Bundle Sheath Diffusive Resistance to CO₂ and Effectiveness of C₄ Photosynthesis and Refixation of Photorespired CO₂ in a C₄ Cycle Mutant and Wild-Type Amaranthus edulis

Abstract

A mutant of the NAD-malic enzyme-type C₄ plant, Amaranthus edulis, which lacks phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells was studied. Analysis of CO₂ response curves of photosynthesis of the mutant, which has normal Kranz anatomy but lacks a functional C₄ cycle, provided a direct means of determining the liquid phase-diffusive resistance of atmospheric CO₂ to sites of ribulose 1,5-bisphosphate carboxylation inside bundle sheath (BS) chloroplasts (r_bs) within intact plants. Comparisons were made with excised shoots of wild-type plants fed 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate, an inhibitor of PEPC. Values of r_bs in A. edulis were 70 to 180 m² s⁻¹ mol⁻¹, increasing as the leaf matured. This is about 70-fold higher than the liquid phase resistance for diffusion of CO₂ to Rubisco in mesophyll cells of C₃ plants. The values of r_bs in A. edulis are sufficient for C₄ photosynthesis to elevate CO₂ in BS cells and to minimize photorespiration. The calculated CO₂ concentration in BS cells, which is dependent on input of r_bs, was about 2,000 μbar under maximum rates of CO₂ fixation, which is about six times the ambient level of CO₂. High re-assimilation of photorespired CO₂ was demonstrated in both mutant and wild-type plants at limiting CO₂ concentrations, which can be explained by high r_bs. Increasing O₂ from near zero up to ambient levels under low CO₂, resulted in an increase in the gross rate of O₂ evolution measured by chlorophyll fluorescence analysis in the PEPC mutant; this increase was simulated from a Rubisco kinetic model, which indicates effective refixation of photorespired CO₂ in BS cells.

In C₄ plants, atmospheric CO₂ is fixed via phosphoenolpyruvate carboxylase (PEPC) in mesophyll cells into C₄ acids, which are transported to bundle sheath (BS) cells where they serve as donors of CO₂ to the C₃ cycle via C₄ acid decarboxylases (Kanai and Edwards, 1999). Photosynthetic carbon metabolism in C₄ plants requires low rates of CO₂ leakage from BS cells for CO₂ to be concentrated around Rubisco in the BS chloroplasts. This favors CO₂ fixation and minimizes photorespiration. However, the BS is not impermeable to gases because there is a need for metabolites to be exchanged between it and the mesophyll cells and for O₂ generated during photosynthesis to be released. This permeability causes CO₂ leakage from BS cells and results in a lower energetic efficiency of the CO₂-concentrating mechanism.

Diffusive resistance of CO₂ into BS cells (r_bs) has been estimated by measuring photosynthetic rates under varying CO₂ concentrations in isolated BS cells (Furbank et al., 1989) and in excised leaves fed a chemical inhibitor of the C₄ cycle (Jenkins et al., 1989a; Brown and Byrd, 1993; Brown, 1997), or by applying models to experimental data related to the magnitude of photorespiration in C₄ plants (He and Edwards, 1996). However, accurate determination of r_bs is difficult; current estimates vary over a wide range from about 15 to 1,400 m² s⁻¹ mol⁻¹ (for review, see He and Edwards, 1996).

BS-diffusive resistance is considered the major component that determines the CO₂ leakage driven by the CO₂ concentration gradient between BS and mesophyll cells. The rate of leakage of CO₂ from the BS cells is equal to the rate of over-cycling of the C₄ pathway (rate of C₄ cycle minus rate of CO₂ fixation by the C₃ cycle). There is considerable variation in estimates of the fraction of CO₂ leakage with various methods, ranging from 0.08 up to 0.5 when expressed as a fraction of C₄ cycle activity (see He and Edwards, 1996). CO₂ leakiness was determined for a number of species using an isotope discrimination method (Henderson et al., 1992) and a method involving analysis of ¹⁴CO₂ release after its fixation (Hatch et al., 1995), with leakiness values ranging from 0.08 to 0.3.

The CO₂ concentration in BS cells is dependent on r_bs, the rate of C₄ pathway over-cycling, and the CO₂ diffusion gradient from BS to mesophyll cells. For a better understanding of C₄ photosynthesis, it would also be valuable to know the actual CO₂ concentration in BS chloroplasts, where CO₂ assimilation takes place. A possible means of estimating this is to combine measurements of CO₂ fixation with information on the kinetic properties of Rubisco. Ribulose 1,5-bisphosphate (RuBP) carboxylation and oxygenation in BS chloroplasts are competing reactions, and information is available on the Rubisco CO₂ to O₂ affinity ratio.

In this study, we have used the PEPC mutant of Amaranthus edulis LaC₄ 2.16 (Dever et al., 1995; Maroco et al., 1998a, 1998b), which has a defective C₄ cycle and requires direct diffusion of atmospheric CO₂ into the BS cells for CO₂ assimilation and growth. In this mutant, the primary carboxylase for fixing atmospheric CO₂ is Rubisco, which is located in the BS chloroplast. The purpose of this work was to use gas exchange measurements on the A. edulis mutant for direct estimation of BS cell resistance to CO₂, and to determine the dependence of r_bs on the developmental stage of the leaf. For comparison, data were also obtained with wild-type plants by feeding the PEPC inhibitor 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (DCDP) to prevent operation of the C₄ cycle. In addition, we determined rates of CO₂ fixation and gross rates of O₂ evolution to analyze the effect of temperature on the cellular conductance to CO₂ and the effects of CO₂, light, and O₂ on partitioning of electron flow and refixation of photorespired CO₂.

RESULTS AND DISCUSSION

Figure 1 outlines important aspects of photosynthesis in the PEPC mutant of A. edulis relative to the experimental approach for determining the BS-diffusive resistance to CO₂ (r_bs). Because the C₄ cycle is inoperative, the mechanism of CO₂ fixation and energy requirements are considered the same as in C₃ plants. Fixation of atmospheric CO₂ in the mutant by Rubisco in the C₃ cycle, requires diffusion of CO₂ from the mesophyll to BS cells. CO₂ is considered the primary species of inorganic carbon diffusing to BS cells and supplying CO₂ to Rubisco. Although CO₂ will be converted rapidly to bicarbonate in the cytosol of mesophyll cells via carbonic anhydrase, the diffusion pathway for bicarbonate will be limited because it is not used by Rubisco and BS cells lack, or have negligible levels of, carbonic anhydrase (Ku and Edwards, 1975). Also, in wild-type A. edulis, where CO₂ is concentrated in the BS cells by the C₄ cycle through C₄ acid decarboxylation, CO₂ is considered the primary form of inorganic carbon leaking from BS to mesophyll cells (Jenkins et al., 1989b). Reaction of O₂ with RuBP in the photosynthetic carbon oxidation cycle in the BS chloroplasts will result in the production of the photorespiratory products glycerate, CO₂, and ammonia through metabolism in mitochondria and peroxisomes. According to the known compartmentalization of carbon assimilation in C₄ plants (Kanai and Edwards, 1999), the only function of mesophyll chloroplasts in carbon assimilation in the mutant may be the conversion of glycerate, the product of photorespiration in BS cells, to triose phosphate and conversion of some of the 3-phosphoglycerate (PGA), generated by Rubisco in BS cells, to triose phosphate.

Figure 1.

Schematic representation of the movement of gases and metabolites in the PEPC mutant of A. edulis. CO₂ diffuses into BS cell chloroplasts where it enters the C₃ cycle. Equilibrium between HCO₃⁻ and CO₂ in mesophyll cell is fast, but it is slow in BS because of lack of carbonic anhydrase activity. Glycerate, ammonia, and CO₂ are generated by the photosynthetic carbon oxidation (PCO) cycle. Glycerate metabolism and partial reduction of PGA in mesophyll cells may account for use of some photochemically generated energy in mesophyll chloroplasts.

BS-Diffusive Resistance to CO₂ (r_bs)

The resistances involved in uptake of atmospheric CO₂ by the mutant are described in Equation 10 (“Materials and Methods”). If the CO₂ response curve of CO₂ assimilation (measured at light saturation) is plotted against the intercellular CO₂ partial pressure (C_i), which equilibrates with the liquid phase at the cell wall, the initial slope of the curve is determined by the average physical liquid phase conductance and by the carboxylation efficiency of Rubisco (Fig. 2). Gas phase resistance to CO₂, r_s, was calculated based on transpiration measurements. Typical values of r_s for the mutant plants were in the range of 5 to 10 m² s⁻¹ mol⁻¹ (2–4 s cm⁻¹). Also, the expected Rubisco CO₂ response curve is shown in Figure 2 in the absence of liquid phase resistance; this demonstrates the contribution of the resistance of RuBP carboxylase (chemical resistance), r_c, to the total CO₂ flux resistance. The Rubisco response was generated taking the maximum velocity of RuBP carboxylase (V_c) as 1.2 times the CO₂-saturated rate of CO₂ fixation (A_max) based on Rubisco extractable activity measurements. Also, there is a decrease in RuBP pool with increasing CO₂, which could account for A_max being lower than V_c of Rubisco if RuBP becomes limiting (as shown later in Fig. 7). In general, r_c for mutant leaves was a minor component (from 10 to 15 m² s⁻¹ mol⁻¹) compared with r_bs (152 m² s⁻¹ mol⁻¹ in Fig. 2).

Figure 2.

Example of calculating BS cell resistance from A versus C_i curves measured at low O₂ (0.3 mbar) and PFD of 1,800 μmol m⁻² s⁻¹ in PEPC mutant. The inverse of the initial slope of A/C_i curve is the sum of the diffusive resistance from the cell wall to the sites of Rubisco and of the chemical RuBP carboxylation resistance. A simulated Rubisco CO₂ response curve without diffusive resistance is shown for comparison. Rubisco resistance can be calculated as K_c/V_c. V_c was taken as A_max.

Figure 7.

CO₂ response for CO₂ assimilation rate (A, ○), gross O₂ evolution rate (J_O2, ▵), RuBP pool size (●), Rubisco activity (⧫), and Rubisco activation state (□) for PEPC mutant A. edulis at two O₂ pressures, 0.3 and 210 mbar. Leaf temperature was 28°C, PFD = 1,400 μmol m⁻² s⁻¹. Each point is from a different leaf of similar age.

Analogous experiments to those in Figure 2 were performed on leaves of different maturity. The value of r_bs increased with plant age and reached its highest value during grain filling when the leaves were pale green, showing early signs of senescence and lower maximum rates of CO₂ fixation (Table I). Although BS resistance was calculated assuming V_c is 1.2 times A_max, a sensitivity analysis taking V_c/A_max = 1.0 and 1.4 showed that this results in a change in the calculated r_bs values of only 3% to 5% in young leaves, and approximately 2% in mature leaves. Interestingly, during the grain filling stage, the leaves still maintained a reasonably high-CO₂ assimilation capacity. The liquid phase resistance from the mesophyll cells to Rubisco in the mutant A. edulis (72–181 m² s⁻¹ mol⁻¹; Table I) is about 70-fold higher than that of C₃ plants (approximately 1–3 m² s⁻¹ mol⁻¹; Evans et al., 1994; Laisk and Loreto, 1996). This high-diffusive resistance in C₄ plants may be attributed to the relatively low BS cell surface area per unit leaf area and structural properties of BS cell walls (Evans and von Caemmerer, 1996). Models of C₄ photosynthesis indicate the rate of CO₂ assimilation under low CO₂ drops rapidly below r_bs values of 50 to 100 m² s⁻¹ mol⁻¹ (Edwards et al., 2000; Laisk and Edwards, 2000; von Caemmerer, 2000).

Table I.

Leaf age-dependent differences in A_max and resistance to CO₂ in the A. edulis PEPC mutant plants

Leaf Description	Amax	Mesophyll to BS Resistance to CO2 (rbs+rc)	Liquid Phase Resistance to CO2 (rbs)
	μmol m−2 s−1	m2 s−1 mol−1
Young, 30% expanded	25.1 ±  1.4	89 ±  16	72.4
Young, 70% expanded	26.6 ±  1.8	103 ±  16	86.3
Mature, vegetative, 100% expanded	30.6 ±  1.0	127 ±  6	113.4
Grain filling stage, 100% expanded	20.6 ±  1.2	201 ±  17	180.8

The mesophyll to BS resistance for CO₂ was calculated as the inverse of the initial slope of A/C_i curves. The sd for mesophyll to BS resistance and A_max was calculated from four independent measurements. r_bs was calculated according to the method used in Figure 2. A_max was the CO₂-saturated rate of photosynthesis, and other conditions of the assay were as in Figure 2.

The resistance observed in the mutant might not reflect the true value of the wild type if the mutation alters the structure of the BS cells. To test this, we used the PEPC inhibitor DCDP, feeding it into the petiole to block the C₄ cycle in wild-type plants (method of Jenkins et al., 1989a). The strong reduction in carboxylation efficiency caused by 4 mm DCDP, without a biphasic response (Fig. 3), suggests that PEPC is almost completely inhibited. CO₂ response curves were measured at 2% (v/v) O₂ before applying DCDP and immediately after photosynthesis declined at ambient CO₂ as PEPC was inhibited (Fig. 3). The results show that the calculated BS resistances are similar to those obtained with the PEPC mutant. We measured A/C_i response curves (C_i determined from analysis of transpiration, which eliminates stomatal resistance) and calculated the resistance from the initial slope with V_c for Rubisco equal to 1.2 A_max estimated from CO₂-saturated rates in the presence of DCDP. In calculating r_bs, it is important to eliminate stomatal resistance and to account for any partial inhibition of Rubisco, and V_c, by DCDP. Jenkins et al. (1989a) calculated a permeability coefficient for CO₂ from the atmosphere to BS cells (the reciprocal for the total diffusive resistance, r_t) from measurements of photosynthetic O₂ evolution in the presence of DCDP at 1.6% (v/v) CO₂ by dividing the photosynthetic rate by the difference between atmospheric CO₂ (C_a) and estimates of C_BS. The calculated value of r_t from the study of Jenkins et al. in A. edulis is 556 s · cm⁻¹ (or 1,373 m² s⁻¹ mol⁻¹), which is about 10-fold higher than values of r_bs in the present study. Because r_t includes stomatal and BS-diffusive resistance, high stomatal resistance in excised leaves could contribute to high r_t values. Our A/C_i response curves with the PEPC mutant saturate sharply at intercellular CO₂ concentrations about 1%, whereas up to 5% (v/v) ambient CO₂ was required in experiments by Jenkins et al. (1989). Also, the calculation of r_t is dependent on input of V_c of Rubisco to calculate C_BS. Using the value of V_c from analysis of wild-type plants (Jenkins et al., 1989), rather than V_c in the presence of DCDP, will also overestimate V_c and the calculated diffusive resistance values (see also He and Edwards, 1996).

Figure 3.

The response of the rates of CO₂ assimilation (A, ○, ●) and gross rate of O₂ evolution from PSII (J_O2, ▴, ▵) on wild type and PEPC mutant with and without feeding DCDP. Measurements were made on leaves of excised plants under 20 mbar O₂. The calculated values of r_bs in wild-type plants in presence of DCDP and in PEPC mutant with and without DCDP are shown. A and B, Wild-type plants grown at 370 μbar CO₂; C and D, wild-type plants grown at 10 mbar CO₂; E and F, PEPC mutant grown at 10 mbar CO₂.

Light microscopy of leaf anatomy indicates that the wild-type plants grown under both 370 μbar and 10 mbar and the PEPC mutant grown under 10 mbar of CO₂ all have Kranz-type leaf anatomy (results not shown; Dever et al., 1995). In wild-type plants grown at 10 mbar of CO₂, the BS cell walls at the intercellular space are very thick relative to the walls of the mesophyll and cross walls (Fig. 4); similar results were obtained with wild type grown under ambient CO₂. In the PEPC mutant, the BS cell walls are also much thicker than those of mesophyll cells, and cross walls include normal plasmodesmata (arrow). This suggests there are no structural differences between mutant and wild type.

Figure 4.

Electron microscopy showing cross sections through interface of mesophyll and BS of leaves of wild type (A) and PEPC mutant (B) with plants grown at 10 mbar CO₂. M, Part of mesophyll cell; BS, part of BS cell; W, cell wall; IS, intercellular air space. Arrows point to plasmodesmata. Scale bar = 0.5 μm. The average thickness of BS cell wall in contact with intercellular air from several sections was 0.34 μm for wild type and 0.32 μm for mutant (n = 3).

Cellular Conductance and the Temperature Dependence of Photosynthesis under Limiting and Saturating CO₂

CO₂ response curves of photosynthesis were measured in wild-type and mutant A. edulis at leaf temperatures from 15°C to 35°C. Figure 5, A and B, describes the temperature response of A_max, determined from CO₂-saturated rates, and cellular conductance for CO₂, g, determined from the initial slope of the net rate of CO₂ assimilation (A/C_i) curves. In this case, conductance instead of resistance (g = 1/r) was used, because it is linearly related to the diffusion flux. In wild-type plants, the cellular conductance for CO₂, g_wt, was a function of liquid phase diffusion and carboxylation by PEPC in the mesophyll cell. For the mutant, g_mut was the total conductance from mesophyll to BS cells, including liquid phase (g_bs) and Rubisco (g_c), as determined earlier.

Figure 5.

Temperature dependence of photosynthetic parameters for wild-type (A) and PEPC mutant (B) A. edulis measured under 0.3 mbar O₂. Shown are internal conductance in the mesophyll for wild type, g_wt (the initial slope of A/C_i curves), the internal conductance in the mutant, g_mut, and the calculated liquid phase-diffusive conductance in the mutant (g_bs) and maximal CO₂ assimilation rate (A_max). C has A_max from A and B plotted in Arrhenius axes (the slope equals −E_a/R). Values of J_O2-net measured under saturating CO₂ (data not shown) were similar to values of A_max.

A_max increased with increasing temperature, with a Q₁₀ value (the factor by which a reaction increases with a 10°C increase in temperature) of 2 for the mutant and 1.9 for the wild type between 20°C and 30°C. The corresponding activation energies were E_a = 13.5 and 11.3 kcal mol⁻¹ (Fig. 5C, calculated between 20°C and 30°C). These values of Q₁₀ and activation energies are as expected if photosynthesis under saturating CO₂ and light is controlled by enzymatic processes. Also, the activation energy for A_max in the mutant (13.5 kcal mol⁻¹) was close to the in vitro Rubisco activation energy of 13 kcal mol⁻¹ (calculated from Jordan and Ogren, 1984). If RuBP is saturating for photosynthesis under high light, the Q₁₀ values obtained would be consistent with Rubisco, rather than a diffusion limitation, being the major limiting factor for light- and CO₂-saturated photosynthesis in the mutant.

In the mutant, g_mut (which includes g_bs and Rubisco conductance) and g_bs had a linear response to increasing temperature. For g_bs, the Q₁₀ values were 1.3 between 20°C and 30°C (Fig. 5B), which coincides with the temperature sensitivity of diffusion of small molecules in solutions that have a Q₁₀ value of 1.3 (Nobel, 1991). The agreement between the measured and expected Q₁₀ value for g_bs provides confidence that we are correctly measuring diffusive resistance of CO₂ to BS cells. Because CO₂ must diffuse to BS cells for fixation in the mutant, the cellular conductance values for the mutant are much lower than for the wild type.

In wild-type plants, CO₂ is fixed initially in mesophyll cells, and the temperature response of the mesophyll conductance, determined from the initial slope of the CO₂ response curve, showed a saturating curve rather than a linear response, indicating that biochemistry is involved (Fig. 5A). The effect of temperature on the initial slope of the A/C_i response in wild-type A. edulis depends on liquid phase diffusion and PEPC in mesophyll cells. The relative insensitivity of the mesophyll conductance in the wild type to temperature indicates control by biochemistry. This could be attributable to regulation of PEPC by temperature-dependent changes in K_m for phosphoenolpyruvate (PEP; the substrate PEP is lower under limiting CO₂ [Leegood and von Caemmerer, 1988]), by allosteric effectors, and/or by covalent modification of the enzyme (phosphorylation/dephosphorylation). There also may be a temperature-dependent effect on PEP because the level is reported to increase with increasing temperature under normal atmospheric levels of CO₂ (Labate et al., 1990).

CO₂ Response and Partitioning of Photochemical Electron Flow

The CO₂ response curves for CO₂ fixation (A), gross rates of O₂ evolution (J_O2), and net rates of O₂ evolution (J_O2-net) of the mutant and wild-type leaves were measured at saturating light (Fig. 6). Measurements of A and J_O2 were made at 210 mbar of O₂, representing current atmospheric levels, and measurements of A, J_O2, and J_O2-net at near zero levels of O₂ (0.3 mbar), with an interest in studying O₂-dependent processes. To overcome the high-diffusive resistance from the atmosphere to the BS cells in the mutant, CO₂ concentrations as high as 2% (20 mbar) were required (Fig. 6, A and C), whereas near atmospheric levels were saturating for the wild type (Fig. 6, B and D). The mutant plants of A. edulis had about 100 times lower initial slopes in A/C_i curves compared with the wild type (Fig. 6, A versus B and C versus D). From various measurements of A_max at 30°C during the course of the study on young to mature leaves, values were usually 30 to 40 μmol m⁻² s⁻¹ in the mutant compared with 40 to 50 μmol m⁻² s⁻¹ in the wild type. It is apparent that rates of A, J_O2, and J_O2-net were very similar in both the mutant and wild type at 0.3 mbar O₂ (Fig. 6, A and B). At 210 mbar of O₂, J_O2 was substantially higher than A in both mutant and wild type (Fig. 6, C and D). This is clearly shown in Figure 6, E and F, where J_O2-A is plotted in response to varying CO₂ at 0.3 and 210 mbar of O₂. J_O2-A can potentially be accounted for by dark-type mitochondrial respiration (R_d), photorespiration (1.5 velocity of RuBP oxygenase [v_o]), photosystem (PS) II-dependent O₂ evolution associated with the Mehler-peroxidase reaction (J_O2Mr), and O₂ evolution associated with nitrogen assimilation (J_O2NA; see Eqs. 3–6).

Figure 6.

The response of the rates of CO₂ assimilation (A, ○), net O₂ evolution (J_O2-net, □), and gross O₂ evolution from PSII (J_O2, ▵) in PEPC mutant and wild-type A. edulis to intercellular CO₂ (C_i) at two oxygen partial pressures, 210 and 0.3 mbar. The CO₂ response curves were measured at PFD = 1,800 μmol m⁻² s⁻¹ and at leaf temperature 29°C.

In the mutant at 0.3 mbar of O₂, it is obvious that most of the PSII activity (J_O2) can be accounted for by CO₂ fixation and that R_d, v_o, J_O2MR, and J_O2NA must be low (Fig. 6, A and E). Because a partial pressure of 0.3 mbar of O₂ in the atmosphere is extremely low, a correspondingly low level is expected in the mesophyll cells within the leaf. However, even under very low external levels of O₂, the O₂ level in the BS cells will increase when O₂ is generated from PSII activity under a high-BS cell-diffusive resistance. At 0.3 mbar of O₂ in the atmosphere and based on the average value for BS cell resistance determined in this study (described later), the calculated level of O₂ in BS cells at CO₂-saturated rates of photosynthesis was about 30 mbar. From Equation 14, if we take A = 30 μmol m⁻² s⁻¹, r_bs = 30 s cm⁻¹ (80 m² s⁻¹ mol⁻¹), a_w = 0.79 (a_w is a constant that takes into account the difference in O₂ and CO₂ diffusivities [at 25°C, a_w = 0.79; Farquhar, 1983]), and b = 0.5, then the concentration of O₂ would be 35 μm (equivalent to about 30 mbar of O₂ in the gas phase; O₂ = 0 + 0.79*0.5*30*A/10 = 35 μm). R_d from measurements in the dark under normal atmospheric conditions is 2 to 3 μmol m⁻² s⁻¹ (data not shown). On average, J_O2NA for nitrate assimilation to Glu is estimated to be about 5% of A, not considering re-assimilation of ammonia from photorespiration (see Edwards and Baker, 1993). Thus, R_d in vascular tissue plus nitrate assimilation could easily account for the difference between J_O2 and A at 0.3 mbar of O₂. Some rise in values of J_O2-A under limiting CO₂ at 0.3 mbar of O₂ would be expected, because A decreases relative to R_d (Fig. 6E). These results indicate that J_O2Mr and v_o must be very low in the mutant under 0.3 mbar of O₂ in the atmosphere.

In the mutant at 210 mbar of O₂, the values of J_O2-A were much greater than at 0.3 mbar of O₂, particularly with decreasing levels of CO₂ (Fig. 6E). The logical explanation for this effect is that the mutant, which lacks a C₄ cycle, has increasing rates of photorespiration under limiting CO₂ just as C₃ plants do (see also Lacuesta et al., 1997; Maroco et al., 1998a), which causes a corresponding increase in J_O2. Under high CO₂ and under 210 mbar of O₂, the mutant may have some additional dark-type respiration in mesophyll cells, resulting in larger values of J_O2-A than at 0.3 mbar of O₂.

In the wild-type plant under 0.3 mbar of O₂ (Fig. 6F), the value of J_O2-A was about 4 μmol m⁻² s⁻¹, which was higher than in the mutant, and was independent of the level of CO₂. As with the mutant, nitrate assimilation and R_d in BS tissue may partly account for the difference. However, the Mehler reaction or photorespiration may also contribute in the wild-type plant. In a recent study, there was evidence for significant Mehler reaction in wild-type A. edulis under rather low levels of O₂ (between 0.2 and 20 mbar; Laisk and Edwards, 1998). In NAD-malic enzyme (NAD-ME) species like A. edulis, the Mehler reaction is proposed to function in mesophyll chloroplasts and contribute to generation of ATP for the C₄ cycle (Furbank and Badger, 1982; Laisk and Edwards, 1998). With increasing CO₂, there may be a rise in the rate of the Mehler reaction as the rate of the C₄ cycle increases, whereas with decreasing CO₂, there may be a rise in photorespiration; together these effects could result in values of J_O2-A being reasonably constant in NAD-ME-type species with varying CO₂ (see Furbank and Badger, 1982).

In the wild-type plant under 210 mbar of O₂, J_O2 was higher than A across the CO₂ response curve (Fig. 6D); the pattern of change with increasing CO₂ is very similar to that of Canvin et al. (1980) from O₂ isotope analysis of photosynthesis in A. edulis. J_O2-A at 210 mbar was greater than at 0.3 mbar of O₂, and a sharp increase in J_O2-A occurred at very low levels of CO₂. Above approximately 0.025 mbar of CO₂, J_O2-A was constant at about 8 μmol m⁻² s⁻¹ (Fig. 6F). As noted above, this can be explained by O₂-dependent photorespiration at extremely low levels of CO₂ and increasing Mehler reaction at higher CO₂. The larger J_O2-A values at high CO₂ under 210 mbar versus 0.3 mbar of O₂ may be accounted for by O₂-dependent dark respiration and/or the Mehler reaction.

To evaluate Rubisco kinetics in mutant leaves relative to A and J_O2, we analyzed RuBP content and Rubisco activity (Fig. 7). With increasing CO₂ from zero up to about 0.8 mbar there was an increase in RuBP content, above which it decreased. The initial extractable activity of Rubisco in the mutant was higher than A_max at saturating CO₂ and decreased slightly at the lower CO₂ concentrations (Fig. 7, C and D). The decrease at low CO₂ correlated with a decrease in the state of activation of the enzyme. In the wild type (results not shown), leaf RuBP content was similar to the mutant at low CO₂ (at CO₂ = 34 μbar, RuBP was 56 μmol m⁻²) and decreased at high CO₂ (at CO₂ = 4.8 mbar, RuBP was 47 μmol m⁻²), which is in close agreement with the values of Leegood and von Caemmerer (1988). Considering a Rubisco active site turnover rate of 2.8 s⁻¹ (Woodrow and Berry, 1988), the number of active sites per leaf area in A. edulis would be about 15 to 20 μmol m⁻². The RuBP concentration across the A/C_i curve at light saturation exceeded the number of active sites by about three times, which suggests the RuBP is at saturating levels. However, the observation that the RuBP concentration decreased at high CO₂ (Fig. 7) and with the known competitive interaction of some chloroplast metabolites with RuBP (e.g. PGA; Servaites and Geiger, 1995), it is possible that A_max is partly limited by RuBP regeneration.

Light Response and Partitioning of Photochemical Electron Flow

The response of photosynthesis to light under very high CO₂ (4%, 40 mbar) in the mutant gave about the same maximal quantum yield for O₂ evolution (J_O2-net, J_O2) as in the wild type (0.064 versus 0.066; Fig. 8, A and B). The quantum yield for O₂ evolution for wild-type A. edulis was higher than measured by Ehleringer and Bjorkman (1977) for CO₂ fixation in NAD-ME-type C₄ species (0.054). Higher values for the wild type may be explained by the use of highly saturating CO₂ and very low O₂, which would restrict O₂-dependent use of energy, and by the fact that O₂ evolution, rather than CO₂ uptake, was measured. In the wild-type plant under very high CO₂, the responses of J_O2 and J_O2-net to increasing light were very similar, indicating there was little photorespiration and Mehler reaction under this condition. It is uncertain why the Mehler reaction would be restricted under such high levels of CO₂ in the wild type. However, in the wild-type plant under 4% (v/v) CO₂, direct diffusion of CO₂ to BS cells would occur, bypassing the C₄ cycle, because in mutant plants, which lack a C₄ cycle, photosynthesis is saturated by 2% (v/v) CO₂.

Figure 8.

Light response of PEPC mutant and wild-type A. edulis O₂ evolution (J_O2-net, □) at highly saturating levels of CO₂ of 40 mbar CO₂ (O₂ = 0.3 mbar), and CO₂ uptake (A, ○) at limiting CO₂ (2 mbar for PEPC mutant and 0.36 mbar for wild type) and 210 mbar O₂ pressure. Leaf temperature was 28°C. Gross rates of O₂ evolution (J_O2, Δ) were calculated from simultaneous fluorescence measurements as described in “Materials and Methods.”

In the mutant, under saturating levels of CO₂, which prevent photorespiration, we would expect maximum quantum yields of O₂ evolution similar to those of C₃ plants. Instead, the values in the mutant were lower than in C₃ plants under saturating CO₂ and similar to those of the A. edulis wild type. This suggests that the absorbed energy, which is used in the wild type in mesophyll chloroplasts for the C₄ cycle (ATP for conversion of pyruvate to PEP, and NADPH to the degree malate is synthesized), may be dissipated as heat in the mutant if there is no other means for using it in carbon assimilation. In the mutant, there is no requirement for energy in mesophyll cells in carbon assimilation, unless part of the PGA and glycerate formed via RuBP carboxylase and oxygenase activities in BS chloroplasts is shuttled to mesophyll cells for reduction (Fig. 1).

In the mutant under CO₂ which is limiting for photosynthesis (2 mbar), and at 210 mbar of O₂, J_O2 was much higher than A, and the maximum quantum yield under limiting light was higher for J_O2 than for A (Fig. 8C). This can be explained by the mutant having a high level of photorespiration and responding like a C₃-type species under limiting CO₂.

In the wild type under atmospheric levels of CO₂ and 210 mbar O₂, the light response curves showed a higher quantum yield (from initial slopes) and a higher light-saturated rate for J_O2 than for A (Fig. 8D). This may be attributed to the Mehler reaction increasing with increasing light and generating ATP for the C₄ cycle under 210 mbar O₂, which could largely account for the difference between J_O2 and A. With the wild-type plant having a functional C₄ cycle, photorespiration and dark-type respiration are expected to be minor components of the difference between J_O2 and A. A previous study indicated that the Mehler reaction is functioning in A. edulis but is insufficient to supply the ATP needed to support the C₄ cycle (Laisk and Edwards, 1998). Thus, both the Mehler reaction and PSI-mediated cyclic electron flow may generate the ATP, with some flexibility in the magnitude of each. In contrast to results under 210 mbar of O₂ (Figs. 6D and 8D), there was no evidence for function of the Mehler reaction under low O₂ (Fig. 8B), which suggests the ATP needed to support the C₄ cycle is provided by PSI-dependent cyclic electron flow.

O₂ Effect on the Partitioning of Photochemical Electron Flow

It is well known that at low-CO₂ concentrations, the rate of CO₂ assimilation in C₄ plants exhibits low sensitivity to O₂. This is in contrast to C₃ plants, where photorespiration greatly reduces the rate of CO₂ assimilation in response to increasing O₂ (Kanai and Edwards, 1999). Measurements of A and J_O2 in mutant plants, in which the C₄ cycle is not functional, provide an opportunity to follow more closely the maximum potential for Rubisco oxygenase and the glycolate pathway to function. This is not possible in normal C₄ leaves, where the CO₂ pump operates.

The responses of A and J_O2 were measured at increasing O₂ concentrations over a range of CO₂-limited concentrations where greater RuBP oxygenase activity is expected for mutant and wild-type plants (Fig. 9). In the wild-type plant at rate-limiting CO₂ levels, high O₂ increased J_O2, but the CO₂ assimilation rates remained relatively unaffected by O₂ (Fig. 9, B and D). The increase in J_O2 with increasing O₂ under low CO₂ suggests an increase in photorespiration through RuBP oxygenase activity. At a given level of O₂, the value of J_O2-A (Fig. 9F) remained about the same with increasing levels of CO₂, which, as discussed earlier, may be attributable to the Mehler reaction partially providing ATP to support the C₄ cycle and increasing with increasing rates of CO₂ fixation.

Figure 9.

Oxygen sensitivity of A. edulis photosynthesis at limiting CO₂ concentrations, 30°C, and PFD = 1,800 μmol m⁻² s⁻¹. The rate of PSII O₂ evolution (J_O2) shows an increase with increasing O₂ concentration and continues at CO₂ = 0 because of re-assimilation of CO₂ released from the photorespiration and from the Krebs cycle.

In the mutant, high O₂ increased J_O2 at the lowest, rate-limiting CO₂ levels, but the CO₂ assimilation rates remained relatively unaffected by O₂ (Fig. 9, A and C). The increased electron transport rate with increasing O₂ under limiting CO₂ suggests an O₂-dependent increase in the RuBP oxygenation rate. At the higher levels of O₂, the difference between J_O2 and A (Fig. 9E) was gradually suppressed by increasing CO₂, which is expected if increasing CO₂ suppresses photorespiration and considering that the mutant does not have a C₄ cycle that could be supported by the Mehler reaction.

For the mutant under limiting CO₂, the activity of J_O2 is expected to be largely accounted for by the sum of the velocity of RuBP carboxylase (v_c) and v_o. We extrapolated J_O2, measured at different O₂ concentrations, to CO₂ = 0 and plotted the resulting values against BS cell O₂ concentration in an effort to evaluate the effect of Rubisco oxygenase activity on J_O2 (Fig. 10). Assuming photorespired CO₂ is re-assimilated with the probability determined by the ratio of Rubisco conductance to BS cell conductance, the expected J_O2 response can be described by the solid line in Figure 10. The experimental points are in good agreement with predicted results based on Rubisco kinetics if re-assimilation is accounted for, except that at the highest O₂ level (480 μm, which is about twice the atmospheric level), the oxygenase activity was lower than expected. Analysis of RuBP content in the leaves of the mutant at low CO₂ (0.4 mbar) showed a decrease of RuBP concentration at rate-limiting CO₂ and with increasing O₂ (Fig. 11). Therefore, we suggest that the lower than expected J_O2 value at O₂ concentration below the K_m for RuBP oxygenase (K_o) value (640 μm) in the mutant (Fig. 10) is the result of RuBP becoming partially limiting for CO₂ assimilation. Very similar results were obtained with wild-type plants (Fig. 11). Also, the response of J_O2 to O₂, under low CO₂, in the wild type is similar to that in A. edulis measured at the CO₂ compensation point by O₂ isotope analysis (Canvin et al., 1980).

Figure 10.

J_O2 for the PEPC mutant of A. edulis from Figure 9 was extrapolated to CO₂ = 0, and the results were plotted against O₂ concentration. The simulated J_O2 shown by the solid line was calculated based on BS O₂ and CO₂ concentration (the latter calculated for each O₂ level considering BS-diffusive resistance) and Rubisco kinetic constants (V_c = 39, K_c = 21 μm, and K_o = 640 μm), where J_O2 equals v_c + v_o (Edwards and Baker, 1993). The rate of CO₂ re-assimilation (at zero external CO₂) is proportional to the ratio of Rubisco conductance and BS-diffusive conductance.

Figure 11.

PEPC mutant and wild-type A. edulis leaf RuBP content (●) versus O₂ concentration with C_i of 0.4 mbar for mutant and 0.025 mbar for wild type. Also, CO₂ assimilation rate (A, ○) and O₂ evolution from PSII (J_O2, ▵) are shown. Leaf temperature was 28°C; light PFD = 1,800 μmol m⁻² s⁻¹.

There was only a small effect of O₂ on the rate of CO₂ assimilation at low-CO₂ concentrations in either the mutant or wild-type A. edulis (Fig. 9). The limited effect of O₂ on CO₂ assimilation in the mutant, where there is no C₄ cycle function, can be explained by effective re-assimilation in the BS cells of the CO₂ that is generated from dark-type respiration and photorespiration. It is also evident that in the wild-type plants, CO₂ re-assimilation at low CO₂ affects assimilation kinetics. In this case, refixation of photorespired CO₂ may occur in mesophyll cells via PEPC, if there is leakage of CO₂, as well as in BS cells. However, the effectiveness of the mutant suggests that, under low CO₂, much of the photorespired CO₂ will be directly refixed in BS cells.

Calculation of CO₂ Concentration and Leakiness of CO₂ in BS Cells of A. edulis

If Rubisco kinetic parameters (V_c, K_c [the K_m for RuBP carboxylase], K_o, and S [the relative specificity factor for carboxylase versus oxygenase]) are known, one can calculate BS CO₂ concentration using a C₄ photosynthesis model (von Caemmerer, 2000). Figure 12 shows calculated BS cell CO₂ concentrations during photosynthesis in wild-type A. edulis leaves under varying intercellular CO₂ concentrations at 210 mbar of O₂ calculated with the model of C₄ photosynthesis. Also, shown in Figure 12 are the calculated values of BS cell leakiness during CO₂ fixation in wild-type plants, which range from approximately 0.2 to 0.3 (from Eqs. 16 and 17, using an r_bs value of 113 m² s⁻¹ mol⁻¹). As increasing ambient levels of CO₂ became saturating for photosynthesis, the calculated levels of CO₂ in the BS cells of mature leaves reached levels of approximately 2,000 μbar, which is about six times that normally occurring in the atmosphere.

Figure 12.

CO₂ response for CO₂ assimilation rate (at leaf temperature of 28°C; PFD = 1,800 μmol m⁻²s⁻¹; ●), calculated CO₂ partial pressure in BS cells (□), and leakiness of CO₂ from BS cells (▵). The CO₂ level in BS cells was calculated according to von Caemmerer (2000), and the leakiness was calculated according to equations 16 and 17 using r_bs value of 113 m² s⁻¹ mol⁻¹. Similar results were obtained from analysis of several experiments on A/C_i responses.

CONCLUDING REMARKS

The PEPC mutant of A. edulis has high light- and CO₂-saturated rates of photosynthesis comparable with those of the wild type. The high CO₂ required for growth and the high-diffusive resistance of the PEPC mutant to CO₂ indicate that physical restraints to gases have been conserved in the C₄ anatomy of the mutant. The value of BS cell resistance to CO₂ increased with leaf age in A. edulis from 72 to 181 m² s⁻¹ mol⁻¹ (Table I). Similar resistance values for mature leaves were obtained with wild-type plants treated with the PEPC inhibitor DCDP. The results suggest there may be developmental changes in BS cells that increase the diffusive resistance to CO₂ by changes in the liquid phase of the cell or cell wall properties. The concentration of CO₂ in BS cells under high rates of photosynthesis is estimated to be about six times current ambient levels, sufficient to largely repress photorespiration. Re-assimilation of CO₂ from dark-type respiration and photorespiration in BS cells is an important component of both PSII activity and consumption of reductive power at very low-CO₂ concentrations in A. edulis.

MATERIALS AND METHODS

Growth Conditions

Mutant plants of Amaranthus edulis LaC₄ 2.16 lacking PEPC activity and protein (Dever et al., 1995) were grown in growth chambers (Econair, Winnipeg, Canada) on fertilized potting soil in 8-L pots (one plant per pot), with a 14/10-h day/night cycle at 28°C/22°C, 50% relative humidity, 10 mbar of CO₂, and an incident photosynthetically quantum flux density (PFD) of 1,200 μmol m⁻² s⁻¹ light. Wild-type plants were grown under the same conditions, except the CO₂ concentration was 370 μbar and 10 mbar in experiments with DCDP and electron microscopy and 370 μbar in all other experiments.

Gas Exchange (A and J_O2-net)

Leaf gas exchange was measured with the FastEst gas system (FastEst, Tartu, Estonia; described in detail in Laisk and Oja, 1998). The system was equipped with a CO₂ analyzer (6251, LI-COR, Lincoln, NE) and a S-3A O₂ ceramic heated zirconium oxide analyzer (Applied Electrochemistry Inc., Sunnyvale, CA). Leaf gas exchange characteristics, net rates of CO₂ fixation (A), C_i, PFD, and leaf temperature were determined as in Laisk and Loreto (1996). For measurements of A under high levels of CO₂, there is an increase in noise to signal ratio in measuring CO₂ with an infrared gas analyzer. To improve measurements of A, sampling time was 0.1 s for 3 min resulting in 1,800 data points, which were averaged. For example, in one experiment A measured at 3% (v/v) CO₂ was 41.9 μmol m⁻² s⁻¹ with a se with n = 1,800 of ± 0.12. The S-3A O₂ analyzer provides an accurate measure of the net rate of O₂ evolution (J_O2-net) under low levels of atmospheric O₂ (less than 10 mbar), independent of CO₂ concentration.

Measurement of Chlorophyll Fluorescence and Calculation of PSII Activity (J_O2)

The yield of PSII was measured by chlorophyll fluorescence using a fluorometer (PAM 101, Walz, Effeltrich, Germany). The gross rate of O₂ evolution from PSII (J_O2) was calculated as:

1

where (F_m′-F_s)/F_m′ is the yield of PSII (e⁻ quanta absorbed), F_s is fluorescence yield of steady-state photosynthesis, F_m′ is maximal fluorescence yield by exposure to a 1-s pulse of 15,000 μmol m⁻² s⁻¹ light and APFD is the absorbed photosynthetic quantum flux density at steady state (Genty et al., 1989). For estimating the relative optical cross section of PS II (Y_II), the method proposed by Laisk and Loreto (1996) was used. Y_II was found by extrapolating a plot of F_s/F_m′ versus quantum yield of O₂ evolution measured with an O₂ electrode (J_O2-net) at different light intensities, to F_s/F_m′ = 0. The measurements were made under a low-O₂ background (0.025%) and high CO₂ so that respiratory uptake of O₂ would be minimized and the O₂ evolution measured would reflect essentially all PSII activity. The calculated values of Y_II for wild-type and mutant plants were 0.44 to 0.55 and 0.41 to 0.48, respectively. For calculations of APFD, the light reflected and transmitted by the leaf was measured using an integrating sphere (Labsphere, North Sutton, NH). For the mutant leaf, the average fractional absorption of incident light was 0.82, whereas for the wild type, the value was 0.85. In mature leaves of mutant plants, the chlorophyll (a+b) content was 156 mg m⁻² compared with 309 mg m⁻² in the ambient CO₂-grown wild type. The fresh weights of the mutant and wild-type leaves were identical, 22 mg cm⁻², but the mutant had less dry weight per leaf area, 2.9 mg cm⁻², compared with 4.4 mg cm⁻² in the wild type (leaves were sampled at midday).

Equations and Calculation of Leaf Photosynthesis Parameters

O₂ Evolution in Mutant and Wild Type

O₂ evolution associated with linear electron transport rate can be expressed as

2

where v_c and v_o are RuBP carboxylation and oxygenation rates, respectively, and J₁ is the use of electrons in other processes (e.g. Mehler reaction and nitrogen reduction). The following equations illustrate the main factors in considering the relationship between J_O2, J_O2-net, and A in C₃ and C₄ plants, because there is no net consumption of reductive power in the C₄ cycle of malic enzyme-type species (see Edwards and Baker, 1993).

3

4

5

6

where R_d = rate of dark-type mitochondrial respiration, J_O2Mr = PSII-dependent O₂ evolution associated with the Mehler-peroxidase reaction, and J_O2NA = O₂ evolution associated with nitrogen assimilation (reduction of nitrate and assimilation to Glu).

Analysis of Parameters of CO₂ Fixation and r_bs in PEPC Mutant

We assume that light-saturated CO₂ uptake in the mutant A. edulis plants is limited primarily by physical diffusive resistance to CO₂ and Rubisco activity (see Fig. 1). Values for the diffusive resistance to CO₂, carboxylation resistance, and RuBP carboxylation and oxygenation velocities can be calculated from leaf gas exchange measurements using the biochemical model of Rubisco developed by Farquhar et al. (1980). According to their model for C₃ photosynthesis, the CO₂ assimilation rate in the mutant can be described according to equation 3 above, where A = v_c − 0.5 v_o − R_d. Assuming Rubisco saturation by RuBP,

7

8

9

where V_c and V_o = maximum carboxylation and oxygenation velocities, respectively, K_c and K_o = carboxylation and oxygenation Michaelis constants, respectively, C_c and O_c = CO₂ and O₂ concentrations at Rubisco active sites, respectively, and S = Rubisco specificity for CO₂ relative to O₂.

Total resistance to CO₂ flux in the mutant, r_t, can be described as the sum of three resistances

10

where r_g is gas phase resistance (boundary layer and stomatal), r_bs is liquid phase resistance (effectively BS resistance), and r_c describes carboxylation resistance of Rubisco. Gas phase resistance, r_g, was calculated from transpiration data. Intercellular CO₂ partial pressure, C_i equals

11

where C_a is ambient CO₂ and A is net CO₂ assimilation rate. Intercellular CO₂ partitions between the gas phase and the cell wall liquid phase, C_w is

12

giving soluble CO₂ where β_c is the CO₂ solubilization coefficient. The CO₂ concentration at the sites of carboxylation in the mutant, C_c is

13

O₂ concentration in the BS chloroplasts can be described as

14

where O₂ concentration at the mesophyll cell wall is

15

a_w is a constant that takes into account the difference in O₂ and CO₂ diffusivities (at 25°C a_w = 0.79; Farquhar, 1983), b is the relative proportion of O₂ evolution in BS cells, O_i is the intercellular concentration of O₂, and β_o is the O₂ partitioning factor between gaseous and liquid phase.

DCDP Feeding Experiments

A leaf petiole was cut under water, and the CO₂ response curve was measured at 2% (v/v) oxygen (Fig. 3, A–C). Water was then replaced by a 4 mm solution of DCDP (PEPC inhibitor). After DCDP caused a decrease of photosynthesis to a stable level under atmospheric levels of CO₂ (about 20 min), the CO₂ response was measured (Fig. 3, B, D, and F). Measurement of photosynthesis on excised leaves can be problematic; however, using the FastEst gas exchange system the A/C_i response curves could be run in 20 min. The maximum rates of CO₂ fixation and response curves of control plants were similar to intact plants.

Calculating BS Cell CO₂ Concentration and Leakiness in Wild-Type Plants

The mechanistic model of C₄ photosynthesis developed by von Caemmerer (2000) was used for estimating concentrations of CO₂ in BS cells. The model requires inputs for Rubisco kinetic parameters. The kinetic constants for A. edulis Rubisco (K_c = 16 μm, K_o = 640 μm, and S = 82 at 25°C) were obtained from the work of Jordan and Ogren (1983) and corrected for temperature according to Woodrow and Berry (1988). V_c was taken equal to the CO₂-saturated assimilation rate at a PFD of 1,800 μmol m⁻² s⁻¹.

Leakiness of CO₂ from BS cells during photosynthesis in wild-type plants was calculated as a fraction of the rate of the C₄ cycle based on the above estimates of BS cell CO₂ concentration and measured values of r_bs in the A. edulis mutant, where the rate of leakage of CO₂ per unit leaf area (L_a) equals

16

with [CO₂]_BS and [CO₂]_i representing the level of CO₂ in BS cells versus the external concentration in the intercellular air space, respectively. Leakiness (L), from BS cells as a fraction of the rate of the C₄ cycle is defined as

17

Measurement of RuBP Content

Leaf metabolism was stopped by quick filling of the leaf chamber with cooled ethanol (approximately −90°C). The frozen leaf (8 cm²) was then removed from the chamber and ground into a fine powder in a small mortar under liquid nitrogen. The powder was then transferred into 3 mL of 1 m HClO₄ and extracted for 15 min. The extract was neutralized with 5 m KOH and centrifuged 3 min at 5,000g. The supernatant was stored under liquid N₂ until analyzed. RuBP content was determined using the method of ¹⁴C incorporation into acid stable product as in Prinsley et al. (1986) using purified Rubisco from tobacco (Nicotiana tabacum).

Rubisco and Chlorophyll Content

For Rubisco activity measurements, the leaves were sampled the same way as for RuBP determination. The frozen leaf powder was then transferred into CO₂-free 100 mm HEPES/KOH buffer (pH = 7.9). The Rubisco extraction buffer (1 mL per cm² of leaf) contained 20 mm MgCl₂, 2 mm EDTA, and 5 mm dithiothreitol. It was then ground and homogenized in a Broeck Tissue Grinder (Corning, Palo Alto, CA) for about 20 s and divided into two parts, the first of which was immediately assayed by injecting 0.1 mL into a 0.9-mL assay medium and running the reaction 30 s at 28°C. This was taken as the in vivo activity of Rubisco. The assay media had the following final composition: 100 mm HEPES/KOH (pH = 7.9), 10 mm Mg²⁺, 2 mm EDTA, 5 mm dithiothreitol, 1 mm RuBP, and 10 mm NaHCO₃ + NaH¹⁴CO₃ with specific activity of 0.5 C_i mol⁻¹. The second assay, which was run after incubating the enzyme preparation 15 min in the presence of 10 mm NaHCO₃, was taken as the fully carbamylated activity. It was shown, by adding purified Rubisco with known activities to leaf material before grinding, that there was no loss of activity attributable to extraction procedures. However, the possibility that some loss of activity may have occurred because of incomplete solubilization of Rubisco from the leaf material cannot be ruled out. The chlorophyll content of the leaves was determined according to Porra et al. (1989) using 80% (v/v) acetone extract.