984 ; Simon & Hatch 1994; Usami et al. 1995 ). Stability at low temperature may result from the presence of cryoprotectants such as PEP and sucrose, or different enzyme structures ( Shirahashi et al. 1978 ; Yamazaki & Sugiyama 1984; Krall et al. 1989 ; Usami et al. 1995 ; Ohta et al. 1997 ). Another possibility is that we may have too few, and perhaps too variable, data points to resolve any thermal breaks below 12 °C. This, and our incomplete recovery of PPDK activity, demonstrate that more work is needed to clarify the controlling role of this enzyme in B. gracilis.
PEPCase capacity does not appear to be a major control because reducing the temperature from 33 to 13 °C primarily affected the CO2-saturated plateau of A, with either no (high-elevation ecotype) or modest (low-elevation ecotype at 13 °C) effect on the initial slope of the CO2 response. Disproportional reductions in PEPCase capacity can reduce CO2-saturated A in C4 plants, but this is accompanied by a reduction in the initial slope of photosynthesis ( von Caemmerer & Furbank 1999). Consistently, we saw no evidence for a disproportional decline in PEPCase activity with temperature reduction, as it remained at least three times the activity of Rubisco and PPDK, and was similarly greater than A at all temperatures. This excess of PEPCase activity over A should be enough to saturate the bundle sheath with CO2 ( von Caemmerer & Furbank 1999).
We also saw little evidence that stomatal control over A increased below the thermal optimum, as the operational Ci remained above the CO2 saturation point of photosynthesis. Stomatal conductance may also be an important limitation, but only below the CO2 saturation point of A (assuming that stomatal conductance is uniform). Temperature change may disproportionately alter stomatal conductance relative to A, causing the operational Ci to change its position relative to the CO2 saturation point, and thus potentially change the degree of stomatal limitation ( Sage & Sharkey 1987). Although the operational Ci did decline with rising temperature when VPD also rose, it only fell below the CO2 saturation point at temperatures above the thermal optimum. Thus any stomatal control that was present in B. gracilis had its effect at high temperature.
While the evidence points to a pronounced Rubisco limitation below 17 °C, Rubisco capacity becomes well in excess of A above this temperature, indicating that photosynthetic control has shifted from Rubisco to one of the other possible limitations that control A in B. gracilis. Between 20 °C and the temperature optimum, this control would likely be PPDK capacity or RuBP regeneration capacity, as the operational Ci corresponds to the CO2-saturated region of the A/Ci response. Above 35 °C, PEPCase activity may become an important limitation, as our estimated reduction in Ci/Ca ratios at high temperature places the operating Ci in the initial slope region of the A/Ci response.
Apparent activation energies can indicate patterns of limitation, but using in vitro values to identify in vivo limitation should be treated with caution unless there are clear supporting reasons ( Berry & Raison 1981). Here, our Ea values were partially consistent with a pattern of Rubisco control at low temperature, although they also indicate shared control by PPDK. Below 17 °C, the Ea of A and Rubisco were similar in the low-elevation plants, as were the Ea of A and PPDK. In high-elevation plants, the Ea of Rubisco was greater than that for A, while PPDK was slightly less. In both ecotypes, the greater Ea of PEPCase than A below 17 °C indicates little control for this enzyme at low temperature. Above 17 °C, the low Ea values of A relative to Rubisco and PPDK indicate that neither Rubisco nor PPDK are the major limiting factors in this temperature range; instead RuBP regeneration or PEPCase capacity may exert the major control over A.
Limitation of C4 photosynthesis by Rubisco capacity at cooler temperatures has been previously proposed ( Caldwell et al. 1977 ; Pearcy 1977), but not pursued in detail. Along with PPDK and PEPCase, Rubisco activity has been well correlated with A in C4 plants ( Usuda et al. 1984 ; Furbank et al. 1997 ), indicating significant control for each of these enzymes at moderate temperature (25–30 °C). Using antisense constructs to each of these enzymes, recent work with Flaveria bidentis and Amaranthus edulis indicates that, at 25 °C, Rubisco accounts for 50–70% of the metabolic control over A at 360 μbar CO2, while PPDK and PEPCase account for only 20–30% ( Dever et al. 1997 ; Furbank et al. 1997 ; von Caemmerer et al. 1997 ). At low CO2 (30 μbar), the control over A by PEPCase in A. edulis rises to 68% ( Dever et al. 1997 ). In response to changing temperature, our results indicate that Rubisco will exert greater control at lower temperature, while PPDK and PEPCase will dominate control at higher temperature. The antisense transformants of Dever et al. (1997) and Furbank et al. (1997) would be useful for evaluating this possibility.
Long-term changes in photosynthetic limitation
While our measurements of gas exchange and enzyme activities do not indicate a PPDK limitation during short-term exposure to low temperature in plants grown at 26/16 °C, they do not necessarily rule out PPDK limitations that might arise following long-term chilling (hours to days). In Echinochloa crus-galli, a widespread C4 weed, prolonged (24 h) exposure to 14/7 °C caused a 15% decrease in both PPDK activity and A ( Potvin et al. 1986 ). We evaluated the possibility of PPDK limitation developing over the long-term by growing B. gracilis at 14/7 °C. As with the short-term low-temperature exposure, the high-elevation B. gracilis plants grown at 14/7 °C showed no depressions in CO2 assimilation below the thermal optimum relative to plants grown at 26/16 °C ( Fig. 7), indicating no appearance of a PPDK limitation during long-term chilling.
Ecotypic differences between low- and high-elevation varieties of B. gracilis
The primary differences between the ecotypes were (1) the low-elevation plants exhibited higher rates of A across a range of temperature than high-elevation plants when grown at 26/16 °C; (2) growth at 14/7 °C slightly enhanced A below 18 °C in the high-elevation ecotype, and slightly depressed it in the low-elevation ecotype; and (3) the Arrhenius plot for Rubisco activity showed a marked break at 17 °C in the high-elevation plants, but not the low-elevation species, indicating there may be changes in Rubisco properties during ecotypic adaptation. These results are consistent with Bowman & Turner’s (1993) observation that a Bouteloua gracilis population from 1400 m in Colorado had higher A than two Rocky Mountain ecotypes from 2600 and 3050 m when all were grown in a common environment. Also, reducing daytime growth temperature from 30 to 20 °C depressed A approximately 20% in the low-elevation ecotype, but not the high-elevation ecotypes ( Bowman & Turner 1993). Matsuba et al. (1997) observed that Spartina anglica from cool temperate salt marshes had higher A across a range of temperatures following 1–4 weeks at lower growth temperature, while Zoysia japonica, a chilling-sensitive species from warm climates, had reduced A following the same treatment. These results indicate that a common acclimatization response in cool-climate C4 plants is to maintain or enhance A when grown at low temperature, while C4 species from warmer climates respond with reductions in A.
Although CO2 assimilation is unaffected by prolonged exposure to chilling temperatures in B. gracilis, the decline in Fv/Fm ratios indicates that slight photoinhibition did occur. A reduction in the Fv/Fm value indicates either damage to photosystem II (PSII) or activation of persistent photoprotection mechanisms ( Genty & Harbinson 1996; Haldimann 1998). The high-elevation plants suffered less photoinhibition than the low-elevation grasses, indicating an increased level of tolerance to low-temperature and high-irradiance conditions. In a similar study, Blowers & Baker (1995) measured the light use efficiency of PSII in Cyperus longus from northern and southern latitudes, and found no difference in photochemical yield between plants grown at 14 and 25 °C. They concluded that low-temperature-induced depressions in A in these ecotypes were not directly related to changes in light use of PSII. Photoinhibition has been extensively studied in Zea mays and shown to result from both PSII reaction centre damage and xanthophyll cycle protection ( Bredenkamp & Baker 1994; Haldimann 1998). Warm varieties are most affected, and varieties originating in high altitude or latitude show greater resistance ( Haldimann 1998; Long 1999). Together, these results indicate that photoinhibition may not be a universal lesion that prevents C4 exploitation of low-temperature habitats.
Is Rubisco an inherent control over C4 performance at low temperature?
In this study, we observed little evidence for damage to the photosynthetic apparatus in B. gracilis exposed to low temperature. As expected by its natural occurrence, our results confirm that it is tolerant of the cold and has overcome chilling sensitivity that may exclude many species of tropical origin from cold climates, regardless of their photosynthetic pathway. Also, our results show no obvious failure of the C4-cycle enzymes in B. gracilis, and photoinhibition does not appear to be a severe problem, at least in the growth conditions used here. Instead, the predominant limitation at low temperature appears to be Rubisco capacity, which establishes a ceiling on C4 photosynthetic performance at low temperature. We propose that this ceiling imposed by Rubisco over C4 photosynthesis may be a critical limitation that acts in concert with inferior quantum yields to restrict C4 success in low-temperature environments.
An important thermal response of Rubisco is the effect of temperature on its CO2 saturation point. Because both the Km for CO2 and Vmax of Rubisco decrease with declining temperature ( Berry & Raison 1981), the amount of CO2 required to saturate Rubisco declines with temperature, such that its CO2 saturation point approaches current atmospheric CO2 levels below 10 °C ( Fig. 8). This has two major consequences. First, little activity of a C4 pump is required for plants to CO2 saturate Rubisco activity. Should there be a disproportional loss of PPDK activity in the cold because of a chilling-induced loss of stability, the consequence at low temperature may be minor because the CO2 requirement of Rubisco is so low. Second, at low temperature, there is little advantage in concentrating CO2 around Rubisco, and attempts to do so would be wasteful in terms of energy and raw materials. Because Rubisco operates close to Vmax in C3 as well as C4 plants at low temperature, the amount of Rubisco becomes an important consideration. Here, C3 plants have a clear advantage over C4 plants at low temperature. Typically, C3 plants contain 2–4 times more Rubisco per unit leaf area than similar C4 plants ( Sage et al. 1987 ; Long 1999). Assuming a Rubisco limitation, this means that, at low temperature, C4 species will always be photosynthetically inferior to C3 species because they do not, or cannot, hold as much Rubisco. In C4 plants, restricting Rubisco to the bundle sheath reduces the cellular volume in which Rubisco can be housed relative to C3 plants. This may limit the amount of Rubisco that C4 plants can accumulate to values below well what similar C3 species could maintain. Such a structural constraint could thus be an inherent limitation that prevents C4 plants from matching the photosynthetic performance of similar C3 plants at low temperature.
If Rubisco capacity is the photosynthetic ceiling on A in C4 plants at low temperature, then a hypothetical acclimatization mechanism would be to increase Rubisco content. Although not evident in B. gracilis, others have reported greater amounts of Rubisco in C4 plants from cool environments ( Long 1999). For example, cool maritime species of C4Atriplex invest about 20% of soluble protein in Rubisco, while Tidestromia oblongifolia from hot desert environments of south-western North America invests less than 10% of its soluble protein in Rubisco ( Berry & Raison 1981). In high-altitude C4 species from central Asia (> 4000 m), Rubisco content is also > 20% of soluble protein, and in one case (Halogeton glomeratus) it is 37%; species from < 2500 m elevation in the same region have less than 10% of soluble protein as Rubisco ( Pyankov & Voznesenskaja (1995). In comparison, C3 species invest over 40% of soluble protein in Rubisco, a proportion that also rises with declining temperature ( Leegood & Edwards 1996).
While acclimatization to the point of equalling C3 photosynthetic performance may be possible if the bundle sheath tissue can accumulate enough Rubisco, it is prob-ably detrimental from an ecological perspective. For C4 species, the greatest photosynthetic advantage occurs above 30 °C because in C3 plants photorespiration is high and Rubisco operates well below CO2 saturation above this temperature. If C4 species accumulate enough Rubisco to match the photosynthetic potential of C3 plants at low temperature, then during warmer episodes of the day and season, the Rubisco capacity would be well in excess of the amount needed to exploit the warmer temperatures. As a consequence, the nitrogen use efficiency of the plant would decline. By expressing high levels of Rubisco, C4 plants could realize modest increases in photosynthetic capacity at low temperature, but this could come at the expense of their nitrogen use efficiency advantage, which may be one of the important factors contributing to C4 success.
This work was supported by grant OGP0154273 from the Natural Science and Engineering Council of Canada to R.F.S. and a Strong-Hull Scholarship to J.P. The assistance of Debbie Tam (N analysis), David Kubien (manuscript comments) and Seth Seegobin (manuscript comments) was much appreciated.
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