Plant Physiol. (1998) 118: 945-955

Estimating the Excess Investment in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Leaves of Spring Wheat Grown under Elevated CO₂¹

Julian C. Theobald, Rowan A.C. Mitchell^*, Martin A.J. Parry, and David W. Lawlor

Biochemistry and Physiology Department, Institute of Arable Crops Research-Rothamsted, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom

	ABSTRACT

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Wheat (Triticum aestivum L.) was grown under CO₂ partial pressures of 36 and 70 Pa with two N-application regimes. Responses of photosynthesis to varying CO₂ partial pressure were fitted to estimate the maximal carboxylation rate and the nonphotorespiratory respiration rate in flag and preceding leaves. The maximal carboxylation rate was proportional to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content, and the light-saturated photosynthetic rate at 70 Pa CO₂ was proportional to the thylakoid ATP-synthase content. Potential photosynthetic rates at 70 Pa CO₂ were calculated and compared with the observed values to estimate excess investment in Rubisco. The excess was greater in leaves grown with high N application than in those grown with low N application and declined as the leaves senesced. The fraction of Rubisco that was estimated to be in excess was strongly dependent on leaf N content, increasing from approximately 5% in leaves with 1 g N m² to approximately 40% in leaves with 2 g N m². Growth at elevated CO₂ usually decreased the excess somewhat but only as a consequence of a general reduction in leaf N, since relationships between the amount of components and N content were unaffected by CO₂. We conclude that there is scope for improving the N-use efficiency of C₃ crop species under elevated CO₂ conditions.

	INTRODUCTION

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The effects of the increasing atmospheric CO₂ concentration on photosynthesis has stimulated an enormous volume of research, which has been extensively reviewed (Lawlor and Mitchell, 1991; Bowes, 1993; Drake et al., 1997). One aspect of particular interest for the possibility of crop improvement is the way in which a change in CO₂ partial pressure alters the balance of limitation between processes that determine the instantaneous A in C₃ plants. At high light, photosynthesis can be limited by the carboxylation capacity or by the light-saturated capacity for regeneration of RuBP, and the optimal use of resources invested in photosynthetic components (i.e. that which gives the maximal rate of photosynthesis) for a given environment occurs when these capacities are balanced (Farquhar and Sharkey, 1994). When CO₂ partial pressure increases, the carboxylation capacity increases more than the light-saturated capacity for RuBP regeneration; therefore, the theoretical optimal balance of investment in photosynthetic components is altered (Sage, 1994; Webber et al., 1994; Medlyn, 1996).

Optimization of these components has considerable implications for N-use efficiency of leaf photosynthesis (Makino et al., 1997), since photosynthetic components involved in determining the carboxylation capacity (Rubisco) and light-saturated RuBP-regeneration capacity (e.g. thylakoid ATP-synthase and the Cyt b/f complex) account for a substantial fraction of leaf N (Evans and Seemann, 1989). For a doubling of CO₂ partial pressure from the current ambient conditions (36 Pa), a 30% to 40% increase in the ratio of light-saturated RuBP-regeneration capacity to carboxylation capacity has been predicted for optimal N-use efficiency (Webber et al., 1994; Medlyn, 1996). Since N is likely to be more limiting to growth at elevated CO₂, this increase in ratio would normally be expected to be achieved by a specific decrease in the amount of Rubisco.

Experimental evidence from gas-exchange studies suggests that such optimization occurs only infrequently in C₃ plants grown at elevated CO₂ (Makino, 1994; Sage, 1994; Medlyn, 1996). However, many studies have found evidence of acclimatory decreases in A and in amount of components on a leaf-area basis in response to growth at elevated CO₂ (for review, see Webber et al., 1994; Drake et al., 1997). Similarly, at subambient CO₂, Rubisco content per unit leaf area may increase (Rowland-Bamford et al., 1991), but there is little evidence for an increase in carboxylation capacity relative to other capacities (Sage and Reid, 1992). It therefore seems that many of the observations reflect a general reduction in all photosynthetic components due to a source-sink imbalance at higher CO₂ rather than to an optimization between photosynthetic components. This is consistent with the hypothesis that the mechanism for the effect of elevated CO₂ on amounts of photosynthetic components is a response to carbohydrate buildup in the leaves (van Oosten and Besford, 1996). Transcription for both a subunit of thylakoid ATP-synthase and the Rubisco small subunit was decreased by cold-girdling, which decreased carbohydrate export from the leaf (Krapp and Stitt, 1995).

In one study (Nie et al., 1995) there was apparent support for an optimization response in wheat crops grown in a free-air CO₂-enrichment facility. While there was no effect of elevated CO₂ on the amount of Rubisco when the leaf emerged, there was a subsequent effect, because Rubisco was decreased relative to other photosynthetic components. However, important recent work (Nakano et al., 1997) has shown that in young rice leaves the effect of elevated CO₂ partial pressure appears to operate entirely via a reduction in leaf N. Since leaves with less N contain a smaller fraction of Rubisco, there is a de facto readjustment in favor of RuBP regeneration compared with carboxylation capacity, which is dependent on N-supply conditions. This result could explain the variability in experimental findings on the occurrence of the theoretical optimization (Sage, 1994), since any rebalancing will depend on whether a reduction in leaf N occurs at elevated CO₂ under the experimental conditions.

The issue is of particular importance for C₃ crop species, because if they do not optimize their investment in photosynthetic components in response to growth CO₂ partial pressure, there may be opportunities to increase the N-use efficiency of photosynthesis by genetic manipulation for the higher CO₂ environments of the future. Specifically, if there is no optimization under agronomic conditions, there will be excess investment in Rubisco relative to RuBP-regeneration capacity at high CO₂ concentrations. Makino et al. (1997) recently demonstrated that N-use efficiency of leaf photosynthesis in rice, measured during short-term exposure to elevated CO₂ concentrations, was greater in lines that had been transformed to specifically reduce the amount of Rubisco compared with the wild type. The extent to which this would be advantageous in plants grown at elevated CO₂ is dependent on how much Rubisco remains in excess when given the opportunity to acclimate. The aim of this study was to estimate the amount of Rubisco that was in excess of the requirement for leaf photosynthesis in wheat plants grown at elevated CO₂ partial pressures and various N-supply treatments under conditions representative of the field. To this end, a large number of gas-exchange measurements were combined with estimations of Rubisco and thylakoid ATP-synthase contents, the latter being chosen as the largest single component of RuBP regeneration (Evans and Seemann, 1989).

	MATERIALS AND METHODS

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Plant Culture

2

Nutrient solution (described by Delgado et al., 1994) was applied twice weekly from approximately 10 to 70 d after sowing; the nitrate concentrations were 12.5 and 4.6 mM for high- and low-N regimes, respectively, for experiments 1 and 2, and 6.25 mM for experiment 3. Nitrate concentrations were varied by substituting chloride. Following eight nutrient applications, the concentrations of solutions were quadrupled in all experiments for the remaining applications. The total N application was equivalent to 8 and 18 g m² in experiment 1, 8 and 21 g m² in experiment 2 for the low- and high-N treatments, and 9.8 g m² in experiment 3. Although the total N applied was similar in experiments 1 and 2, the nature of the application regimes were different, since the last application was 30 d before anthesis in experiment 1 and 8 d before anthesis in experiment 2. The exact nature of the N-application regimes was not critical for the hypotheses being tested, but this change between experiments was made to induce a wider range of leaf N contents in experiment 2. Plants were watered daily with demineralized water and grown with day/night temperature regimes of 20°C/7°C from the start of tillering until maturity (before this time the temperatures were 12°C/6°C), a 16-h photoperiod, and constant artificial PPFD of 580 µmol quanta m² s¹ at plant height. To minimize positional effects, boxes were moved within and between chambers every 10 to 11 d. To mimic a continuous crop with an even canopy structure, reflective screens were placed from the soil surface to the top of the canopy around the arrays. Gaps caused by destructive sampling were filled with bags containing plants of the same treatment.

Gas-Exchange Measurement and Analyses

2

1

The first measurement of each leaf was when the ligule had just become visible. Light-saturated photosynthesis was estimated at a p_i of approximately 2, 4, 7, 10, 14, 25, 50, 60, and 68 Pa for each leaf. In addition, the sensitivity of leaf photosynthesis to changing O₂ partial pressure from ambient (20 kPa) to 2 kPa was determined at a p_a of 70 Pa. Light-saturated photosynthesis was assumed to be determined by the RuBP-saturated kinetics of Rubisco for p_i <35 Pa (Makino et al., 1988), and points in this region were fitted to Equation 1 below. In about 10% of cases this procedure gave a poor fit (r² < 0.90), in which case the fit was restricted to points with a p_i < 25 Pa, which invariably increased r² to >0.90. The responses were fitted assuming a finite g_w (Loreto et al., 1992) so that A_g is given by:

(1)

where K is the effective Michaelis-Menten constant for CO₂ at 20 kPa O₂, * is the photorespiratory compensation point, V_cmax is the maximal carboxylation rate, and R_d is the rate of nonphotorespiratory respiration. A similar equation was derived by von Caemmerer and Evans (1991). The values of K and * at 20°C were assumed to be constant for all leaves and were taken from the literature: K of 47.0 Pa is the value for wheat at 25°C taken from Makino et al. (1988); temperature dependence is from A.J. Keys, (unpublished data) and Machler et al. (1980); and * of 3.4 Pa is from Brooks and Farquhar (1985). The value of g_w cannot practically be estimated by fitting A  p_i responses (von Caemmerer et al., 1994); therefore, three different approaches were tried for this parameter for all response curves. In the first, g_w was assumed to have a constant value for all leaves; in the second, it varied between leaves such that the CO₂ partial pressure at the site of carboxylation was 0.55 of atmospheric ambient (i.e. 20 Pa when p_a = 36 Pa; Farquhar and Sharkey, 1994); in the third, an initial value of 5.0 µmol m² s¹ Pa¹ at the time of leaf emergence, which declined as the leaf senesced to a value of 2.0 µmol m² s¹ Pa¹, was used based on the dependence on leaf age found for wheat flag leaves (Loreto et al., 1994). Since the last approach gave the best r² values (>0.95 for 96% and >0.99 for 74% of curves), all of the data presented from the 508 response curves in the three experiments were obtained using this approach. (However, because g_w was not measured, the sensitivity of our conclusions to the assumed value of g_w is addressed in ``Discussion''.) For each A  p_i response curve, nonlinear regression (Genstat5, Rothamsted Experimental Station, Hertfordshire, UK) was used to estimate the values of only two parameters in Equation 1, V_cmax and R_d.

For any observed value of A at a given p_i, the minimum value of V_cmax required to achieve this (V_cmax,req) from Equation 1 is:

(2)

The amount of apparent excess investment in Rubisco (r_xs) compared with that needed to maintain the A is given by:

(3)

where k_cat is the maximal rate of carboxylation per active site on fully activated Rubisco, 8 is the number of active sites per molecule, and 0.55 is the molecular mass (g µmol¹) for Rubisco. Equations 2 and 3 were used to estimate the theoretical excess in Rubisco content compared with that required to maintain the observed assimilation rate with the p_a set at the elevated CO₂ growth value. Thus, Equation 2 was used with the observed p_i and A values corresponding to p_a = 70 Pa for experiments 1 and 2 and p_a = 100 Pa for experiment 3. In only 4% of the A  p_i responses were these estimated values of r_xs less than 0, and these were within the error of the fitting procedure, suggesting that the assumed values of K, *, and g_w were reasonable.

The k_cat for Rubisco was estimated by the coefficient relating V_cmax to the Rubisco content, estimated by linear regression forced through the origin, and converted to moles of CO₂ per mole active site per second. The k_cat for thylakoid ATP-synthase can be estimated from the relationship between A_g,70 and ATP-synthase content. The rate of ATP synthesis (V_ATP) required to maintain a A at p_a = 70 Pa was reported previously (eq. 16.27, Farquhar and von Caemmerer, 1982):

(4)

where p_c is the CO₂ partial pressure at the site of carboxylation, given by p_i  A/g_w. Since estimates of p_c did not vary much, V_ATP was well approximated by 3.68(A_g,70). Linear regression forced through the origin of A_g,70 on the amount of thylakoid ATP-synthase gives a coefficient that is therefore approximately proportional to k_cat. This is multiplied by the 3.68 factor and by 0.555 g µmol¹ to convert to moles of ATP per mole of ATP-synthase per second.

Biochemical Assays

The amount of Rubisco was determined from the binding of [¹⁴C]CABP using a modified method of Yokota and Canvin (1985). One-half of the blade from each leaf sample (approximately 50 mm²) was homogenized in 2 mL of a 50 mM Bicine buffer, pH 7.5, containing 20 mM MgCl₂, 1 mM EDTA, and 50 mM -mercaptoethanol. Part of this homogenate was removed for the determination of chlorophyll and soluble protein content. The remainder was clarified at 10,000g for 3 min, and 200 µL of the supernatant was incubated for 15 min in 100 µL of a 4× activating buffer containing 400 mM Bicine, pH 8.0, 80 mM MgCl₂, 40 mM NaHCO₃, and 200 mM -mercaptoethanol. To this 40 µL of 1 M Na₂SO₄, 35 µL of water, and 25 µL of 2.3 mM [¹⁴C]CABP (37 GBq mol¹) were added. Protein was precipitated by adding 288 µL of 60% (w/v) PEG, which was left to stand on ice for 30 min, followed by clarification at 10,000g for 10 min. The pellet was washed twice in 20% (w/v) PEG and then resuspended in 1 mL of 1% (v/v) Triton X-100 and left overnight at room temperature prior to the addition of scintillant and counting (model 2500 TR liquid-scintillation analyzer, Packard Instruments, Meriden, CT).

Initial, total, and maximal activities of Rubisco were determined using the method of Parry et al. (1997). Rubisco from leaves (40-50 mm²) was rapidly extracted in 1 mL of CO₂-free buffer containing 50 mM Bicine, pH 8.0, 20 mM MgCl₂, 1 mM EDTA, and 50 mM -mercaptoethanol. Extracts were clarified at 10,000g for 2 min and the supernatant was immediately assayed for Rubisco activity. Initial activity was determined at 25°C by adding 20 µL of extract to 980 µL of a CO₂-free assay buffer containing 100 mM Bicine, pH 8.2, and 20 mM MgCl₂ to which [¹⁴C]NaHCO₃ (4.6 kBq µmol¹) and RuBP had been added to concentrations of 10 and 0.4 mM, respectively, immediately prior to adding the extract. Total activity was determined at 25°C by incubating 20 µL of extract for 3 min in 980 µL of the same assay buffer without RuBP and with free CO₂ to allow for the carbamylation of all available active sites. The assay was started by adding RuBP to 0.4 mM as above.

For determination of maximal activity, 100 µL of extract was incubated for 30 min in an equal volume of 400 mM Na₂SO₄ at 4°C to remove any tight-binding inhibitors that may have been present. This was followed by centrifugation at 400g for 2 min at 4°C using Sephadex G25 medium (Pharmacia) equilibrated with extraction buffer in a small polystyrene column (Pierce). Maximal activity was determined using the protocol for total activity, beginning with the 3-min incubation in the absence of RuBP. All assays were stopped after 1 min by adding 100 µL of 10 M formic acid to liberate any unfixed CO₂ and thereafter evaporated to dryness. Determination of ¹⁴C was by liquid-scintillation spectrometry.

The content of CF₁ - and -subunits was quantified in a selection of the remaining leaf halves by extraction in a 50 mM Bicine buffer, pH 7.6, which contained 20 mM EDTA, 1 mM MgCl₂, and 50 mM -mercaptoethanol. Again, part of the homogenate was removed for the determination of chlorophyll and soluble protein, and each sample was clarified at 10,000g for 3 min. Samples were solubilized at a SDS to protein ratio of 4:1 in 250 mM Tris buffer, pH 7.6, containing 250 mM DTT, 10% (w/v) SDS, 0.2% (w/v) bromphenol blue, and 10% (w/v) glycerol. Denatured CF₁ was electrophoresed on a 12% discontinuous gradient of SDS-polyacrylamide minigel (Bio-Rad), and separated proteins were electroblotted for 90 min at 1.8 mA cm² onto a PVDF membrane (Millipore) in a cell (TransBlot, Bio-Rad) containing 25 mM Tris, pH 8.3, 192 mM Gly, and 20% methanol. The membrane was blocked and developed with a chemiluminescent detection system (Aurora, ICN) using primary polyclonal antibody against the - and -subunits of CF₁ and the manufacturer's protocol.

Bands visualized onto radiographic film were scanned and quantified using a software package (SigmaGel, Jandel Scientific, Sausalito, CA). CF₁ standard run alongside samples was prepared to approximately 90% purity from 10-d-old wheat seedlings using the method of Moase and Green (1981). The standard was further purified on a Sephacryl S-300 column (Pharmacia) that had been preequilibrated with several washes of buffer containing 40 mM Tricine-NaOH, pH 8.0, 1 mM ATP, and 1 mM EDTA. Crude CF₁ was applied to the column and eluted using a 0.1 to 0.5 M NaCl gradient. Total CF₁ obtained was assessed at 98% purity, of which 63% represented the - and -subunits. Assuming that all CF₁ - and -subunits are present in the thylakoid ATP-synthase complex, the amount of this complex was estimated by multiplying CF₁  and  content by 1.61 g ATP-synthase per g CF₁  and  (Moase and Green, 1981).

	RESULTS

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The As measured at 70 Pa p_a and high light corrected for nonphotorespiratory respiration (A_g,70) are shown in Figure 1 for various leaves in experiments 1 and 2. In each case, the first point for each leaf corresponds approximately to full leaf expansion (ligule just visible), and there was no significant effect of growth-CO₂ conditions on A_g at this time. When there were significant effects (e.g. at 15 d after anthesis in experiment 2), growth in elevated CO₂ decreased the A. The effect of the N treatment was more pronounced in flag leaves in experiment 2 than for the other leaves in experiments 1 and 2. The pattern of effects was very similar for the carboxylation capacity, V_cmax, as estimated from A  p_i responses (Fig. 2). Again, elevated CO₂ treatment did not affect leaves at full expansion or shortly afterward but did induce a more rapid subsequent decline.

View larger version (31K): [in this window] [in a new window]   Figure 1. The estimated rate (see text) of Ag,70 versus time as days relative to anthesis in wheat leaves in two experiments. Data for flag leaves are presented in separate graphs from preceding leaves for clarity. Plants were grown in different N-application and CO2 environments, as denoted in the key. For all plants, photosynthesis was measured at a leaf temperature of 20°C, a PPFD of 1500 µmol quanta m2 s1, and a leaf-to-air vapor-pressure deficit of 1.5 kPa. Error bars represent SE of differences between mean values shown from the CO2 × N analysis of variance for each occasion (n = 6).

View larger version (30K): [in this window] [in a new window]   Figure 2. The estimated rate (see text) of Vcmax versus time as days relative to anthesis for wheat leaves in two experiments. Treatments were as described in Figure 1. Error bars are defined as in Figure 1.

The relationship between A_g measured at 70 Pa CO₂ partial pressure and the ATP-synthase content of the same leaf samples is shown in Figure 3. The line shown is a linear regression through the origin (r² = 0.65), and from the slope, k_cat was estimated using Equation 4 at 200 mol ATP mol¹ ATP-synthase s¹ (SE = 6). The relationship between V_cmax estimated from A  p_i responses and Rubisco content of the same leaf samples was close to the theoretical proportionality (Fig. 4). The linear regression through the origin (r² = 0.83) gave an overall k_cat estimate of 2.79 s¹ (SE = 0.06).

View larger version (22K): [in this window] [in a new window]   Figure 3. Rate of Ag,70 versus ATP-synthase content for flag 1 and flag leaves in experiment 1. Photosynthesis was measured at a leaf temperature of 20°C, a PPFD of 1500 µmol PPFD m2 s1, and a leaf-to-air vapor-pressure deficit of 1.5 kPa. Plants were grown with a total application of eight (circles) and 18 (squares) g N m2 under 36 (open symbols) and 70 (closed symbols) Pa CO2. The line is a linear regression forced through the origin: y = 158x (r2 = 0.65).

View larger version (20K): [in this window] [in a new window]   Figure 4. Rate of estimated (see text) Vcmax versus Rubisco content for the first four occasions of flag leaves from experiment 2. Conditions and treatments were as described in Figure 3. The line is a linear regression forced through the origin: y = 40.6x (r2 = 0.83).

Chlorophyll, Rubisco, and ATP-synthase content were related to leaf N content (Fig. 5). Chlorophyll was approximately proportional to the leaf N content (Fig. 5b), whereas Rubisco had a positive x intercept (Fig. 5a), suggesting that the fraction of leaf N represented by Rubisco increases as leaf N content increases. ATP-synthase against leaf N (Fig. 5d) had a smaller x intercept than for Rubisco. Figure 6 shows the relationship between ATP-synthase and Rubisco content determined in the same flag1 and flag leaves of various ages in the different treatments. It is clear that Rubisco and ATP-synthase are not simply proportional and that the mass ratio of Rubisco to ATP-synthase increases from about 3.8 at low Rubisco content to 5.75 at high Rubisco content. There were no significant effects of N or CO₂ treatment on this relationship.

View larger version (17K): [in this window] [in a new window]   Figure 5. Amounts of Rubisco and chlorophyll, chlorophyll a/b ratio, and ATP-synthase content versus total leaf N content. For ATP-synthase (d), points are the means of three N values versus the means of three ATP-synthase values in leaves of the same occasion and treatment, with SE shown for both axes of each. Plants were grown at either low (circles) or high (squares) N applications and at 36 (open symbols) or 70 Pa CO2 (closed symbols). For Rubisco (a), y = 1.944x  0.684 (r2 = 0.92). For chlorophyll (b), y = 0.364x (r2 = 0.75). For ATP-synthase (d), y = 0.15x  0.04 (r2 = 0.69).

View larger version (22K): [in this window] [in a new window]   Figure 6. The relationship between ATP-synthase and Rubisco contents measured in the same leaf (flag1 or flag) of experiment 1. The line is a linear regression: y = 0.14x + 0.08 (r2 = 0.68). Symbols are as described in Figure 5.

Given the relationship between V_cmax and Rubisco content (Fig. 4), Equation 3 was used to estimate the apparent excess Rubisco content compared with that required to maintain the observed rate of photosynthesis at 70 Pa atmospheric CO₂ (Fig. 7). For all leaves and for both experiments, the excess was consistently less at low N. Where elevated CO₂ treatment had a significant effect (flag1 leaves in experiment 1 and flag leaves in all experiments), it decreased the excess. In some cases (e.g. experiment 2: low N, 70 Pa, flag leaves 15 d after anthesis), the estimated excess was not significantly greater than 0. It is also possible to use Equation 3 to estimate excess Rubisco at ambient CO₂. This was only significantly different from 0 for the last three points for high-N flag leaves in experiment 2, decreasing from approximately 0.4 to approximately 0.2 g m² (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 7. The estimated excess (see text) of Rubisco at 70 Pa CO2 atmospheric partial pressure versus time as days relative to anthesis for various wheat leaves from two experiments. Treatments were as described in Figure 1. Error bars are defined as in Figure 1.

In experiment 3 leaves used for gas exchange were freeze-clamped at high light and at their growth p_a and the activity of isolated Rubisco was determined in vitro, which was related to the Rubisco content. In Figure 8 the in vitro activities are compared with the V_cmax required to achieve the observed A measured at the growth CO₂ partial pressure, estimated from the gas-exchange parameters using Equation 2. Total activity refers to that determined after complete activation of the enzyme and maximal activity after treatment to remove any inhibitors bound to the active site (Parry et al., 1997). The initial and total activities show a distinct curvilinear relationship, with the amount determined by [¹⁴C]CABP binding, whereas maximal activity is proportional, giving an estimated k_cat of 3.1 s¹ at 25°C. Although the relationships between total and maximal activity and Rubisco content are independent of growth-CO₂ treatment, the lines for initial activity for the two treatments diverge at high Rubisco content. A similar pattern is seen for the estimates of required V_cmax, which diverge at higher Rubisco contents.

View larger version (15K): [in this window] [in a new window]   Figure 8. Estimates of in vivo and in vitro Rubisco activity versus Rubisco content in flag leaves of different ages measured between 22 d before anthesis and 18 d after anthesis in experiment 3. Plants were grown at 36 (open symbols) and 100 (closed symbols) Pa CO2 partial pressure. Top, Vcmax required to maintain observed A at a PPFD of 1500 µmol quanta m2 s1 and the growth CO2 partial pressure estimated from the A  pi response using Equation 2. Bottom, Initial, total, and maximal in vitro activities determined at 25°C of Rubisco isolated from leaves freeze-clamped under a PPFD of 1500 µmol quanta m2 s1 and the growth atmospheric CO2 partial pressure. Lines are regressions forced through the origin. Separate regressions were used for the two treatments where this improved the r2 value (dashed lines); otherwise a single regression was used (solid line). Second-order linear regressions were used where this improved the r2 value (gas-exchange, elevated-CO2 treatment, and initial and total, both treatments); otherwise, first-order regression was used.

	DISCUSSION

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A  p_i Responses

Amounts of Photosynthetic Components

1

1

The relationship between the estimated in vivo V_cmax and the Rubisco content used to derive the estimate of Rubisco k_cat of 2.8 s¹ at 20°C (Fig. 4) was strong (r² = 0.83). The value is in line with in vivo estimates for tobacco of 3.5 s¹ (25°C; von Caemmerer et al., 1994) and for wheat of 3.3 s¹ (23°C; Evans and Austin, 1986) and an in vitro estimate for wheat of 3.0 s¹ (25°C; Makino et al., 1988), given the temperature dependence that would be expected to increase the value by 40% going from 20°C to 25°C (Machler et al., 1980; Makino et al., 1988). The fraction of leaf N invested in Rubisco increases with leaf N content from 21% to 26% in the range 1 to 2 g N m² (Fig. 5d) in a manner similar to previous findings for wheat flag leaves of varying age (Makino et al., 1988; Lawlor et al., 1989).

The relationship between ATP-synthase and Rubisco content determined in experiment 1 (Fig. 6) was such that the ratio of Rubisco to ATP-synthase increased with increasing Rubisco content independently of treatment. Since the amounts of these components are expected to reflect carboxylation and RuBP-regeneration capacities (supported by the data in Figs. 3 and 4), this suggests that carboxylation capacity increases relative to RuBP-regeneration capacity in leaves with high N content and that this is not affected by elevated CO₂. This is in close agreement with the results of Nakano et al. (1997) using rice. Also, a field experiment on wheat revealed that elevated CO₂ did not affect the ratio of Rubisco to other components, including CF₁, at flag leaf emergence but did induce a decrease in this ratio during grain fill (Nie et al., 1995), which could be interpreted as a more rapid decline in leaf N in the elevated-CO₂-grown leaves.

Excess Investment in Rubisco

2

2

View larger version (23K): [in this window] [in a new window]   Figure 9. The fraction of Rubisco estimated to be in excess of that required to maintain A at 70 Pa atmospheric CO2 partial pressure versus total leaf N content, for all leaves in experiments 1 and 2. Plants were grown at low (circles) and high (squares) N applications and at 36 (open symbols) or 70 (closed symbols) Pa CO2. The solid line is linear regression of all points (r2 = 0.41; n = 87). Dotted lines are equivalent regressions for same data when recalculated with gw = 2 and 6 µmol m2 s1 Pa1 (r2 = 0.56 and 0.45, respectively).

The estimates of excess Rubisco refer to the conditions used for the determination of steady-state photosynthesis. The conditions of high light intensity and moderate temperature used are those under which Rubisco would be expected to be the least in excess; therefore, the excess would presumably be greater under variable field conditions at elevated CO₂. (However, this conclusion would not hold for conditions that decrease stomatal conductance, such as water stress.)

Sensitivity to g_w

2

1

1

1

1

2

1

1

2

Rubisco Activation

The pattern of V_cmax,req was similar to initial activity, with elevated CO₂ points lying increasingly below ambient CO₂ points as Rubisco content increased, although the absolute initial in vitro activities were much lower. The apparent decrease in activation state (initial activity/maximal activity) with increasing Rubisco content in ambient CO₂ leaves to 60% to 70% was unexpected, since it has been assumed that all Rubisco is utilized at high light and p_a = 36 Pa, for which there is good evidence (Makino et al., 1988; Sage et al., 1990) and which resulted in a close relationship between estimated V_cmax and Rubisco content (Fig. 4). Part of the decrease in activation state is associated with an inhibitor, as demonstrated by the difference between total and maximal activity (Parry et al., 1997), but initial activity was also somewhat lower than total. Although the conditions used were similar to those used in many other studies, initial activity is known to be affected by pretreatment of the extraction buffer and by the time taken to determine activity (Sage et al., 1993), which may have lowered absolute initial activities in our material, but the difference between treatments probably reflects the in vivo state.

The in vitro maximal activity of Rubisco was closely correlated (r² = 0.98) with its amount, and the estimated k_cat of 3.1 s¹ was the same as that estimated previously for wheat at 25°C (Makino et al., 1988). However, even the maximal activity was somewhat less (20% lower at 1.5 g m² Rubisco) than the V_cmax,req for ambient leaves (Fig. 8); estimates of Rubisco activity are often higher in vivo than in vitro (von Caemmerer et al., 1994).

	CONCLUSIONS

We have found evidence that there is excess investment in Rubisco over that required for maintaining A at elevated CO₂ and that this excess is greater at high N. Long-term growth at elevated CO₂ does reduce this excess somewhat but apparently only as an indirect consequence of elevated CO₂ causing a decreased leaf N content, which agrees with the findings of Nakano et al. (1997). There is therefore no evidence of a direct mechanism optimizing the balance between carboxylation and RuBP-regeneration capacities in response to long-term growth at elevated CO₂. Makino et al. (1997) observed an increase in A per unit leaf N in rice plants genetically manipulated to reduce Rubisco expression, which was measured under conditions of short-term exposure to elevated CO₂. In agreement with the relationship shown in Figure 9, they found the greatest advantage in leaves with high N content. The work presented here suggests that most of this advantage would be preserved if the plants were grown under conditions of elevated CO₂, since there is still excess investment in Rubisco under these conditions. There is therefore potential for improving the adaptation of crop plants to growth at elevated CO₂.

	FOOTNOTES

   Received April 28, 1998; accepted August 13, 1998.

	ABBREVIATIONS

Abbreviations: A, photosynthetic rate. A_g, gross photosynthetic rate. CABP, 2-carboxyarabinitol-1,5-bisphosphate. CF₁, thylakoid coupling factor 1. g_w, conductance for diffusion of CO₂ from the intercellular space to the carboxylation site. p_a, external CO₂ partial pressure. p_i, internal CO₂ partial pressure. RuBP, ribulose-1,5-bisphosphate.

	ACKNOWLEDGMENTS

The kind gift of antisera against CF₁ from Dr. J.C. Gray (University of Cambridge, UK) is gratefully acknowledged. We thank Dr. P.J. Andralojc for assistance and advice with the ATP-synthase work and S.P. Driscoll and V.J. Mitchell for technical assistance.

	LITERATURE  CITED

Top Abstract Introduction Methods Results Discussion References

Bowes G (1993) Facing the inevitableplants and increasing atmospheric CO₂. Annu Rev Plant Physiol Mol Biol 44: 309-332 [CrossRef][Web of Science]

Brooks A, Farquhar GD (1985) Effect of temperature on the CO₂/O₂ specificity of ribulose-1,5- bisphosphate carboxylase oxygenase and the rate of respiration in the lightestimates from gas-exchange measurements on spinach. Planta 165: 397-406 [CrossRef][Web of Science]

Delgado E, Mitchell RAC, Parry MAJ, Driscoll SP, Mitchell VJ, Lawlor DW (1994) Interacting effects of CO₂ concentration, temperature and nitrogen supply on the photosynthesis and composition of winter-wheat leaves. Plant Cell Environ 17: 1205-1213 [CrossRef]

Drake BG, Gonzalez-Meler MA, Long SP (1997) More efficient plants: a consequence of rising atmospheric CO₂? Annu Rev Plant Physiol Mol Biol 48: 609-639 [CrossRef][Web of Science]

Evans JR, Austin RB (1986) The specific activity of ribulose-1,5-bisphosphate carboxylase in relation to genotype in wheat. Planta 167: 344-350 [CrossRef]

Evans JR, Seemann JR (1989) The allocation of protein nitrogen in the phototsynthetic apparatus: costs, consequences and control. In W Briggs, ed, Photosynthesis. Alan R Liss, New York, pp 183-205

Farquhar GD, von Caemmerer S (1982) Modelling of photosynthetic response to environmental conditions. In OL Lange, PS Nobel, CB Osmond, H Ziegler, eds, Physiological Plant Ecology, Vol 12. Springer-Verlag, Berlin, pp 549-587

Farquhar GD, Sharkey TD (1994) Photosynthesis and carbon assimilation. In KJ Boote, JM Bennett, TR Sinclair, GM Paulson, eds, Physiology and Determination of Crop Yield. American Society of Agronomy, Madison, WI, pp 187-210

Fromme P, Graber P (1989) Heterogeneity of ATP-hydrolyzing sites on reconstituted CF₀CF₁. FEBS Lett 259: 33-36 [CrossRef]

Habash DZ, Paul MJ, Parry MAJ, Keys AJ, Lawlor DW (1995) Increased capacity for photosynthesis in wheat grown at elevated CO₂the relationship between electron-transport and carbon metabolism. Planta 197: 482-489

Krapp A, Stitt M (1995) An evaluation of direct and indirect mechanisms for the sink-regulation of photosynthesis in spinachchanges in gas-exchange, carbohydrates, metabolites, enzyme-activities and steady-state transcript levels after cold-girdling source leaves. Planta 195: 313-323 [Web of Science]

Lawlor DW, Konturri M, Young AT (1989) Photosynthesis by flag leaves of wheat in relation to protein, ribulose bisphospate carboxylase activity and nitrogen supply. J Exp Bot 40: 43-52 [Abstract/Free Full Text]

Lawlor DW, Mitchell RAC (1991) The effects of increasing CO₂ on crop photosynthesis and productivitya review of field studies. Plant Cell Environ 14: 807-818 [CrossRef]

Lawlor DW, Mitchell RAC, Franklin J, Mitchell VJ, Driscoll SP, Delgado E (1993) Facility for studying the effects of elevated carbon-dioxide concentration and increased temperature on crops. Plant Cell Environ 16: 603-608

Loreto F, Dimarco G, Tricoli D, Sharkey TD (1994) Measurements of mesophyll conductance, photosynthetic electron-transport and alternative electron sinks of field-grown wheat leaves. Photosynth Res 41: 397-403 [CrossRef]

Loreto F, Harley PC, Dimarco G, Sharkey TD (1992) Estimation of mesophyll conductance to CO₂ flux by three different methods. Plant Physiol 98: 1437-1443 [Abstract/Free Full Text]

Machler F, Keys AJ, Cornelius MJ (1980) Activation of ribulose bisphosphate carboxylase purified from wheat leaves. J Exp Bot 31: 7-14 [Abstract/Free Full Text]

Makino A (1994) Biochemistry of C₃-photosynthesis in high CO₂. J Plant Res 107: 79-84

Makino A, Mae T, Ohira K (1988) Differences between wheat and rice in the enzymic properties of ribulose-1,5-bisphosphate carboxylase oxygenase and the relationship to photosynthetic gas-exchange. Planta 174: 30-38 [CrossRef][Web of Science]

Makino A, Shimada T, Takumi S, Kaneko K, Matsuoka M, Shimamoto K, Nakano H, Miyao-Tokutomi M, Mae T, Yamamoto N (1997) Does decrease in ribulose-1,5-bisphosphate carboxylase by antisense RbcS lead to a higher N-use efficiency of photosynthesis under conditions of saturating CO₂ and light in rice plants? Plant Physiol 114: 483-491 [Abstract]

Medlyn BE (1996) The optimal allocation of nitrogen within the C₃ photosynthetic system at elevated CO₂. Aust J Plant Physiol 23: 593-603

Moase EH, Green BR (1981) Isolation and properties of chloroplast coupling factor from wheat. Eur J Biochem 119: 145-150 [Medline]

Nakano H, Makino A, Mae T (1997) The effect of elevated partial pressures of CO₂ on the relationship between photosynthetic capacity and N content in rice leaves. Plant Physiol 115: 191-198 [Abstract]

Nie GY, Long SP, Garcia RL, Kimball BA, Lamorte RL, Pinter PJ, Wall GW, Webber AN (1995) Effects of free-air CO₂ enrichment on the development of the photosynthetic apparatus in wheat, as indicated by changes in leaf proteins. Plant Cell Environ 18: 855-864 [CrossRef]

Parry MAJ, Andralojc PJ, Parmar S, Keys AJ, Habash D, Paul MJ, Alred R, Quick WP, Servaites JC (1997) Regulation of Rubisco by inhibitors in the light. Plant Cell Environ 20: 528-534 [CrossRef]

Rowland-Bamford AJ, Baker JT, Allen LH, Bowes G (1991) Acclimation of rice to changing atmospheric carbon-dioxide concentration. Plant Cell Environ 14: 577-583 [CrossRef]

Sage RF (1994) Acclimation of photosynthesis to increasing atmospheric CO₂the gas-exchange perspective. Photosynth Res 39: 351-368 [CrossRef]

Sage RF, Reid CD (1992) Photosynthetic acclimation to sub-ambient CO₂ (20 Pa) in the C3 annual Phaseolus vulgaris L. Photosynthetica 27: 605-617

Sage RF, Reid CD, Moore BD, Seemann JR (1993) Long-term kinetics of the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase oxygenase activity in plants with and without 2-carboxyarabinitol 1-phosphate. Planta 191: 222-230 [Web of Science]

Sage RF, Sharkey TD, Seemann JR (1990) Regulation of ribulose-1,5-bisphosphate carboxylase activity in response to light intensity and CO₂ in the C₃ annuals Chenopodium album and Phaseolus vulgaris. Plant Physiol 94: 1735-1742 [Abstract/Free Full Text]

Sicher RC, Bunce JA (1997) Relationship of photosynthetic acclimation to changes of Rubisco activity in field-grown winter wheat and barley during growth in elevated carbon dioxide. Photosynth Res 52: 27-38 [CrossRef]

Sicher RC, Kremer DF, Bunce JA (1995) Photosynthetic acclimation and photosynthate partitioning in soybean leaves in response to carbon-dioxide enrichment. Photosynth Res 46: 409-417

van Oosten JJ, Besford RT (1996) Acclimation of photosynthesis to elevated CO₂ through feedback-regulation of gene-expressionclimate of opinion. Photosynth Res 48: 353-365 [CrossRef]

von Caemmerer S, Evans JR (1991) Determination of the average partial-pressure of CO₂ in chloroplasts from leaves of several C₃ plants. Aust J Plant Physiol 18: 287-305 [Web of Science]

von Caemmerer S, Evans JR, Hudson GS, Andrews TJ (1994) The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in-vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco. Planta 195: 88-97 [Web of Science]

Webber AN, Nie GY, Long SP (1994) Acclimation of photosynthetic proteins to rising atmospheric CO₂. Photosynth Res 39: 413-425 [CrossRef]

Yokota A, Canvin DT (1985) Ribulose bisphosphate carboxylase oxygenase content determined with [C-14]carboxypentitol bisphosphate in plants and algae. Plant Physiol 77: 735-739 [Abstract/Free Full Text]

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