Variation in the k_cat of Rubisco in C₃ and C₄ plants and some implications for photosynthetic performance at high and low temperature

Abstract

The capacity of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) to consume RuBP is a major limitation on the rate of net CO₂ assimilation (A) in C₃ and C₄ plants. The pattern of Rubisco limitation differs between the two photosynthetic types, as shown by comparisons of temperature and CO₂ responses of A and Rubisco activity from C₃ and C₄ species. In C₃ species, Rubisco capacity is the primary limitation on A at light saturation and CO₂ concentrations below the current atmospheric value of 37 Pa, particularly near the temperature optimum. Below 20 °C, C₃ photosynthesis at 37 and 68 Pa is often limited by the capacity to regenerate phosphate for photophosphorylation. In C₄ plants, the Rubisco capacity is equivalent to A below 18 °C, but exceeds the photosynthetic capacity above 25 °C, indicating that Rubisco is an important limitation at cool but not warm temperatures. A comparison of the catalytic efficiency of Rubisco (k_cat in mol CO₂ mol⁻¹ Rubisco active sites s⁻¹) from 17 C₃ and C₄ plants showed that Rubisco from C₄ species, and C₃ species originating in cool environments, had higher k_cat than Rubisco from C₃ species originating in warm environments. This indicates that Rubisco evolved to improve performance in the environment that plants normally experience. In C₄ plants, and C₃ species from cool environments, Rubisco often operates near CO₂ saturation, so that increases in k_cat would enhance A. In warm‐habitat C₄ species, Rubisco often operates at CO₂ concentrations below the K_m for CO₂. Because k_cat and K_m vary proportionally, the low k_cat indicates that Rubisco has been modified in a manner that reduces K_m and thus increases the affinity for CO₂ in C₃ species from warm climates.

Amaranthus, Chenopodium, CO₂ response, C₃ and C₄ photosynthesis, Rubisco, temperature response.

Introduction

The evolution of C₄ photosynthesis resulted in the localization of Rubisco into the bundle‐sheath compartment where CO₂ concentration is higher than the surrounding atmosphere (Edwards et al., 1985). At moderate temperatures of 20–30 °C, Rubisco in C₄ plants encounters CO₂ concentrations close to saturation, and the potential for photorespiration is low (von Caemmerer and Furbank, 1999). By contrast, CO₂ concentrations in C₃ plants at 20–30 °C are near the K_m for CO₂ of Rubisco, and photorespiration can be high (Jordan and Ogren, 1984; Sharkey, 1988). As a result of the high CO₂ environment that allows Rubisco to function near CO₂ saturation, C₄ species at 20–30 °C require one‐third to one‐quarter as much Rubisco as C₃ species to maintain a given CO₂ assimilation rate (Brown, 1978; Sage et al., 1987; Long, 1999). This reduced requirement explains the 60–80% lower Rubisco content of C₄ plants relative to C₃ species of similar life form (Long, 1999). In many C₄ species, the kinetic properties of Rubisco are also modified from the ancestral C₃ isozyme to yield a type with kinetics similar to CO₂‐concentrating algae. For example, the k_cat and K_m of Rubisco from C₄ grasses can be twice that of C₃ grasses and dicots, indicating that C₄ plants evolved a type of Rubisco that more efficiently utilizes high CO₂ (Yeoh et al., 1980, 1981; Seemann et al., 1984; Sage and Seemann, 1993).

At moderate temperatures, the control of Rubisco and other limitations over photosynthetic capacity are well understood, and robust theoretical models of C₃ and C₄ photosynthesis are now available (Harley and Sharkey, 1991; von Caemmerer and Furbank, 1999; von Caemmerer, 2000). Below moderate temperatures, the understanding of photosynthetic limitations is less certain In early work, Rubisco was suggested to limit C₄ photosynthesis below 20 °C (Björkman and Pearcy, 1971; Caldwell et al., 1977; Pearcy, 1977). Subsequent research emphasized chilling sensitivity and limitations arising from cold‐lability of light‐harvesting and C₄‐cycle enzymes (reviewed in Leegood and Edwards, 1996). Recent work using C₄ plants from cooler climates has shown that deficiencies in light‐harvesting and PPDK do not appear following chilling, indicating that C₄ photosynthesis is not inherently limited by these processes (Simon and Hatch, 1994; Leegood and Edwards, 1996; Matsuba et al., 1997; Long, 1999). Instead, as indicated by early work, Rubisco appears to exert high control over net CO₂ assimilation (A) at low temperature in chilling‐tolerant C₄ species (Pittermann and Sage, 2000, 2001). In chilling‐tolerant C₃ species, the capacity of starch and sucrose synthesis to regenerate P_i for photophosphorylation commonly limits A at low temperatures (8–15 °C), but primarily when plants were grown at moderate conditions (Sage and Sharkey, 1987; Labate and Leegood, 1988; Labate et al., 1990). When plants are grown in cool conditions, the capacity for P_i regeneration apparently increases as indicated by the recovery of O₂ and CO₂ sensitivity of A (Cornic and Louason, 1980; Huner et al., 1986, 1993; Mawson et al., 1986; Holaday et al., 1992, 1993; Paul et al., 1990), and the control over A can shift to either Rubisco capacity or electron transport capacity (Mawson and Cummins, 1989; Holaday et al., 1992; Leegood and Edwards, 1996).

Similarly, at temperatures above 30 °C, the controlling role of Rubisco over C₃ and C₄ photosynthesis is uncertain. From gas exchange analyses, C₃ leaves exposed to high temperature often maintain intercellular CO₂ levels that fall on the initial slope of the photosynthetic CO₂ response curve (Sage and Sharkey, 1987; Sage et al., 1990a, 1995). This is a region commonly thought to reflect a limitation in Rubisco capacity (von Caemmerer, 2000). Alternatively, in the C₃ weed Chenopodium album, whole‐chain electron transport exhibits an identical temperature optimum as photosynthesis, indicating RuBP regeneration capacity above the temperature optimum may be a principal limitation on A (Sage et al., 1995). Rubisco also deactivates at high temperature in C₃ species (Weis, 1981; Kobza and Edwards, 1987; Sage et al., 1990a; Vu et al., 1997). This could be a regulatory response to a limitation in RuBP regeneration, as proposed for C. album (Sage et al., 1995). Alternatively, as shown for wheat, cotton and tobacco, it may be a direct effect of high temperature on the carbamylation state, in which case Rubisco capacity declines, lowering A in the process (Feller et al., 1998; Law and Crafts‐Brandner, 1999; Sharkey et al., 2001). In C₄ plants, Rubisco does not appear to limit A at elevated temperature because its in vitro capacity is well in excess of the net photosynthetic rate (Pittermann and Sage, 2000, 2001).

To understand plant performance in the field, it is necessary to have a comprehensive picture of Rubisco performance across a range of temperatures. In addition, because the modern flora evolved in atmospheres that were substantially different from those of today, consideration of Rubisco use and photosynthetic temperature responses across a range of CO₂ concentrations is necessary. This report reviews the current understanding of Rubisco control over C₃ and C₄ photosynthesis at different temperature and CO₂ conditions. New data on photosynthetic and Rubisco temperature responses are presented, including results that show the k_cat of Rubisco varies between C₃ plants originating in cool versus warm habitats.

Materials and methods

Plant material and culture

Plants were grown in four locations, (1) the greenhouse of the University of Georgia, Athens (UGA); (2) in fields in the vicinity of Athens, Georgia USA (Athens); (3) outdoors in 20 l pots of soil at the roof‐top garden of the Botany Department, University of Toronto (UTB) or (4) unmanaged fields near the campus of the University of Toronto (UT). Samples for Rubisco assay were collected during summer months at 25–35 °C by removing 2–4 cm² of leaf material and freezing it immediately in liquid nitrogen. Samples were stored in liquid nitrogen or in a −70 °C freezer until assay. Leaves were sampled at light intensities above 1000 μmol m⁻² s⁻¹. All cultivated plants were well fertilized, using standard greenhouse fertilizer mixes. All plants sampled in field locations were selected to have a vigorous appearance and deep green colour.

Plants sampled for Rubisco assay were Amaranthus retroflexus (seed from Prague, Czech Republic; C₄; UTB, sampled at 30 °C); Arachis hypogea (escaped peanut, C₃, Athens, 30 °C); Capsicum chinense (Habanero chile pepper, C₃, Athens, 30 °C); Chenopodium album (C₃; Prague seed, UTB, 28 °C); Cynodon dactylon (Bermuda grass, C₄, Athens, 35 °C), Digitaria sanguinalis (crabgrass, C₄, Athens, 35 °C); Flaveria trinervia (Harold Brown collection No. 84‐1, C₄, UGA, 33 °C); Flaveria pringlei (Harold Brown collection No. 85‐207; C₃, UGA, 33 °C; Gossypium hirsutum (cotton, cv. Coker 201, C₃; 30 °C); Oryza sativa (rice, cv. IR42, C₃, UGA 30 °C); Muhlenbergia montanum (mountain muhly, grown from culms collected at Kenosha Pass, Colorado; C₄, UTB, 30 °C); Portulaca oleracea (C₄, Athens, 35 °C); Poa arctica (Hoosier Pass Colorado source; C₃, UTB, 25 °C); Poa pratensis (bluegrass, C₃, UT, 25 °C); Pueraria lobata (kudzu, C₃, Athens, 30 °C); Solanum tuberosum (potato cv. Eigen, UGA, 25 °C); Zoysia japonica (C₄, Athens, 35 °C).

Gas exchange analysis

The temperature and CO₂ response of photosynthesis was determined for Amaranthus retroflexus and Chenopdium album plants grown outdoors in 20 l pots at the roof‐top garden at UTB. The plant material originated from seed collected at the north‐eastern outskirts of Prague, Czech Republic, 50° N latitude. All gas exchange responses were measured using a temperature‐regulated, null‐balance gas exchange system (Pittermann and Sage, 2000). To determine the temperature response of the net rate of CO₂ assimilation (A), leaves were first equilibrated at the measurement CO₂ level and 25–30 °C. Leaf temperature was then raised to the maximum measurement temperature near 40 °C, and lowered in 4–5 °C steps to 5–10 °C. Gas exchange parameters were determined at each step after a 15–30 min equilibration period. The rate of net CO₂ assimilation was determined at approximately 27 °C before and after the elevation in temperature to the maximum measurement value. If the two measurements did not correspond, the measurement was discontinued. The CO₂ response of photosynthesis was determined by first equilibrating leaves in air at the measurement temperature, after which CO₂ was reduced to the minimum measurement level. Gas exchange parameters were determined as CO₂ was increased in a series of steps to maximum measurement values. Measurements were conducted just above the light saturation point for A. At temperatures above 20 °C, the light saturation point was greater than 1500 μmol m⁻² s⁻¹. Below 20 °C, the light saturation point declined with reductions in temperature and the measurement light intensity was reduced by an equivalent amount as described earlier (Pittermann and Sage, 2000).

Rubisco assay

To determine the temperature response of the activity of fully activated Rubisco, leaves were rapidly ground in a pH 8.0 extraction buffer (Pittermann and Sage, 2000). The extract was then centrifuged at 5000 g for 60 s, and the Rubisco in the supernatant was allowed 5 min to reach full activation. Rubisco activity was determined by assaying the rate at which ¹⁴CO₂ was incorporated into acid‐stable products during a 30 or 60 s incubation period (Sage and Seemann, 1993; Sage et al., 1993). The assay buffer was 100 mM Bicine (pH 8.2) with 1 mM Na‐EDTA, 5 mM dithiothreitol, 28 mM MgCl₂, 15 mM [¹⁴C]NaHCO₃ (20 Bq nmol⁻¹), 2 mM ATP, 1.5 mM ribose 5‐phosphate, 1 unit ml⁻¹ ribose‐5‐phosphate isomerase, and 0.1–0.3 units ml⁻¹ ribulose‐5‐phosphate kinase. RuBP (1.5 mM) was generated in vitro by isomerization and phosphorylation of ribose 5‐phosphate. The drift in pH with temperature was less than ±1.5 pH units between 4 °C and 40 °C, which did not significantly affect measured activity values. Assays were conducted at a range of temperatures between 0 °C and 45 °C using water baths to control leaf temperature. Four to five baths were used to minimize the time between grinding and assaying of Rubisco. At most, 30 min elapsed between a grind and the final assay from a given extraction. No loss of Rubisco activity was observed over this period if the extract was left on ice prior to assay.

To minimize problems associated with incubation time after extraction and exchange of ¹⁴C between the assay buffer and the atmosphere, the assay buffer remained covered at 0 °C except during a 120 s period to equilibrate with the water bath temperature. To minimize variation between assay days, which can be a problem due to differences in the ¹⁴C source material, pairs of species giving the more interesting comparisons were assayed at the same time using the same extraction and assay buffer. For example, samples of the C₃ and C₄Flaveria species were assayed on the same day in an alternating sequence. This ensured that any differences observed were due to differences in the species rather than the assay conditions.

Rubisco content was determined by measuring the binding of [¹⁴C]‐carboxyarabinitol bisphosphate (CABP) to the Rubisco catalytic sites, with rabbit anti‐Rubisco antibodies being used to precipitate the CABP‐Rubisco complexes (Sage and Seemann, 1993). The amount of [¹⁴C]CABP bound to Rubisco was estimated using scintillation spectroscopy after filtration through a supor‐450 filter (Gelman Science, Ann Arbor, MI 48106). The binding stoichiometry of CABP to Rubisco was assumed to be 6.5 CABP molecules per molecule of Rubisco (Butz and Sharkey, 1989). The k_cat of Rubisco (mol CO₂ mol⁻¹ Rubisco catalytic sites s⁻¹) was determined by dividing the activity of Rubisco by the content determined for a given leaf sample. To ensure the assay was consistent over the experimental period, the k_cat of bean (Phaseolus vulgaris) was routinely determined. If the bean k_cat shifted from previously determined values (Sage et al., 1993), the assay was halted until the problem was rectified.

Results

Temperature and CO₂ responses in Chenopodium album and Amaranthus retroflexus

In the C₃ species Chenopodium album exposed to Pleistocene levels of CO₂ (18.5 Pa), the net CO₂ assimilation rate (A) was moderately affected by temperature below 20 °C, and it exhibited a broad temperature optimum between 20 °C and 30 °C (Fig. 1). At higher CO₂ levels, the temperature response of A below 25 °C was more pronounced, exhibiting a Q₁₀ near 2. Below 18 °C, A was stimulated by rising CO₂ concentration between 18 and 38 Pa, but not above 38 Pa. Above 20 °C, the relative stimulation of A by rising CO₂ was increased below 38 Pa, and became pronounced above 38 Pa. In the C₄ plant Amaranthus retroflexus, the temperature sensitivity of A was similarly enhanced by rising CO₂ above 20 °C as observed in the C₃ species (Fig. 1B). Below 18 °C, CO₂ increase had little effect on the temperature response of A in contrast to the marked response observed above 20 °C.

In C. album, the V_max of Rubisco measured in vitro was well in excess of the CO₂ assimilation rate observed at the same temperature, with the discrepancy rising as temperature increased (Fig. 1A). Using an estimate of Rubisco activity at 25 °C and the activation energy determined for C. album (Table 1), A was modelled assuming Rubisco capacity was the only limitation. At 38 Pa, modelled A corresponded to observed A at optimal temperatures, but was greater than observed A at sub‐optimal temperatures. At 18.5 Pa, the modelled temperature response of A exhibited a similar response as measured A above 10 °C. At 68 Pa, by contrast, modelled A was greater than the measured A at all temperatures. In A. retroflexus, in vitro activities of Rubisco correspond to the observed rates of A at temperatures below 15–20 °C at all measurement CO₂ levels, but are well in excess of A above 20 °C (Fig. 1B).

In C. album and A. retroflexus, the response of A to variation in intercellular CO₂ (C_i) was similarly affected by temperature (Fig. 2). Temperature had a relatively small effect on the initial slope of the A/C_i response in both species, but rising temperature markedly stimulated the CO₂‐saturated rate of A (Figs 2, 3). As a result, the CO₂ saturation point of A increased with temperature in both photosynthetic types (Fig. 2). The operating C_i (the intercellular CO₂ partial pressure corresponding to a given ambient CO₂ partial pressure) increased slightly with declining temperature in both species, as indicated by the arrows for C. album or circled data points for A. retroflexus in Fig. 2.

Fig. 1. 

The temperature response of the net rate of photosynthetic CO₂ assimilation (open symbols) and the maximum activity of Rubisco (filled symbols) in the C₃ plant Chenopodium album and the C₄ plant Amaranthus retroflexus. The measurement level of CO₂ in Pa is indicated beside each curve. In C. album, different symbols reflect representative measurements from single leaves. In A. retroflexus, symbols are the averages (±SE) from 2–4 measurements, except for data at 18 Pa where N=1. For C. album, light grey lines are modelled photosynthesis rates assuming Rubisco activity is limiting the rate of net CO₂ assimilation. The modelled responses were generated using WIMOVAC 4.01 (Humphries and Long, 1995), assuming (a) V_cmax=135 μmol m⁻² s⁻¹ (this study), (b) k_c=40.7 Pa and k_o=28 Pa (after spinach at 25 °C; von Caemmerer and Quick, 2000), (c) the C. album activation energy in Table 1, (d) a dark respiration activation energy of 46.39 kJ mol⁻¹ (Bernacchi et al., 2001) and (e) activation energies for V_o, k_c and k_o from Atriplex glabriuscula above 15 °C (von Cammerer and Quick, 2000).

Fig. 2. 

The response of the net rate of CO₂ assimilation to variation in intercellular CO₂ partial pressure in A. retroflexus (filled symbols) and C. album (open symbols). Measurement temperatures are indicated beside each curve. Measurements for a given species were determined using the same leaf on the same day. The operating C_i (the intercellular CO₂ partial pressure corresponding to ambient partial pressure) at 18 and 36 Pa are indicated for A. retroflexus by symbols enclosed in grey circles and for C. album by the arrows beside each curve. The smaller operating C_i on each curve correspond to measurements at 18 Pa.

Fig. 3. 

(A) The temperature response of the initial slope of the CO₂ response curve of A. retroflexus (black and white symbols) and C. album (grey symbols). Each type of symbol represents measurements from an individual plant. (B) The temperature response of the CO₂‐saturated rate of photosynthesis in A. retroflexus (filled circles), and C. album (grey circles). 95% confidence intervals are shown for the regressions of A. retroflexus responses.

Table 1. 

The k_catand activation energy of Rubisco from a variety of C₃ and C₄ plants

For individual species, k_cat data are mean±SE, N=3–6, except for C. album where N=1 or 2. Group k_cat averages are calculated using individual measurements from all species (mean±SD, N=15–31). Activation energies are calculated from the slope of the log(k_cat) versus T as described earlier (Berry and Raison, 1981), with the group average (±SE) being calculated from the species values shown. Letters indicate differences between functional groups (cool habitat C₃, warm habitat C₃, or C₄) at P=0.05 (One‐way ANOVA with Tukey's means test using Sigmastat 2.0).

	kcat (mol CO2 mol−1 sites s−1)					Activation energy (kJ mol−1)
	16 °C	28 °C	40 °C
C3 plants from cool habitats
Poa arctica	1.53±0.09	4.03±0.26	10.24±0.78	56.1
Solanum tuberosum (potato)	1.21±0.05	3.76±0.39	8.20±0.36	50.6
Poa pratensis	1.10±0.05	3.01±0.18	6.26±0.61	53.2
Chenopodium album	1.68±0.19	3.71 (N=1)	8.16±1.56	60.9
Group average	1.34±0.26 b	3.55±0.54 b	8.14±1.82 b	55.2±2.2 a
C3 plants from warm habitats
Gossypium hirsutum (cotton)	0.88±0.06	2.84±0.23	6.79±0.34	60.4
Pueraria lobata (kudzu)	0.74±0.03	2.51±0.19	6.49±0.46	66.6
Flaveria pringlei	1.04±0.04	2.96±0.10	6.05±0.21	53.8
Arachis hypogea (peanut)	0.61±0.06	2.13±0.25	5.13±0.66	65.1
Oryza sativa (rice)	0.80±0.03	2.69±0.09	6.41±0.29	68.8
Capsicum chinense	0.64±0.04	1.99±0.23	4.33±1.60	51.5
Group average	0.78±0.17 a	2.46±0.52 a	5.76±1.22 a	61.0±2.9 a
C4 plants
Muhlenbergia montanum	1.73±0.13	4.93±0.25	10.56±0.59	57.7
Cynodon dactylon	2.11±0.28	5.39±0.39	11.81±1.13	50.1
Flaveria trinerva	1.44±0.09	4.08±0.19	8.35±0.29	53.5
Digitaria sanguinalis	1.85±0.06	4.29±0.36	8.58±0.18	50.3
Zoysia japonica	1.48±0.14	4.05±0.40	8.25±0.74	51.2
Portulaca oleracea	1.96±0.08	5.94±0.34	12.81±0.46	60.6
Amaranthus retroflexus	1.63±0.08	5.40±0.23	13.09±0.51	68.0
Group average	1.73±0.32 c	4.86±0.89 c	10.46±2.43 c	55.9±1.8 a

Temperature responses of Rubisco in a variety of C₃ and C₄ species

The k_cat of Rubisco at 16, 28 and 40 °C was consistently larger in C₄ relative to C₃ species (Table 1). C₃ species originating from cool habitats exhibited Rubisco k_cat values that were approximately 24% below that of C₄ species, but 40–70% above the k_cat values observed in C₃ species from warm habitats. Activation energies ranged between 50 and 70 kJ mol⁻¹ in the 17 species. No statistical differences in activation energy were observed between species differing in photosynthetic pathway or temperature environment although the majority of C₃ species from warm habits exhibited activation energies greater than 60 kJ mol⁻¹, while most cool‐temperate C₃ species, and C₄ species, had Rubisco with activation energies below 60 kJ mol⁻¹. The Q₁₀ for all species ranged between 2.2 and 2.9 except for rice at temperatures below a breakpoint, where the Q₁₀ was 4.1 (Fig. 4).

In all 17 species, the k_cat of Rubisco increased exponentially with rising temperature between 5 °C and 40 °C (see Fig. 4 for four examples). Rubisco from C₄ species commonly exhibited a higher k_cat at a given temperature than C₃ species. There was little evidence of a thermal break in the corresponding Arrhenius plots with the exception of rice, which exhibited a sharp break at 22 °C (Fig. 5). The Arrhenius responses slopes of the species were similar, except for the rice response below 20 °C. The important difference between the Arrhenius response was the y‐axis intercept was lower in warm‐habitat C₃ species. Thus, the difference in Rubisco between warm‐habitat C₃ and the other two groups is not in the activation energy, but in the turnover capacity of the enzyme at a given temperature.

The difference between Rubisco from C₃ plants of warm and cool habitats is well demonstrated by a comparison of the temperature response of Capsicum chinense and potato, Solanum tuberosum (Fig. 6). Capsicum chinense, the Habanero chile pepper, originated in the lowland tropics of South America and grows best above 30 °C (RF Sage, personal observation). Potatoes originated in the high montane environments of South America and grow exclusively in cool climates or seasons where daytime temperatures rarely rise above 30 °C (Evans, 1993). Both are in the Solanaceae. In Habanero peppers, the k_cat of Rubisco is lower than potato at all temperatures (Fig. 6A). Arrhenius plots also show the downward shift in the response of Rubisco in Habanero pepper compared to potato; however, the slopes of the responses were similar, as were the estimated activation energies (Table 1; Fig. 6B).

Fig. 4. 

The k_cat of fully‐activated Rubisco as a function of temperature in two pairs of C₃ and C₄ species that were assayed at approximately the same time using a common extraction and assay buffer. Means±SE, N=3–5.

Fig. 5. 

Arrhenius plots for the temperature response of Rubisco k_cat presented in Fig. 3. Filled symbols are from C₄ plants; open symbols from C₃ plants. The species shown are Amaranthus retroflexus (filled circles), Chenopodium album (open circles), Flaveria trinervia (filled squares), Flaveria pringlei (open squares), Cynodon dactylon (filled diamonds), rice (Oryza sativa, open diamonds), Muhlenbergia montanum (filled inverse triangles), and Poa arctica (open inverse triangles). Regressions are presented for the four cool habitat C₃ species (dashed grey line), the six warm habitat C₃ species (solid grey line) and the seven C₄ species (solid black line) presented in Table 1. The dotted line indicates the response of rice above a distinct break point in the response.

Fig. 6. 

(A) The k_cat of the C₃ Solanaceae species Solanum tuberosum (potato) and Capsicum chinense (Habanero pepper). (B) The Arrhenius plots corresponding to responses in (A).

Discussion

Photosynthetic limitation as a function of temperature in C₃ and C₄ plants

In the current atmosphere, the net rate of CO₂ assimilation is often equivalent between ecologically similar C₃ and C₄ species at 25–30 °C. Photosynthesis is typically greater in C₃ species below this temperature, but greater above it in C₄ species (Pearcy et al., 1981; Edwards et al., 1985; Sage and Pearcy, 1987, 2000). Increases in atmospheric CO₂ are commonly thought to stimulate A in C₃ species, but have a minimal effect in C₄ species (Cure and Acock, 1986; Wand et al., 1999, 2001). This impression is biased, however, because research has emphasized CO₂ responses above recent atmospheric CO₂ levels of 33–37 Pa (Morison and Lawlor, 1999). When temperature responses at low CO₂ are considered, C₄ photosynthesis exhibits a similar response to rising CO₂ as C₃ species, in that the CO₂ stimulation of A is minor at cool temperature while it is substantial at high temperature (Fig. 1). This interaction between low CO₂ and temperature is important when considering the responses of C₃ and C₄ vegetation to CO₂ variation in prehistoric times. During the past 2 million years, CO₂ levels have varied between 18 and 28 Pa during glacial to interglacial cycles (Sage and Pearcy, 2000). Because C₄ species largely occur in warmer habitats where greater CO₂ sensitivity of A is expected, substantial reduction of C₄ photosynthesis per unit area likely followed a decline in CO₂ to 18 Pa during past glacial episodes.

The response of A to intercellular CO₂ (C_i) is widely used to evaluate mechanisms controlling photosynthetic responses to environmental variation (Sharkey, 1985). In both C₃ and C₄ species, analysis of A/C_i responses show the CO₂‐saturated rate of A is markedly enhanced by rising temperature, while the initial slope has a small temperature response (Fig. 3; Kirschbaum and Farquhar, 1984; Sage and Sharkey, 1987; Sage et al., 1990a). Consequently, the CO₂ saturation point increases with rising temperature such that at ambient CO₂ levels below 40–50 Pa, the operating C_i corresponds to the initial slope region of the A/C_i response where CO₂ sensitivity is high. Below 20 °C, the CO₂ saturation point of both C₃ and C₄ photosynthesis declines to values that are below current air levels of CO₂, such that the operating C_i falls on or near the CO₂‐saturated plateau. Hence, A has little response to rising CO₂ at lower temperatures.

The biochemical limitations controlling A have been well described at moderate temperatures and robust theoretical models are available to interpret A/C_i responses in terms of the biochemical controls (von Caemmerer, 2000; see also the Wimovac photosynthesis program of Humphries and Long, 1995). At saturating light levels, the initial slope region of the A/C_i response is thought to reflect Rubisco limitations on A in C₃ plants, and PEP carboxylase limitations in C₄ plants (von Caemmerer and Furbank, 1999; von Caemmerer, 2000). The CO₂‐saturated rate of A reflects RuBP regeneration limitations in C₃ plants, and either Rubisco, RuBP regeneration, and PEP regeneration limitations in C₄ plants (Furbank et al., 1997; von Caemmerer and Furbank, 1999; von Caemmerer, 2000). The interpretation of the biochemical controls over the A/T response is less clear than the A/C_i response, although analysis of Rubisco activity and A/C_i responses at a range of temperatures allows for enough interpretation to contrast differences in the mechanisms controlling C₃ and C₄ temperature responses. In C. album, Rubisco activity in vitro is markedly greater than A at all but the lowest measurement temperatures (Fig. 1). This must be the case in C₃ plants because in vivo, Rubisco activity in air is depressed below the Rubisco activity measured in vitro by the deficiency of substrate CO₂, the presence of 21% O₂, and possible depressions in the activation state of Rubisco (von Caemmerer and Farquhar, 1981; Sage et al., 1987, 1990b). CO₂ levels in the chloroplast are reduced about 50% with respect to air levels of CO₂ by diffusion restrictions in the boundary layer, stomata and mesophyll tissue (Evans and Loreto, 2000). Thus, in the current atmosphere, the CO₂ concentration in the C₃ chloroplast is near the K_m of Rubisco at 25 °C, and below it above 30 °C. When enzyme kinetics, diffusion restrictions, O₂ concentration, and CO₂ supply are accounted for, predicted A calculated from the Rubisco capacity measured in vitro is often equal to measured A (von Caemmerer and Farquhar 1981, 1984; von Caemmerer and Quick, 2000). In C. album at 38 Pa CO₂, modelled A, assuming Rubisco activity was limiting, was similar to measured A at the temperature optimum (Fig. 1). In accordance, the A/C_i response shows the operating C_i occurs in the initial slope region at the temperature optimum (about 31 °C) in C. album, indicating high Rubisco control over A.

Below the temperature optimum of C. album at 38 Pa, modelled A is greater than measured A, and A is minimally affected by CO₂ enrichment above 38 Pa below 18 °C. This possibly reflects a ceiling on A established by a limitation in the capacity of starch and sucrose synthesis to regenerate P_i for photophosphorylation (Sage and Sharkey, 1987). A P_i‐regeneration limitation is generally indicated by a lack of stimulation of A by rising CO₂ at CO₂ levels below 100 Pa where photorespiration occurs (Sharkey, 1985; Harley and Sharkey, 1991). If Rubisco or RuBP regeneration capacity were limiting, A would respond positively to increases in CO₂, because a higher CO₂ concentration inhibits photorespiration (Sharkey, 1988). The appearance of a strong CO₂ response at the temperature optimum indicates the limitation on A at high CO₂ shifts from P_i regeneration to Rubisco capacity or one of the components limiting the capacity for RuBP regeneration (Sage and Sharkey, 1987). Results presented here and in other studies indicate Rubisco is not limiting A at elevated CO₂ because modelled Rubisco‐limited A is in excess of observed A, and the operational C_i occurs on the plateau of the A/C_i response at all temperatures (von Caemmerer and Farquhar, 1981, 1984; Kirschbaum and Farquhar, 1984; Sage and Sharkey, 1987; Sage et al., 1990a). In addition, the capacity for electron transport in C. album has a similar temperature optimum as A at high CO₂, indicating the capacity of RuBP regeneration is limiting A at warmer temperatures and high CO₂ (Sage et al., 1995).

At 18 Pa CO₂, modelled A in C. album has a similar response to temperature as measured A, except below 10 °C. A Rubisco limitation is generally assumed to predominate in C₃ plants at low CO₂ (von Caemmerer, 2000) and this is consistent with results presented here. The operating C_i corresponds to the initial slope of the A/C_i response at 23 °C and 31 °C, indicating a Rubisco limitation is present at warmer temperatures. The relatively flat temperature response of A at low CO₂ is consistent with a Rubisco limitation, as is the low temperature effect on the initial slope of the A/C_i response (Kirschbaum and Farquhar, 1984; Sage et al., 1995). The reason for the flat temperature response of Rubisco‐limited A, and hence the initial slope of the A/C_i response, is that the K_m for CO₂ and V_max of Rubisco both have a similar Q₁₀ (Berry and Raison, 1981). When this occurs, Rubisco activity at CO₂ levels below the K_m has a Q₁₀ near 1.

In C₄ plants, the ability of the leaf to concentrate CO₂ around Rubisco allows Rubisco in vivo to function close to its CO₂‐saturated rate, as it does in vitro during the assay (Edwards et al., 1985; von Caemmerer and Furbank, 1999). Hence, when Rubisco capacity is limiting, its in vivo activity should correspond to the observed rate of A. This was the case at cooler temperatures, but not at temperatures above 20 °C. Notably, A at all CO₂ levels exhibited a similar value below 16 °C, which corresponded to the measured Rubisco activity. These observations indicate that Rubisco capacity is a common limitation on A in A. retroflexus at lower temperatures. By contrast, at temperatures approaching and exceeding the temperature optimum, Rubisco capacity is well in excess of observed A, indicating Rubisco is non‐limiting. Instead, the capacity for RuBP or PEP regeneration is a more likely limitation in cases where A is CO₂ saturated (von Caemmerer and Furbank, 1999). If the temperature rise is high enough to cause the operational C_i to drop below the CO₂ saturation point, PEP carboxylase activity could also become an important limitation on A at warmer temperature.

Two montane C₄ grasses from the Rocky mountains, USA (Bouteloua gracilis and Muhlenbergia montanum) exhibited similar responses of A and Rubisco activity below 18 °C as observed in A. retroflexus (Pittermann and Sage, 2000, 2001). All three species have some degree of cold tolerance that is associated with occurrence at high altitude or northern latitude (Pittermann and Sage, 2000, 2001). Together, the results from these species indicate that Rubisco is a widespread limitation on A in cold‐tolerant C₄ plants in low temperature, but not at elevated temperature. If so, then photosynthetic acclimation and adaptation to low temperature in C₄ species could involve an enhancement in Rubisco content to remove this limitation. Pittermann and Sage examined this possibility in B. gracilis and M. montana, but saw no evidence that ecotypes from higher altitude, nor plants grown in chilling conditions, enhanced Rubisco capacity (Pittermann and Sage, 2000, 2001). By contrast, Osmond et al. noted that C₄Atriplex species from cold climates have proportionally greater Rubisco contents than C₄ species from warm climates, indicating there may be some compensation for limiting Rubisco capacity at low temperature (Osmond et al., 1982).

In the experiments presented here, the vapour pressure difference between leaf and air was maintained below 1.5 kP except at high temperature. Under these conditions, A. retroflexus and C. album operate at high C_i/C_a ratios for their respective photosynthesis types, and stomata show little response to temperature. In natural environments, VPD usually increases with rising temperature (Schulze and Hall, 1982). This will usually reduce stomatal conductance and C_i/C_a such that the operating C_i is more likely to correspond to the initial slope region of the A/C_i response (Sage and Sharkey, 1987). At the biochemical level, this will increase the temperature at which Rubisco controls A in C₃ plants and PEP carboxyase controls A in C₄ plants, and will result in higher CO₂ responsiveness of A than may be otherwise expected.

The significance of differences in the k_cat of Rubisco

Previous work has shown that Rubisco from most C₄ plants has a higher catalytic turnover rate at 25–30 °C than most C₃ plants (expressed either as molar activity or k_cat; Seemann et al., 1984; Sage and Seemann, 1993). Associated with this is a tendency for C₄ Rubisco to have a lower specificity for CO₂ relative to O₂ than Rubisco from C₃ species (Badger, 1987; von Caemmerer and Quick, 2000). These results demonstrate that Rubisco in most C₄ plants has evolved into a type that is better adapted to exploit the high‐CO₂ conditions in the bundle sheath compartment (Seemann et al., 1984; Badger, 1987). Unlike the trends between C₃ and C₄ Rubisco, however, no trend in catalytic performance has previously been identified between Rubisco from C₃ species of different habitats, although it is recognized that the k_cat of Rubisco can vary substantially between C₃ species (Seemann et al., 1984; Evans and Austin, 1986). Here, enough species have been characterized to identify a pattern where warm‐habitat C₃ plants have a lower k_cat for Rubisco than cool‐habitat C₃ species. Consistently, Rubisco k_cat was 24–39% lower in the C₃ species from warm habits (cotton, Phaseolus bean and soybean) than cool habit species (spinach and wheat) (Seemann et al., 1984).

A higher k_cat of Rubisco from C₄ species is advantageous because less enzyme is required for a given photosynthesis rate if CO₂ levels around Rubisco are near saturation (Seemann et al., 1984). Increased catalytic turnover is associated with a reduced affinity (increased K_m) for CO₂, so the cost of the higher k_cat in C₄ plants is lower affinity and specificity for CO₂ (Badger, 1987; von Caemmerer and Quick, 2000). Because Rubisco in C₄ plants operates in a very high CO₂ environment, the cost of reduced affinity for CO₂ is negligible and the advantages of the higher k_cat are substantial. In C₃ plants, by contrast, Rubisco operates in a low CO₂ environment, and thus there is greater advantage in having a higher relative specificity, but this comes at the cost of reduced turnover capacity (Badger, 1987). Although appropriate for moderate temperature, this interpretation is limited because it does not consider the differences in Rubisco kinetics at high and low temperature, which often are the conditions in which species function. By considering temperature effects on k_cat, the advantage of higher k_cat in C₃ species from cool habitats can be identified.

As temperature falls, the CO₂ concentration required to saturate Rubisco declines (Fig. 7), such that below 10 °C, enough CO₂ is present in plants to allow the enzyme to function near the CO₂ saturation point (Fig. 8). As the CO₂ saturation point approaches the operating C_i, the V_max of Rubisco become an important determinant of in vivo activity. For species in cool conditions, increasing the k_cat of Rubisco will therefore increase photosynthesis and nitrogen use efficiency (Pittermann and Sage, 2000). Photorespiratory costs associated with decreased affinity are offset because cooler temperatures directly increase CO₂ specificity of Rubisco and the solubility of CO₂ relative to O₂ (Jordan and Ogren, 1984). At high temperature (>30 °C), by contrast, the K_m for CO₂ (k_c) and the CO₂ saturation point rise substantially such that the operating C_i corresponds to CO₂ levels below the k_c, even at atmospheric CO₂ levels greater than that of today (Figs 7, 8). In addition, the low atmospheric CO₂ content of recent evolutionary time enhances the potential for photorespiration at elevated temperature (Sharkey, 1988; Sage, 1999). Thus, selection pressure for greater CO₂ affinity by Rubisco may have been high for species that grow in warm to hot environments.

Prior surveys have generally assessed only one component of Rubisco kinetics, for example, k_c (Yeoh et al., 1980, 1981), k_cat (Seemann et al., 1984; Sage et al., 1993); or relative specificity (Parry et al., 1989; Kane et al., 1994; Kent and Tomany, 1995). Because of variation in these parameters reported for the same species used in the different studies, it is difficult to obtain a clear picture of possible trade‐offs between k_cat and affinity in C₃ plants. One of the best comparisons of Rubisco is the literature review by von Caemmerer and Quick (von Caemmerer and Quick, 2000). This shows that plants of cool habitat (Atriplex glabriuscula, Lolium perenne, spinach, and wheat) have k_c of 33–62 Pa (11–21 μM), and apparent Michaelis constants in the presence of O₂, k_c(1+O/k_o), of 49.5–110 Pa. Plants of warm habits (rice, soybean and tobacco) have K_m of 23–34.4 Pa and Michaelis constants of 41.8–71.3 Pa. Relative specificity exhibited no clear trend. In addition, a recent report shows the CO₂ compensation point in the absence of mitochondrial respiration (Γ*) decreases in Taraxacum officinale plants grown in warm environments, indicating there is some adjustment in Rubisco properties to maintain catalytic efficiency as growth temperature changes (Bunce, 2000).

In summary, the results presented here indicate there is microevolutionary adjustments of Rubisco properties to enhance performance in the temperature environment to which a plant is adapted. For C₃ plants in cool environments, this may be the expression of a type of Rubisco similar to C₄ plants in terms of k_cat and activation energy. In C₃ plants from warm to hot habitats, the Rubisco may be a form with slower turnover capacity at CO₂ saturation, but a greater affinity for CO₂ at the CO₂ and temperature conditions in which the plant grows. To validate these possibilities, careful biochemical measurements are required on a range of species, particularly those from markedly contrasting climates.

Fig. 7. 

The effect of temperature on the modelled estimate of the amount of CO₂ required for Rubisco to operate at 80% of V_max. The value of 80% V_max was selected as the comparison value because this is where activity versus substrate plots exhibit marked diminishing returns. (Modelled for spinach Rubisco according to von Caemmerer and Farquhar, 1981, using kinetic constants from Jordan and Ogren, 1984.) (Reprinted from Pittermann and Sage, 2000.)

Fig. 8. 

The modelled CO₂ response of the gross photosynthesis rate (in mol CO₂ mol⁻¹ Rubisco catalytic sites s⁻¹) for a Rubisco with a k_cat of 3.12 mol mol⁻¹ s⁻¹ at 25 °C. The dark shaded band shows the CO₂ partial pressure in chloroplasts of C₃ plants in the current atmosphere. The light grey shading indicates the range of CO₂ normally found in the bundle sheath of C₄ plants. (Modelled according to von Caemmerer and Farquhar, 1981, using kinetic constants from spinach (Jordan and Ogren, 1984).)

I thank the following individuals for technical assistance on this project: Paul Alloway, Professor Harold Brown, and Jarmila Pittermann. This research was supported by grant No. OGP0154273 from the National Science and Engineering Council of Canada.