The Role of Phosphoenolpyruvate Carboxylase during C₄ Photosynthetic Isotope Exchange and Stomatal Conductance^,

Abstract

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) plays a key role during C₄ photosynthesis and is involved in anaplerotic metabolism, pH regulation, and stomatal opening. Heterozygous (Pp) and homozygous (pp) forms of a PEPC-deficient mutant of the C₄ dicot Amaranthus edulis were used to study the effect of reduced PEPC activity on CO₂ assimilation rates, stomatal conductance, and ¹³CO₂ (Δ¹³C) and C¹⁸OO (Δ¹⁸O) isotope discrimination during leaf gas exchange. PEPC activity was reduced to 42% and 3% and the rates of CO₂ assimilation in air dropped to 78% and 10% of the wild-type values in the Pp and pp mutants, respectively. Stomatal conductance in air (531 μbar CO₂) was similar in the wild-type and Pp mutant but the pp mutant had only 41% of the wild-type steady-state conductance under white light and the stomata opened more slowly in response to increased light or reduced CO₂ partial pressure, suggesting that the C₄ PEPC isoform plays an essential role in stomatal opening. There was little difference in Δ¹³C between the Pp mutant (3.0‰ ± 0.4‰) and wild type (3.3‰ ± 0.4‰), indicating that leakiness (φ), the ratio of CO₂ leak rate out of the bundle sheath to the rate of CO₂ supply by the C₄ cycle, a measure of the coordination of C₄ photosynthesis, was not affected by a 60% reduction in PEPC activity. In the pp mutant Δ¹³C was 16‰ ± 3.2‰, indicative of direct CO₂ fixation by Rubisco in the bundle sheath at ambient CO₂ partial pressure. Δ¹⁸O measurements indicated that the extent of isotopic equilibrium between leaf water and the CO₂ at the site of oxygen exchange (θ) was low (0.6) in the wild-type and Pp mutant but increased to 0.9 in the pp mutant. We conclude that in vitro carbonic anhydrase activity overestimated θ as compared to values determined from Δ¹⁸O in wild-type plants.

The enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC) utilizes bicarbonate () to catalyze the β-carboxylation of PEP, to form the four-carbon acid oxaloacetate (Andreo et al., 1987; Chollet et al., 1996; Lepiniec et al., 2003; Izui et al., 2004). In higher plants, PEPC plays the anaplerotic role of replenishing the citric acid cycle intermediates, oxaloacetate and malate, which are required for nitrogen assimilation and amino acid biosynthesis. The synthesis of malate through PEPC activity has also been implicated in cytosolic pH regulation (Lepiniec et al., 1994; Britto and Kronzucker, 2005). In photosynthetic tissues, PEPC participates in the exchange of CO₂ and water between a leaf and the atmosphere through its role in guard cell metabolism. Additionally, in C₄ species PEPC catalyzes the first carboxylation reaction of C₄ photosynthesis in the mesophyll cells (Kanai and Edwards, 1999).

C₄ plants generally have high PEPC activity in the cytosol of mesophyll cells, allowing for the accumulation of four-carbon acids that subsequently diffuse into the bundle sheath cells (BSCs) for decarboxylation (Kanai and Edwards, 1999; Furbank et al., 2000). The specialized biochemistry and leaf anatomy of C₄ plants results in CO₂ partial pressure (pCO₂) around the site of Rubisco severalfold higher than current atmospheric levels, significantly reducing the rates of photorespiration (Hatch, 1987).

Theoretical models of ¹³CO₂ isotope discrimination (Δ¹³C) during C₄ photosynthesis have been developed that link Δ¹³C to the ratio of intercellular to ambient pCO₂ and bundle sheath leakiness (φ) defined as the fraction of CO₂ fixed by PEPC that subsequently leaks out of the BSCs and is not fixed by Rubisco (Farquhar, 1983). Leakiness is a measure of the efficiency of C₄ photosynthesis and has been estimated with concurrent measurements of leaf gas exchange and Δ¹³C in a number of C₄ species (Henderson et al., 1992). It has been shown that φ increases in transgenic Flaveria bidentis with reduced Rubisco content, demonstrating that the balance between C₃ and C₄ cycle activity influences φ (von Caemmerer et al., 1997a, 1997b; Cousins et al., 2006a). C¹⁸OO discrimination (Δ¹⁸O) during C₄ photosynthesis, which is largely determined by the residence time of CO₂ within a leaf and the number of hydration reactions per CO₂ molecule, is influenced by changes in the carbonic anhydrase (CA) activity (Cousins et al., 2006b) and the capacities of the C₄ and C₃ cycles.

In higher plants, independent of the photosynthetic pathway, PEPC participates in guard cell metabolism. Stomatal opening is achieved through the accumulation of high levels of solutes in guard cell vacuoles. The accumulation of potassium ions requires anions (such as malate or chloride) to provide charge balance and to maintain the membrane potential. Malate produced via PEPC is believed to contribute substantially to the maintenance of the proton and charge balance in these cells during stomatal opening (Allaway, 1973; Outlaw and Lowry, 1977). The production of malate in guard cells is thought to be directly linked to carbon metabolism as PEP, the substrate for carboxylation, originates mainly from carbon skeletons derived from starch breakdown in the guard cell chloroplast (Vavasseur and Raghavendra, 2005). Additionally, the amount of PEPC in guard cells of C₃ plants has been shown to be an order of magnitude greater than in mesophyll cells when expressed on a protein basis (Cotelle et al., 1999).

The concentration of malate inside guard cells correlates with stomatal aperture in epidermal strips but it was also shown that the influence of malate is dependent on the availability of chloride (van Kirk and Raschke, 1978; Willmer and Fricker, 1996). The role of PEPC in stomatal opening was also confirmed in epidermal strips of C₃ plants using a PEPC inhibitor (Parvathi and Raghavendra, 1997; Asai et al., 2000). At the whole leaf level, the use of antisense and overexpression of PEPC in Solanum tuberosum also suggested that malate accumulation is involved in stomatal function (Gehlen et al., 1996). This showed that rates of stomatal opening increased in plants overexpressing PEPC and decreased in plants with reduced levels of PEPC. However, low PEPC levels had no effect on steady-state stomatal conductance and overexpression of PEPC had only a marginal effect (Gehlen et al., 1996).

Isotope analysis of atmospheric carbon CO₂ has become an important tool for monitoring changes in the global exchange of CO₂ (Flanagan and Ehleringer, 1998; Yakir and Sternberg, 2000). However, to interpret the atmospheric CO₂ isotopic signature requires an understanding of the isotopic fractionation steps associated with specific processes during leaf gas exchange (Yakir and Sternberg, 2000). Here we used the PEPC-deficient mutants of the C₄ dicot Amaranthus edulis (Dever et al., 1995, 1996, 1997) to asses the contribution of PEPC activity on photosynthetic isotope exchange and stomatal conductance. Previous work on these plants has shown that the heterozygous (Pp) and the homozygous (pp) PEPC mutants contain approximately 50% and 2% of wild-type PEPC activity, respectively (Dever et al., 1996, 1997; Maroco et al., 1997, 1998a, 1998b; Kiirats et al., 2002). These mutants are a nice comparison to earlier isotope work on the C₄ dicot F. bidentis that had high rates of PEPC and low CA due to antisense silencing of CA (Cousins et al., 2006a, 2006b). We have used the PEPC-deficient A. edulis mutants to address three distinct questions. (1) How does a reduction in PEPC activity affect Δ¹³C and bundle sheath leakiness? (2) Does the increased ratio of CA to PEPC activity affect the isotopic equilibrium between leaf water and CO₂ and hence Δ¹⁸O? (3) What are the effects of reduced PEPC activity on stomatal conductance?

RESULTS

Steady-State Gas-Exchange and Enzyme Activities

Under our growth conditions, which contained 9.8 mbar of CO₂, both the heterozygous (Pp) and homozygous (pp) PEPC mutants had similar total nitrogen per leaf area and leaf mass per area as compared to wild-type plants (Table I). Concurrent measurements of Δ¹³C and Δ¹⁸O and gas exchange were made by directly coupling a mass spectrometer to the outlet of a portable leaf gas-exchange system via a gas permeable silicone membrane (Cousins et al., 2006a, 2006b). This allowed simultaneous measurements of leaf gas exchange and the ¹³C/¹²C or ¹⁸O/¹⁶O ratios of the CO₂ in the air stream without prior purification of the CO₂. Under ambient CO₂ concentrations (531 μbar), net CO₂ assimilation rates in the Pp and pp mutants were 78% and 10% of wild-type plants, respectively (Table I). Compared to the wild type, the ratio of intercellular to atmospheric pCO₂ (p_i/p_a) was higher and stomatal conductance (g_s) lower in the pp mutant at high light and ambient CO₂ (531 μbar), whereas the differences between the wild-type and Pp mutant were not significant. PEPC activity determined on whole leaf extracts was 42% and 3% of wild-type in Pp and pp mutants, respectively, whereas Rubisco activity was not significantly different between the wild-type and pp mutant (Table I). The total extractable CA activity expressed as the rate constant k_CA (μmol m⁻² s⁻¹ bar⁻¹) was similar in all plants (Table I). The k_CA was determined from leaf extracts using mass spectrometry to measure the rates of ¹⁸O₂ exchange from labeled ¹³C¹⁸O₂ to (Badger and Price, 1989; Cousins et al., 2006a, 2006b). Leaf CA activity (CA_leaf), determined as the product of k_CA and the mesophyll pCO₂ (p_m), increased in the pp mutant due to the low rates of net CO₂ assimilation that caused p_m to be greater in these plants (Table I). The values of p_m were calculated as p_m = p_i − A/g_m, where A is the net CO₂ assimilation rate and g_m is the CO₂ conductance from the intercellular air space to the site of PEPC carboxylation (assumed to equal 1 mol m⁻² s⁻¹ bar⁻¹; Cousins et al., 2006a).

Table I.

Gas exchange and leaf characteristics of wild-type A. edulis and PEPC mutants

Net CO₂ assimilation rate (A), the ratio of intercellular to ambient CO₂ partial pressures (p_i/p_a), stomatal conductance (g_s), carbon isotope discrimination (Δ¹³C), oxygen isotope discrimination (Δ¹⁸O), PEPC and Rubisco activity, the rate constant of leaf CA (k_CA mol m⁻² s⁻¹ bar⁻¹), leaf CA activity (CA_leaf), the extent of isotopic equilibrium, leaf mass per area (LMA), and total leaf nitrogen per area in wild-type (WT) and heterozygous (Pp) and homozygous (pp) mutant A. edulis plants. Gas-exchange measurements were made at a pCO₂ of 531 μbar, a pO₂ of 48 mbar, irradiance of 2,000 μmol quanta m⁻² s⁻¹, and a leaf temperature of 30°C. Calculations of θ were made with g_m = 1 mol m⁻² s⁻¹ bar⁻¹. Shown are the means ± se of measurements made on three to five leaves from separate plants. nd indicates measurements were not determined. Statistical analysis was conducted using an ANOVA and different letters indicate significant differences between plants at P < 0.05. *, CA _leaf = k_CAp_m and p_m was calculated from A and p_i using a conductance of CO₂ from the intercellular air space to the site of PEPC carboxylation equal to 1 mol m⁻² s⁻¹ bar⁻¹.

Measurements	WT	Pp	pp
Photosynthetic parameters
A (μmol m−2 s−1)	40.9 ± 1.6a	32.1 ± 1.7b	4.1 ± 0.4c
pi/pa	0.39 ± 0.06a	0.45 ± 0.07a	0.82 ± 0.02b
gs (mol m−2 s−1)	0.28 ± 0.04a	0.25 ± 0.06a	0.09 ± 0.01b
Δ13C ‰	3.3 ± 0.4a	3.0 ± 0.5a	16.2 ± 3.0b
Δ18O ‰	16.8 ± 1.8a	20.9 ± 3.1a	207.4 ± 50.8b
Enzyme activities
PEPC (μmol m−2 s−1)	144.9 ± 8.8a	61.0 ± 4.6b	3.8 ± 0.4c
Rubisco (μmol m−2 s−1)	44.2 ± 3.2a	nd	48.1 ± 3.2a
kCA (mol m−2 s−1 bar−1)	6.1 ± 0.4a	5.3 ± 0.7a	6.4 ± 1.0a
CAleaf* (μmol m−2 s−1)	783 ± 153a	831 ± 113a	2818 ± 64b
Isotopic equilibrium (θ)
Predicted (Eq. 12)	0.98 ± 0.01a	0.98 ± 0.01a	1.00 ± 0.00a
Measured (Eq. 11)	0.62 ± 0.02a	0.62 ± 0.07a	0.91 ± 0.05b
Leaf parameters
Total leaf N (mmol m−2)	132 ± 23a	120 ± 10a	130 ± 4a
LMA (g m−2)	34.0 ± 2.9a	38.5 ± 3.2a	30.8 ± 1.6a

¹³CO₂ and C¹⁸OO Discrimination

Carbon isotope discrimination (Δ¹³C) decreased slightly but not significantly in the Pp mutant as compared with the wild type (Table I). In the pp mutant the value of Δ¹³C was approximately 5-fold higher than in wild-type and Pp plants, and p_i/p_a was 0.82 compared to 0.39 in the wild type (Table I; Fig. 1). The values of Δ¹⁸O were not significantly different between the wild-type and the Pp plants but Δ¹⁸O was 12-times higher in the pp mutant compared to the wild type (Table I; Fig. 2). The proportion of CO₂ in isotopic equilibrium with water at the site of oxygen exchange (θ) predicted from in vitro CA assays (Eq. 12) was substantially higher in the wild-type and Pp plants than the θ values estimated from Δ¹⁸O measurements (Eq. 11; Table I). However, θ calculated from in vitro CA assays (Eq. 12) and Δ¹⁸O measurements (Eq. 11) were similar in the pp mutant (Table I). The Δ¹⁸O increased with p_m/p_a as predicted from Equation 8, but the measured values of Δ¹⁸O were less than those predicted at full isotopic equilibrium for the wild-type and Pp mutant (Fig. 2). The pp mutant had a high p_m/p_a but wild-type levels of extractible CA activity and the measured values of Δ¹⁸O were closer to the predicted values of Δ¹⁸O, compared to wild-type plants (Table I; Fig. 2). The theoretical line of full isotopic equilibrium in Figure 2 was calculated using Equation 8, assuming an average value of the oxygen isotope composition of CO₂ at the site of exchange during photosynthesis (Δ_ea), taken from all plants in Table II, of 39.2‰. The values of Δ_ea were not significantly different between the plants (Table II).

Figure 1.

Carbon isotope discrimination (Δ¹³C) as a function of the ratio of intercellular to ambient pCO₂ (p_i/p_a) in wild-type and mutant A. edulis plants. The dashed line represents the theoretical relationship of Δ¹³C₄ and p_i/p_a during C₄ photosynthesis where φ = 0.24, using Equation 6. The solid lines represent the theoretical relationship of Δ¹³C₃ and p_i/p_a using the C₃ model (Eq. 3). Gas-exchange conditions are as in Table I. Each point represents the means ± se of measurements made on three to five leaves from separate plants from wild type (▪), Pp mutant (▴), and pp mutant (•).

Figure 2.

Oxygen isotope discrimination (Δ¹⁸O) as a function of the ratio of mesophyll cytosolic to ambient CO₂ partial pressure (p_m/p_a). p_m was calculated with g_m = 1 mol m⁻² s⁻¹ bar⁻¹. The line represents the theoretical relationship of Δ¹⁸O and p_m/p_a at full isotopic equilibrium where a = 7.7‰ and Δ_ea = 33.7‰ (Eq. 8) and the CO₂ supplied to the leaf had a δ¹⁸O of 24‰ relative to VSMOW. Symbols are as in Figure 1 and measurement conditions are as in Table I. The inset shows the expanded scale of Δ¹⁸O for the wild-type and Pp plants.

Table II.

Oxygen isotope exchange parameters of wild-type A. edulis and PEPC mutants

The δ¹⁸O of water at the site of evaporation (δ_e), ¹⁸O enrichment of CO₂ compared to the atmosphere at the site of exchange in full oxygen isotope equilibrium (Δ_ca), the oxygen isotope composition of CO₂ at the site of exchange during photosynthesis (Δ_ea), the ratio of the vapor pressure in the atmosphere to the leaf intercellular spaces (e_a/e_i), the residence time of CO₂ in the aqueous phase within the leaf (τ s⁻¹), and the intercellular pCO₂ (p_i) in wild-type (WT) and heterozygous (Pp) and homozygous (pp) mutant A. edulis plants. Source water was −5.3 ± 0.3. Measurement conditions as in Table I. Shown are the means ± se of measurements made on three to five leaves from separate plants. Statistical analysis was conducted using an ANOVA and different letters indicate significant differences between plants at P < 0.05.

Plants	δe	Δca	Δea	ea/ei	τ s−1	pi
	‰	‰	‰
WT	24.0 ± 0.8a	20.6 ± 0.6a	36.9 ± 1.4a	0.41 ± 0.03a	1.9 ± 0.1a	166 ± 19a
Pp	24.7 ± 0.9a	21.6 ± 3.0a	37.6 ± 1.5a	0.39 ± 0.03a	2.8 ± 0.2a	199 ± 29a
pp	29.9 ± 0.9b	38.8 ± 1.1b	43.2 ± 1.7a	0.21 ± 0.03b	16.5 ± 2.7b	431 ± 14b

The δ¹⁸O of water at the site of evaporation (δ_e) was similar in the wild-type and Pp plants but significantly more enriched in the pp plants (Table II). The ¹⁸O enrichment of CO₂ compared to the atmosphere at the site of exchange in full oxygen isotope equilibrium (Δ_ca) and the ratio of the water vapor pressure in the atmosphere to the leaf intercellular spaces (e_a/e_i) were also similar in the wild-type and Pp plants but were different in the pp plants (Table II). The residence time of CO₂ in the aqueous phase within the leaf (τ = p_m/F_in) and the intercellular pCO₂ were greater in the pp plants compared to the wild-type and Pp plants (Table II).

Stomatal Response

Online measurements of Δ¹³C and Δ¹⁸O leaf gas exchange at 531 μbar CO₂ partial pressure indicated that steady-state leaf conductance in the homozygous pp mutant was reduced compared to the wild type under these conditions (Table I). Further analysis showed that stomatal conductance (g_s) under growth conditions (9.8 mbar CO₂, 400 μmol quanta m⁻² s⁻¹, air humidity 29–32 mmol mol⁻¹, leaf temperature of 30°C) was higher in the pp mutant (0.7 ± 0.1 mol m⁻² s⁻¹) compared to wild type (0.3 ± 0.1 mol m⁻² s⁻¹; Fig. 3). However, g_s declined in the pp mutant and increased in the wild type when plants were rapidly shifted from growth conditions to similar conditions with low CO₂ (364 μbar; Fig. 3). To compare the response of g_s under laboratory conditions the pp and wild-type plants were dark adapted overnight in ambient CO₂ (364 μbar) and gas exchange was measured the following day. The two genotypes showed no difference in g_s under steady-state conditions in the dark before the onset of illumination (Fig. 4). However, the homozygous pp mutant had an approximately 3-times lower rate of stomatal opening (Fig. 4) in response to light under 364 μbar CO₂ than the wild type and g_s after 90 min in the light was only 41% of wild-type values (Fig. 4). In spite of the difference in steady-state conductance, both the wild-type and the pp mutant reached half their maximal conductance within approximately 13 min of the onset of illumination (Fig. 4). Long-term (5 h) measurements of g_s under the same conditions showed that the difference in stomatal conductance between the wild-type and pp mutant plants was maintained (data not shown).

Figure 3.

Stomatal conductance for wild-type and pp mutant plants under growth conditions (indicated by arrow) at 400 μmol quanta m⁻² s⁻¹, leaf temperature of 30°C, and the leaf chamber humidity was 29.6 ± 0.6 and 32.4 ± 0.4 mmol mol⁻¹ for the wild-type and pp mutant, respectively. Plants were subsequently transferred from the growth cabinets at time zero and a leaf was immediately placed into the gas-exchange chamber under growth conditions except the CO₂ concentration was 360 μbar instead of 9.8 mbar. The leaf chamber humidity was maintained at 30.01 ± 0.01 for both the wild-type and pp mutant. Data are the means of measurements of four different wild-type and five different pp mutants, error bars represent se; wild type (○) and pp mutant (•).

Figure 4.

Light induction of stomatal conductance (A) and net CO₂ assimilation (B) in wild-type and the pp mutant. Plants were grown under 9.8 mbar CO₂ and then transferred to ambient CO₂ in the dark overnight. Leaf gas exchange was measured for several minutes prior to the start of illumination at 2,000 μmol quanta m⁻² s⁻¹ (indicated by the arrow). The rate of stomatal opening for the wild-type and pp mutant was 7.8 ± 1.9 and 2.4 ± 0.5 (mmol water m⁻² s⁻²), respectively. The ambient CO₂ partial pressure was maintained at 364 μbar for the duration of the measurements. Data are the means of measurements of six different plants, error bars represent se; wild type (○) and pp mutant (•).

To examine whether the guard cells in the pp mutant were sensitive to changes in pCO₂, leaf gas exchange was measured under steady-state conditions (364 μbar CO₂, 2,000 μmol quanta m⁻² s⁻¹ and a vapor-pressure difference of 10 mbar) and then the pCO₂ was dropped to 48 μbar. Compared to the wild type, the pp mutant had a lower initial rate of stomatal opening and steady-state conductance reached only half of wild-type values (Fig. 5). As in the response to light, the halftime of stomatal opening to CO₂ was similar in both types of plants (14.1 ± 0.9 min and 11.2 ± 0.7 min, for wild-type and pp mutant, respectively; Fig. 5).

Figure 5.

A, The net change in stomatal conductance over time after a step change from 364 μbar to 48 μbar CO₂, normalized by the initial conductance in 364 μbar CO₂. Steady-state conductance at low CO₂ was 0.83 ± 0.08 (wild type) and 0.41 ± 0.05 (pp mutant) mol water m⁻² s⁻¹ and the rate of stomatal opening for the wild-type and pp mutant was 20.2 ± 4.5 and 11.9 ± 2.3 (mmol water m⁻² s⁻²), respectively. Illumination was kept at 2,000 μmol quanta m⁻² s⁻¹ for the duration of the experiment. B, The change in CO₂ assimilation rate over time at low CO₂ partial pressure is shown for comparison. Data are the means of measurements of four and five different plants, respectively, for the wild-type (○) and homozygous pp mutant (•). Error bars represent se.

Epidermal PEPC Content and Stomatal Density

In agreement with previous reports (Dever et al., 1995, 1996, 1997; Maroco et al., 1998b) and our activity measurements (Table I), leaf tissue of the pp mutant showed a large decrease in the content of PEPC, detected either by Coomassie staining or by immunoblot (Fig. 6). An epidermal fraction showed a protein profile similar to that of whole leaves and was identical in wild-type and pp mutant with the exception of the band corresponding to PEPC. The pp mutant epidermis contained 53% ± 4% of wild-type PEPC as determined by western analysis on three wild-type and four pp mutant plants (Fig. 6 shows one such representative western). The number of stomata per unit leaf area was greater in the pp mutant (63% and 77% greater than in the wild type on the adaxial and abaxial sides, respectively); however, the stomatal index remained unchanged (Table III).

Figure 6.

Soluble protein profile of leaf discs and epidermis of A. edulis wild-type (WT) and pp mutant. A, Coomassie Blue-stained SDS-PAGE gel of soluble leaf protein from leaf and epidermal (epi) fraction. B, Immunoblot of PEPC and the large subunit of Rubisco (RbcL), shown as a loading control. Thirty micrograms of total protein were loaded per lane. The epidermal PEPC protein content of the pp mutant was 53% ± 4% of wild type. The relative abundance of epidermal PEPC protein in the pp mutant compared to wild type was determined from immunoblot labeling of three wild-type and four pp mutant epidermal extractions. Shown here is one representative blot.

Table III.

Stomatal density and index of wild-type A. edulis and PEPC pp mutants

Stomatal index and stomatal density per leaf area in wild-type and homozygous mutant A. edulis. Shown are the means ± se of counts taken on leaves from three (wild type) or four (pp mutant) plants. Statistical analysis was conducted using a Student's t test and different letters indicate significant differences between wild type (WT) and pp plants at P < 0.1.

Leaf Side	Plant	Stomatal Density	Stomatal Index (Stomata/Total Epidermal Cells)
		mm−2
Adaxial	WT	144 ± 11a	0.22 ± 0.01a
	pp	233 ± 35b	0.22 ± 0.01a
Abaxial	WT	123 ± 7a	0.23 ± 0.01a
	pp	218 ± 35b	0.22 ± 0.01a

DISCUSSION

¹³CO₂ Isotope Discrimination in the PEPC Mutants

The low activity of PEPC caused rates of net CO₂ assimilation in the heterozygous (Pp) and the homozygous (pp) PEPC mutant to be significantly less than wild-type plants (Table I) when measured under ambient CO₂ (531 μbar) concentrations as previously reported (Dever et al., 1997, 1998; Maroco et al., 1998a, 2000; Kiirats et al., 2002). Limited activity of PEPC during C₄ photosynthesis causes a decrease in the initial CO₂ carboxylation reaction and reduces the capacity of the C₄ pump to concentrate CO₂ within the BSCs (von Caemmerer, 2000; Cousins et al., 2006a). In the model of C₄ carbon isotope discrimination (Eq. 6 in “Materials and Methods”) the main factors that influence Δ¹³C are changes in the intercellular to ambient CO₂ partial pressures (p_i/p_a), the fraction of CO₂ fixed by PEPC that subsequently leaks out of the BSC (φ), and the combined fractionation of PEPC and the isotopic equilibrium during dissolution of CO₂ and conversion to bicarbonate (b₄), which is dependent on the ratio of PEPC to CA activity (Farquhar, 1983). During C₄ photosynthesis decreases in φ and b₄ are both predicted to cause values of Δ¹³C to decrease (Farquhar, 1983). The values of Δ¹³C and p_i/p_a in the Pp mutant were not significantly different from the wild-type plants even though the Pp plants had lower rates of net CO₂ assimilation (Table I). This implies that neither φ nor the b₄ value was significantly different between wild-type and Pp plants. We conclude that although photosynthetic rates were lower in the Pp mutant compared to wild type, the Δ¹³C remained constant because the balance in the C₃ and C₄ cycles was not altered.

The very low PEPC activity in the pp plants (Table I) severely inhibited the initial carboxylation step of the C₄ photosynthetic pathway causing the rates of net CO₂ assimilation to decrease considerably relative to wild type (Table I; Fig. 4B). The value of p_i/p_a increased in the pp plants compared to wild type and the Pp plants (Table I) and according to the Δ¹³C model during C₄ photosynthesis (Eq. 6) a decrease in p_i/p_a leads to a decrease in Δ¹³C at φ values of less than approximately 30% (Fig. 1). However, values of Δ¹³C were dramatically higher in the pp plant compared to the wild-type and Pp plants (Figs. 1) even though p_i/p_a was higher in the pp plants and the b₄ would be close to −5.7 (Table I; Eq. 7). This implies that the simplistic model of C₄ isotope exchange presented here does not provide an accurate description of the processes contribution to Δ¹³C in the pp plants (see below).

C₃-Like Isotope Discrimination in the pp Mutant

As reported previously, the defective C₄ cycle in the pp plants necessitates the direct diffusion of atmospheric CO₂ into the BSC for CO₂ assimilation (Dever et al., 1995, 1997; Maroco et al., 1998a, 1998b; Kiirats et al., 2002) and the instantaneous Δ¹³C in these plants is more accurately described by the Δ¹³C₃ model for C₃ photosynthesis with a low conductance of CO₂ diffusion to the site of Rubisco carboxylation (g_i) within the BSC (see below). Assuming that the discriminations associated with photorespiration and respiration are negligible (see Eq. 1), we estimated an average CO₂ conductance from the intercellular air space to the site of Rubisco carboxylation within the BSC (g_i) of 33 ± 10 mmol m⁻² s⁻¹ in the pp plants (Eq. 5). This value of g_i is toward the high end of the range reported in the literature from various C₄ plants and approximately 4-times higher than the g_bs value reported by Kiirats et al. (2002) in their experiments with the pp plants. Mathematical modeling of the C₄ photosynthetic pathway suggests that such high conductance values need to be matched with high biochemical capacity to maintain low values of leakiness (for discussion see von Caemmerer, 2003).

To estimate the effect of photorespiratory and respiratory fractionation on our estimates of g_i we used discrimination factors for photorespiration (f = 10‰) and for respiration (e = −6‰) reported from the literature (Gillon and Griffiths, 1997; Ghashghaie et al., 2003; Igamberdiev et al., 2004). Our measurements were made at low O₂ partial pressures so that the expected fractionation from photorespiration is only about 0.17‰ at a Γ_* of 9.25 μbar. The effect of respiratory fractionation is between −1.67‰ to 1‰. It therefore appears that neither photorespiration nor respiration can easily account for the high Δ¹³C values measured in the pp plants and the major contributing factor to the high Δ¹³C values in these plants is the direct diffusion of CO₂ from the intercellular air spaces to the site of Rubisco carboxylation with the BSC.

Determining absolute values of g_i in C₄ plants and BSC leakiness is difficult as these parameter are effected by numerous factors including growth and measurement conditions (Henderson et al., 1992; Cousins et al., 2006a; Kubásek et al., 2007). Potentially g_i can be influenced by changes in the diffusivity of CO₂ across cell walls, the cytoplasm in the mesophyll and BSC, and across plasma and chloroplast membranes (von Caemmerer and Furbank, 2003). Additionally, the BSC surface area to leaf area ratio can also influence g_i; however, we saw no differences in leaf vein density in the pp plants (data not shown), indicating that the BSC surface area on a leaf area basis had not increased. However, because the pp plants rely on the direct diffusion of CO₂ into the BSC to sustain photosynthesis, the internal conductance of CO₂ may be greater than in wild-type plants, increasing the CO₂ concentration within the BSC.

C¹⁸OO Isotope Discrimination and CO₂/Water Isotopic Equilibrium

In a leaf, the oxygen isotope composition of CO₂ is determined by the isotope composition of leaf water at the site of evaporation (δ_e) and CA activity. The exchange of ¹⁸O between CO₂ and water is facilitated by CA, which catalyzes the interconversion of CO₂ and bicarbonate (), and high CA activity will increase the proportion of CO₂ in isotopic equilibrium with the water. Based on calculated values of δ_e (Table II) the C¹⁸OO discrimination (Δ¹⁸O) was low in the wild-type plants compared to the high levels of leaf CA (CA_leaf; Table I). The extent of isotopic equilibrium (θ) measured from Δ¹⁸O (Eq. 11) was also low in the wild-type plants relative to θ estimated from CA activity using Equation 12 (Table I). These findings are similar to previous work with another C₄ dicot, F. bidentis, which also had low Δ¹⁸O and θ measured from Δ¹⁸O compared to the high rates of CA_leaf (Cousins et al., 2006b). This further suggests that the total leaf CA activity in C₄ dicots does not represent the CA activity associated with the CO₂-water oxygen exchange that influences Δ¹⁸O (Cousins et al., 2006b).

The value of θ is related to the mean number of hydration reactions a CO₂ molecule experiences inside a leaf. This in turn is the product of residence time (τ = p_m/F_in) and the hydration constant of leaf CA (k_CA), where p_m is the mesophyll pCO₂ and F_in is the gross flux of CO₂ into the leaf (Eq. 12). The low photosynthetic rates increase the residence time of CO₂ as p_m increases (Tables I and II). Therefore, the number of hydration reactions per CO₂ increases when rates of net CO₂ assimilation are reduced by low PEPC activity. The amount of CA, expressed as the rate constant, was similar in the wild-type and PEPC mutants (Table I); however, under similar gas-exchange conditions the CA_leaf activity in the PEPC mutants were higher (Table I). The increase in CA_leaf in these plants is attributed to an increase in substrate availability for CA due to the lack of photosynthetic CO₂ drawdown caused by the low PEPC activity. The increase in θ in the pp plants suggests that in wild-type A. edulis the CA_leaf activity does not allow full isotopic equilibrium between the CO₂ and water within the leaf under steady-state conditions.

Our study suggests that in C₄ species leaf CA activity cannot readily be used as an indicator of the extent of ¹⁸O equilibration as has had been suggested by Gillon and Yakir (2001). This might be because not all of the CA located in mesophyll cytosol in C₄ species is available at the site of CO₂ water exchange compared to C₃ species where CA is located in chloroplasts that appress intercellular airspaces (Poincelot, 1972; Ku and Edwards, 1975). Alternatively, the extent of ¹⁸O equilibration may be miscalculated if the estimated values of δ_e do not accurately describe the isotopic composition of water at the site of oxygen exchange between leaf water and CO₂.

Stomatal Conductance in Response to Light and Atmospheric CO₂

The pp mutant had low stomatal conductance (g_s) during steady-state gas-exchange conditions (at 531 μbar CO₂) relative to the Pp mutant and wild-type plants (Table I). Additionally, g_s in the pp mutant decreased but increased in the wild type when leaves were rapidly transferred from the high CO₂ (9.8 mbar) growth conditions into air (364 μbar CO₂) at a constant leaf chamber humidity (Fig. 3). The higher g_s under elevated CO₂ reported here is consistent with previous reports that g_s is generally greater under superelevated CO₂ (above 4.0 mbar) compared to air CO₂ concentrations (see review and references within Wheeler et al., 1999). Low g_s in the pp mutant at ambient CO₂ (364 or 531 μbar) is different from previous publications where stomatal conductance was generally not affected when the photosynthetic capacity was reduced due to antisense silencing of either Rubisco or Rubisco activase in the C₄ dicot F. bidentis (von Caemmerer et al., 1997b, 2005). In C₄ plants PEPC initiates carbon fixation in mesophyll cells but also plays an important role in providing malate as a counter ion and an osmoregulator in the guard cells to help counterbalance the large influx of potassium ions during stomatal opening (Vavasseur and Raghavendra, 2005). The content of PEPC in the pp mutant was reduced at both the whole leaf level as well as in the epidermal tissue, indicating that the C₄ PEPC gene is the same as the guard cell gene in A. edulis (Table I; Fig. 6). Therefore, reduced PEPC activity in the pp mutant not only limited photosynthetic rates but likely impaired the accumulation of malate in the guard cells. Studies with epidermal peels have demonstrated strong correlations between stomatal opening and malate accumulation in guard cells (Allaway, 1973; Pearson, 1973; van Kirk and Raschke, 1978) and with the use of a PEPC inhibitor (DCDP) it has also been shown that stomatal opening is restricted when PEPC activity is reduced in epidermal strips (Parvathi and Raghavendra, 1997). In epidermal strips, the importance of malate as a counter ion to K⁺ is influenced by the availability of chloride (van Kirk and Raschke, 1978; Schnabl and Raschke, 1980; Willmer and Fricker, 1996). The low g_s and epidermal PEPC content in the pp mutants show that maximum stomatal opening is dependent on PEPC activity and presumably malate in the guard cells in vivo.

In the dark g_s was similar in pp mutant and wild type at air CO₂ concentrations (364 μbar), but the rate of opening during the light induction was slow in the pp mutant and the stomata were able to maintain only a third of the conductance under steady-state conditions compared to wild-type plants (Fig. 4). These findings provide further support that PEPC is necessary for stomatal opening in response to light (Asai et al., 2000). Interestingly, both the wild-type and pp mutants reached half their maximal g_s rate at similar times (Fig. 4), indicating that the perception of changing light conditions was not inhibited in the pp mutant, only that the stomata could not open as quickly and were unable to establish high rates of conductance.

Stomatal conductance increased in both the wild-type and the pp mutant in response to lowering pCO₂ (Fig. 5). However, g_s was slower to respond to the shift in CO₂ in the pp mutants and did not reach similar rates as in the wild-type plants (Fig. 5). The pp mutant can therefore sense the change in CO₂ availability but lacks the ability to achieve maximal values of g_s in response to conditions that normally stimulate g_s. As with the light response, even though g_s in the pp mutant did not reach similar values to the wild-type plants, the values of g_s increased about three times in response to low CO₂ availability in both plants (Fig. 5).

The shifts in g_s in response to changing light and pCO₂ did not correlate with the changes in net CO₂ assimilation in the pp plants (Fig. 5). For example, there was only a slight increase in net CO₂ assimilation from the dark to light transition in the pp plants but stomatal conductance increased about 8 times (Fig. 4). This increase in g_s during the light induction was less than in the wild-type plants but was still significant. Changes in net CO₂ assimilation were also minor in response to CO₂ in the pp plants (Fig. 5) but g_s was approximately 3-times greater under the lower CO₂ concentrations (Fig. 5). Although it has been demonstrated that there is a tight correlation between g_s and photosynthetic capacity in both C₃ and C₄ plants (Wong et al., 1985), the use of antisense and photosynthetic mutants indicates that under certain conditions this relationship may not hold (for review, see von Caemmerer et al., 2004a).

The low g_s in the pp mutant could have been attributed to reduced stomatal density compared to wild-type plants; however, stomatal density was approximately 1.5-times greater in the pp mutant, both adaxial and abaxial, than in the wild type (Table III). In fact stomatal conductance was higher in the pp mutant compared to the wild type under the 9.8 mbar CO₂ growth conditions (Fig. 3), which may in part be due to the alleviation of PEPC limitation on g_s in the pp mutant by high CO₂ availability coupled with the higher stomatal density in the pp mutant (Table II). The stomatal index in these two plants was similar, indicating that the increase in stomata in the pp plants was due to a general increase in the number of total epidermal cells (Table III). The increase in stomatal density may help alleviate the BSC CO₂ limitation in the pp plants that rely on direct fixation of atmospheric CO₂ by Rubisco.

CONCLUSION

The reduction in PEPC activity in A. edulis reduced rates of net CO₂ assimilation and Δ¹³C and Δ¹⁸O were dramatically increased in the homozygous PEPC mutant (pp). The high Δ¹³C value in the pp plants is likely caused by the direct diffusion of CO₂ from the intercellular air spaces to the site of Rubisco carboxylation within the BSC. The isotopic equilibrium between leaf water and the intercellular pCO₂ appears to be overestimated by in vitro measurements of total leaf CA activity compared to isotopic equilibrium determined from Δ¹⁸O measurements in wild-type plants. Lower stomatal conductance under steady-state conditions and the slower responses of stomata to changing light and CO₂ conditions in the pp mutant corresponded with reduced PEPC content in the epidermal tissue, implicating the C₄ isoform of PEPC in controlling stomatal movement.

MATERIALS AND METHODS

Growth Conditions

Seeds from the F₂ population of Amaranthus edulis LaC4 2.16 mutant deficient in PEPC activity (Dever et al., 1995, 1997) and from the corresponding wild type were grown under 9.8 mbar of CO₂ in a controlled environment growth cabinet at an irradiance of 400 μmol quanta m⁻² s⁻¹ at plant height and air temperature of 27°C during the day and 18°C at night, with a day length of 14 h. Plants were grown in 5 L pots in garden mix with 2.4 to 4 g Osmocote/L soil (15/4.8/10.8/1.2 N/P/K/Mg + trace elements: B, Cu, Fe, Mn, Mo, Zn; Scotts Australia Pty Ltd.) and watered daily. The mutant plants were screened by gas exchange and PEPC activity (see below).

Gas-Exchange Measurements

Online ¹³CO₂ and C¹⁸OO Discrimination

The uppermost fully expanded leaves were placed into the leaf chamber of the LI-6400 portable gas-exchange system (LI-COR) and equilibrated under measurement conditions for a minimum of 1.5 h (Cousins et al., 2006a, 2006b). Air entering the leaf chamber was prepared by using mass flow controllers (MKS instruments) to obtain a gas mix of 909 mbar dry N₂ and 48 μbar O₂. A portion of the nitrogen/oxygen air was used to zero the mass spectrometer to correct for N₂O and other contaminants contributing to the 44, 45, and 46 peaks. Pure CO₂ (δ¹³C_VPDB = −29‰, and δ¹⁸O_VSMOW = 24‰) was added to the remaining air stream to obtain a CO₂ partial pressure of approximately 531 μbar. The isotopic CO₂ used for measurements was similar to that used in the controlled environments. Low oxygen (48 mbar) was used to minimize contamination of the 46 (mass-to-charge ratio) signal caused by the interaction of O₂ and N₂ to produce NO₂ with the mass spectrometer source. Simultaneous measurements of leaf gas-exchange and isotope discrimination were determined by linking the LI-6400 gas-exchange system to a mass spectrometer through an ethanol/dry ice water trap and a thin, gas permeable silicone membrane (Cousins et al., 2006a, 2006b; Griffiths et al., 2007).

Stomatal Responses

To characterize the stomatal response of A. edulis plants, gas-exchange measurements were made with the LI-6400 portable gas-exchange system on two different sets of plants grown under identical conditions, young plants with an average of 10 leaves/plant and older plants with 20 to 25 leaves/plant. Data from the two sets of plants were combined in the final figures and tables. Light was provided by a red/blue LED light source (LI-6400-02B, LI-COR). Plants were dark adapted overnight at ambient CO₂ conditions and then the uppermost expanded leaf was equilibrated in the gas-exchange leaf chamber at 364 μbar in the dark for 20 min. The blue/red light source was then turned on to give an irradiance of 2,000 μmol m⁻² s⁻¹ and maintained at that level for the rest of the experiment. After the initial 90 min in the light the CO₂ concentration in the leaf chamber was decreased to 48 μbar and maintained for a further 90 min. Leaf chamber humidity and temperature were kept at 18 to 20 mmol mol⁻¹ and 25°C, respectively, for the duration of the measurements. Flow rate over the leaf was 500 μmol s⁻¹.

Steady stomatal conductance measurements were also made under the elevated CO₂ growth conditions as described above by bringing the gas-exchange system into the growth chamber. Leaves were clamped into the leaf chamber and the growth chamber air (9.8 mbar of CO₂) was allowed to flow over a leaf illuminated with 400 μmol quanta m⁻² s⁻¹ at a leaf temperature of 30°C. The leaf chamber humidity was not controlled but was similar between the wild-type and pp mutant 29.6 + 0.6 and 32.4 + 0.4 mmol mol⁻¹, respectively. Plants were subsequently transferred from the elevated CO₂ growth cabinets to air and a leaf was immediately placed into a gas-exchange chamber under the same measurement conditions except the CO₂ concentration was 360 μbar and the leaf chamber humidity was controlled at 30.01 + 0.01 for both the wild-type and pp mutant (Fig. 3).

Determination of Stomatal Numbers

Stomatal numbers were determined from the same or similar leaves as used for gas-exchange measurements, from silicone rubber impressions taken from both sides of the leaves (von Caemmerer et al., 2004a). Stomata and epidermal cells were counted from positives made from the impressions with nail polish, in 10 different fields of view per leaf, with a compound microscope using a magnification of 200-fold. Digital photographs of each field were taken and cells counted with the publicly available Image J software (http://rsb.info.nih.gov/ij/).

Calculations of ¹³CO₂ Discrimination

To calculate the conductance to CO₂ diffusion from intercellular airspace to the site of Rubisco carboxylation in the BSCs in the pp mutant we used the model of C₃ carbon isotope discrimination (Δ¹³C₃) developed by Farquhar et al. (1982). This model is given as:

(1)

where p_a, p_i, and p_c represent the pCO₂ of the air surrounding the leaf, in the intercellular air spaces and at the site of Rubisco carboxylation, respectively; a (4.4‰) is the fractionation during diffusion of CO₂ in air, a_i is the combined fractionation due to dissolution and diffusion of CO₂ in water (1.8‰), and the fractionation by Rubisco is b₃ = 30‰ (Roeske and Oleary, 1984). Γ_* is the CO₂ compensation point in the absence of day respiration. R_d is the rate of mitochondrial respiration, e and f are the discrimination factors of respiration and photorespiration with respect to the average carbon composition associated with respiration and photorespiration, respectively, and k is the Rubisco carboxylation efficiency (Farquhar et al., 1982; Evans et al., 1986). The carboxylation efficiency is given by

(2)

where V_cmax denotes the maximal Rubisco activity, K_c and K_o are the Michaelis Menten constants for CO₂ and O₂, respectively, and O stands for O₂ partial pressure.

The discrimination that would occur if the partial pressure of CO₂ in the chloroplast equals the intercellular pCO₂ and ignoring fractionations associated with respiration and photorespiration is usually given by

(3)

Subtracting Equation 3 from Equation 1 shows that the difference between the Δ_i and the measured Δ¹³C₃ is inversely proportional to the conductance to CO₂ diffusion from the intercellular airspace to the site of Rubisco carboxylation (g_i; Evans et al., 1986; von Caemmerer and Evans, 1991; Evans and von Caemmerer, 1996):

(4)

such that g_i can be estimated after rearranging Equation 4 from

(5)

The model of C₄ carbon isotope discrimination (Δ¹³C₄) from Farquhar (1983) was used to determine which factors in the model would influence Δ¹³C₄ consistent with our experimental data in the Pp mutant and wild-type plants. The simplified model predicts that

(6)

where s (1.8‰) is the fractionation during CO₂ leakage from the BSCs. The combined fractionation of PEPC and the isotopic equilibrium during dissolution of CO₂ and conversion to bicarbonate (b₄) was calculated as (Farquhar, 1983)

(7)

indicating that the fractionation when CO₂ and are not at equilibrium depends on the rate of CO₂ hydration (V_h) and the rate of PEP carboxylation (V_p).

Calculations of C¹⁸OO Isotope Discrimination

Discrimination against C¹⁸OO (Δ¹⁸O) when water and CO₂ at the site of exchange are at full isotopic equilibrium (θ = 1) can be predicted as (Farquhar and Lloyd, 1993)

(8)

where a′ is the diffusional discrimination (7.7‰) and ɛ is calculated as p_m/(p_a − p_m) (Fig. 3, solid line). The ¹⁸O enrichment of CO₂ compared to the atmosphere at the site of exchange in full oxygen isotope equilibrium with the water was calculated as

(9)

(Cernusak et al., 2004). The equilibrium fractionation between water and CO₂ (ɛ_w) was 40.17‰ at 30°C (Cernusak et al., 2004).

The δ¹⁸O of water at the sites of evaporation within a leaf (δ_e) can be estimated from the Craig and Gordon model of evaporative enrichment (Craig and Gordon, 1965; Farquhar and Lloyd, 1993)

(10)

where e_a and e_i are the vapor pressures in the atmosphere and the leaf intercellular spaces. δ_a and δ_t are the isotopic composition of water vapor in the air and transpired by the leaf, respectively. The kinetic fractionation during diffusion of water from leaf intercellular air spaces to the atmosphere (ɛ_k) and the equilibrium fractionation between liquid water and water vapor (ɛ⁺) was calculated according to Cernusak et al. (2004) and Cousins et al. (2006b). During the online gas-exchange measurements we let the leaves remain in steady-state conditions for a minimum of 1.5 h and under those conditions the value of δ_t is equal to the isotopic composition of source water, the water taken up by the plant (δ_s = −5.3 ± 0.3; Harwood et al., 1998).

The proportion of CO₂ in isotopic equilibrium with water at the site of oxygen exchange (θ) can be estimated from

(11)

where Δ_ca is the oxygen isotope composition of CO₂ at the site of exchange during photosynthesis (Gillon and Yakir, 2000a; Cousins et al., 2006b).

It has been suggested that the extent of θ in a leaf can also be calculated from in vitro CA assays coupled with the unidirectional flux of CO₂ into the leaf (Gillon and Yakir, 2000a, 2000b, 2001) from the equation initially developed by Mills and Urey (1940):

(12)

where CA_leaf/F_in represents the mean number of hydration reactions for each CO₂ molecule inside the leaf (Gillon and Yakir, 2001). Leaf CA activity (CA_leaf) is determined as the product of the CA hydration rate constant (k_CA, mol m⁻² s⁻¹ bar⁻¹) and the mesophyll pCO₂ (p_m). The rate constant k_CA is calculated from in vitro measurements of CA activity in leaf extracts (see below). The gross influx of CO₂ into a leaf (F_in = g_t p_a; where g_t is the total conductance of CO₂ from the atmosphere to the site of CO₂-water oxygen exchange; Gillon and Yakir, 2000a), as well as p_m determine the residence time (τ = p_m/F_in) of CO₂ within the leaf. The relationship of CA_leaf/F_in indicates that conditions that influence p_a, p_m, g_t, or k_CA can alter the value of θ.

Enzyme Activities

Enzyme activities were determined on approximately 1 cm² discs taken from the same leaves used for gas exchange. Leaf samples were collected after the gas-exchange measurements and subsequently frozen in liquid nitrogen and stored at −80°C. Tissue was ground on ice in 600 μL of extraction buffer (50 mm HEPES-KOH, pH 7.4, 10 mm dithiothreitol, 1% polyvinylpolypyrrolidone, 1 mm EDTA, and 0.1% Triton) with 20 μL of protease inhibitor cocktail (Sigma) and briefly centrifuged. PEPC activity was determined by placing 20 μL of leaf extract in 1 mL of assay buffer (100 mm EPPS-NaOH pH 8.0, 20 mm MgCl₂, 1 mm EDTA, 0.2 mm NADH, 5 mm Glc-6-P, 1 mm NaHCO₃, and 12 units of malate dehydrogenase). The reaction was initiated with 4 mm PEP and the rate of NADH consumption was determined by the absorbance change at 340 nm.

CA activity was measured on leaf extracts using mass spectrometry to measure the rates of ¹⁸O₂ exchange from labeled ¹³C¹⁸O₂ to (Badger and Price, 1989; von Caemmerer et al., 2004b; Cousins et al., 2006a, 2006b). Measurements of leaf extracts were made at 25°C with a subsaturating total carbon concentration of 1 mm. The hydration rates were calculated from the enhancement in the rate of ¹⁸O loss over the uncatalyzed rate and the nonenzymatic first order rate constant was applied (pH 7.4, appropriate for the mesophyll cytosol). The CA activity was reported as a first order rate constant k_CA (mol m⁻² s⁻¹ bar⁻¹) and k_CAp_m gives the in vivo CA activity at that particular cytosolic pCO₂.

Epidermal Preparations

A fraction enriched in epidermal tissue was prepared by adapting the method of Kopka et al. (1997). A young expanding leaf was picked, the major veins were removed and discarded, and the rest was blended with 250 mL of chilled distilled water with a Sorvall Omni Mixer blender at maximum speed, with four pulses of 30 s each and waiting 30 s between pulses. The resulting epidermal fragments were rinsed with 300 mL of chilled distilled water on a 100 to 149 μm Nytal mesh to rid them of contaminating mesophyll cells. The epidermal fragments were drained of excess water, disrupted by grinding with mortar and pestle in liquid nitrogen for 3 min, and stored at −80°C until later use. The resulting fraction was highly enriched in epidermis compared to mesophyll cells (less than 1 mesophyll cell per mm² epidermal fragment was observed under the compound microscope). Different blending regimes and filtration methods were ineffective in decreasing the contamination of epidermal fragments with bundle sheaths, so that the final preparations routinely contained a 10% to 20% contamination with bundle sheaths and vascular tissue as determined visually with the light microscope.

Protein Extraction and Immunoblotting

Soluble proteins from 1.28 cm² leaf discs or 100-mg of epidermal fragments were extracted on ice in 0.7 mL of extraction buffer containing 50 mm HEPES-KOH, pH 7.8, 5 mm MgCl₂, 2 mm EDTA, 5 mm dithiothreitol, 1% (w/v) polyvinylpolypyrrolidone, 0.1% (v/v) Triton X-10, and 4% (v/v) of protease inhibitor cocktail, using a 2-mL glass homogenizer. Samples were centrifuged in a cooled microcentrifuge at maximum speed for 4 min. The green pellet was discarded and the supernatant was brought to a final concentration of SDS of 2% (w/v) and heated to 65°C in a water bath for 10 min.

Protein concentration in the samples was determined with the bicinchoninic acid method (BCA Protein Assay kit, Pierce) prior to addition of SDS. Samples were prepared for gel loading by adding 0.25 volumes of Bio-Rad XT sample buffer (Bio-Rad). Thirty micrograms of total protein were loaded per gel well. Proteins were separated by electrophoresis on NuPAGE Bis-Tris precast gels (4%–12% acrylamide concentration, Novex) using the manufacturer-specified buffer system and blotted onto nitrocellulose membranes. Blots were probed with polyclonal antibodies raised against tobacco (Nicotiana tabacum) Rubisco and recombinant maize (Zea mays) PEPC, and with anti-Ig G alkaline phosphatase conjugate (Bio-Rad) as secondary antibody. Blots were developed using the AttoPhos fluorescence substrate system (Promega). Epidermal PEPC in the pp mutant was compared to wild type using Image Quant (Molecular Dynamics) to determine the relative abundance of the PEPC protein labeled by immunoblot from extractions prepared from three individual wild-type and four individual pp mutants.

Statistical Analysis

An ANOVA was conducted and Student's t test in STATISTICA (version 6.0 StatSoft). Tukey's honestly significant difference tests were used for post hoc comparisons.