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BIOENERGETICS AND PHOTOSYNTHESIS

The Role of Phosphoenolpyruvate Carboxylase during C₄ Photosynthetic Isotope Exchange and Stomatal Conductance^1,[OA]

Molecular Plant Physiology Group (A.B.C., I.B., M.R.B., A.I., S.v.C) and Australian Research Council Centre of Excellence in Plant Energy Biology (A.B.C., M.R.B.), Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2601, Australia; Department of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, United Kingdom (P.J.L.); and Department of Animal and Plant Sciences, Robert Hill Institute, University of Sheffield, Sheffield S10 2TN, United Kingdom (R.C.L.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
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 (¹³C) and C¹⁸OO (¹⁸O) 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 ¹³C between the Pp mutant (3.0 ± 0.4) and wild type (3.3 ± 0.4), indicating that leakiness (), 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 ¹³C was 16 ± 3.2, indicative of direct CO₂ fixation by Rubisco in the bundle sheath at ambient CO₂ partial pressure. ¹⁸O measurements indicated that the extent of isotopic equilibrium between leaf water and the CO₂ at the site of oxygen exchange () 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 as compared to values determined from ¹⁸O in wild-type plants.

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 (¹³C) during C₄ photosynthesis have been developed that link ¹³C to the ratio of intercellular to ambient pCO₂ and bundle sheath leakiness () 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 ¹³C in a number of C₄ species (Henderson et al., 1992). It has been shown that increases in transgenic Flaveria bidentis with reduced Rubisco content, demonstrating that the balance between C₃ and C₄ cycle activity influences (von Caemmerer et al., 1997a, 1997b; Cousins et al., 2006a). C¹⁸OO discrimination (¹⁸O) 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 ¹³C 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 ¹⁸O? (3) What are the effects of reduced PEPC activity on stomatal conductance?

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

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 ¹³C and ¹⁸O 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).

Carbon isotope discrimination (¹³C) decreased slightly but not significantly in the Pp mutant as compared with the wild type (Table I). In the pp mutant the value of ¹³C 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 ¹⁸O were not significantly different between the wild-type and the Pp plants but ¹⁸O 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 () predicted from in vitro CA assays (Eq. 12) was substantially higher in the wild-type and Pp plants than the values estimated from ¹⁸O measurements (Eq. 11; Table I). However, calculated from in vitro CA assays (Eq. 12) and ¹⁸O measurements (Eq. 11) were similar in the pp mutant (Table I). The ¹⁸O increased with p_m/p_a as predicted from Equation 8, but the measured values of ¹⁸O 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 ¹⁸O were closer to the predicted values of ¹⁸O, 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 (_ea), taken from all plants in Table II , of 39.2. The values of _ea were not significantly different between the plants (Table II).

View larger version (8K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Carbon isotope discrimination (13C) as a function of the ratio of intercellular to ambient pCO2 (pi/pa) in wild-type and mutant A. edulis plants. The dashed line represents the theoretical relationship of 13C4 and pi/pa during C4 photosynthesis where = 0.24, using Equation 6. The solid lines represent the theoretical relationship of 13C3 and pi/pa 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 ().

View larger version (10K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Oxygen isotope discrimination (18O) as a function of the ratio of mesophyll cytosolic to ambient CO2 partial pressure (pm/pa). pm was calculated with gm = 1 mol m–2 s–1 bar–1. The line represents the theoretical relationship of 18O and pm/pa at full isotopic equilibrium where a = 7.7 and ea = 33.7 (Eq. 8) and the CO2 supplied to the leaf had a 18O 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 18O for the wild-type and Pp plants.

Stomatal Response

Online measurements of ¹³C and ¹⁸O 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).

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   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 ().

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   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 ().

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   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.

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 ).

View larger version (52K): [in this window] [in a new window] [as a PowerPoint slide]   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.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

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 ¹³C 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 (), 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 and b₄ are both predicted to cause values of ¹³C to decrease (Farquhar, 1983). The values of ¹³C 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 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 ¹³C 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 ¹³C model during C₄ photosynthesis (Eq. 6) a decrease in p_i/p_a leads to a decrease in ¹³C at values of less than approximately 30% (Fig. 1). However, values of ¹³C 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 ¹³C 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 ¹³C in these plants is more accurately described by the ¹³C₃ 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 _* 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 ¹³C values measured in the pp plants and the major contributing factor to the high ¹³C 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 (_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 _e (Table II) the C¹⁸OO discrimination (¹⁸O) was low in the wild-type plants compared to the high levels of leaf CA (CA_leaf; Table I). The extent of isotopic equilibrium () measured from ¹⁸O (Eq. 11) was also low in the wild-type plants relative to 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 ¹⁸O and measured from ¹⁸O 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 ¹⁸O (Cousins et al., 2006b).

The value of 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 ( = 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 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 _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

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
The reduction in PEPC activity in A. edulis reduced rates of net CO₂ assimilation and ¹³C and ¹⁸O were dramatically increased in the homozygous PEPC mutant (pp). The high ¹³C 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 ¹⁸O 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

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

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 (¹³C_VPDB = –29, and ¹⁸O_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 (¹³C₃) 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). _* 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 _i and the measured ¹³C₃ 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 (¹³C₄) from Farquhar (1983) was used to determine which factors in the model would influence ¹³C₄ 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 (¹⁸O) when water and CO₂ at the site of exchange are at full isotopic equilibrium ( = 1) can be predicted as (Farquhar and Lloyd, 1993)

(8)

where a' is the diffusional discrimination (7.7) and 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 (_w) was 40.17 at 30°C (Cernusak et al., 2004).

The ¹⁸O of water at the sites of evaporation within a leaf (_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. _a and _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 (_k) and the equilibrium fractionation between liquid water and water vapor (⁺) 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 _t is equal to the isotopic composition of source water, the water taken up by the plant (_s = –5.3 ± 0.3; Harwood et al., 1998).

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

(11)

where _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 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 ( = 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 .

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.

	ACKNOWLEDGMENTS

 
We thank Dr. Spencer Whitney for the Rubisco and Dr. Tsuyoshi Furumoto for PEPC antibodies. We are thankful to Dr. Louisa Dever for the original isolation of the LaC4 2.16 PEPC-deficient mutant of A. edulis and Jessica Janek for her technical assistance.

Received June 5, 2007; accepted September 3, 2007; published September 7, 2007.

	FOOTNOTES

 
¹ This work was supported in part by a National Science Foundation international postdoctoral fellowship (to A.B.C.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Peter J. Lea (p.lea{at}lancaster.ac.uk).

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.103390

^* Corresponding author; e-mail asaph.cousins{at}anu.edu.au.

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