A single serine to alanine substitution decreases bicarbonate affinity of phosphoenolpyruvate carboxylase in C4Flaveria trinervia.

Phosphoenolpyruvate (PEP) carboxylase (PEPc) catalyzes the first committed step of C4 photosynthesis generating oxaloacetate from bicarbonate (HCO3-) and PEP. It is hypothesized that PEPc affinity for HCO3- has undergone selective pressure for a lower KHCO3 (Km for HCO3-) to increase the carbon flux entering the C4 cycle, particularly during conditions that limit CO2 availability. However, the decrease in KHCO3 has been hypothesized to cause an unavoidable increase in KPEP (Km for PEP). Therefore, the amino acid residue S774 in the C4 enzyme, which has been shown to increase KPEP, should lead to a decrease in KHCO3. Several studies reported the effect S774 has on KPEP; however, the influence of this amino acid substitution on KHCO3 has not been tested. To test these hypotheses, membrane-inlet mass spectrometry (MIMS) was used to measure the KHCO3 of the photosynthetic PEPc from the C4Flaveria trinervia and the non-photosynthetic PEPc from the C3F. pringlei. The cDNAs for these enzymes were overexpressed and purified from the PEPc-less PCR1 Escherichia coli strain. Our work in comparison with previous reports suggests that KHCO3 and KPEP are linked by specific amino acids, such as S774; however, these kinetic parameters respond differently to the tested allosteric regulators, malate and glucose-6-phosphate.

Introduction

Phosphoenolpyruvate (PEP) carboxylase (PEPc) catalyzes the irreversible carboxylation of PEP using bicarbonate (HCO3−) to form the four-carbon sugar, oxaloacetate (OAA). In plants, this reaction generally influences stomatal conductance (Parvathi and Raghavendra, 1997; Cousins ), seed development (Sangwan ; O’Leary ), pH regulation (Davies, 1986; Britto and Kronzucker, 2005), and the balance between carbon and nitrogen metabolism by providing intermediates for the tricarboxylic acid (TCA) cycle (Rademacher ; Plaxton and Podestá, 2006). In mesophyll cells of C4 plants, PEPc catalyzes the first committed step of C4 photosynthesis by providing OAA that is subsequently modified to other four-carbon compounds before entering bundle sheath cells for decarboxylation, releasing CO2 at the site of Rubisco (Hatch ; von Caemmerer and Furbank, 2003).

Higher plants contain multiple PEPc-encoding (ppc) genes comprising a multigene family where most of the genes encode a non-photosynthetic C3 PEPc (Christin and Besnard, 2009). C4 plants obtained a modified PEPc isoform to power C4 photosynthesis through mutations to a native ppc coding region (Christin ; Rosnow ) and upstream promoter region (Schaffner and Sheen, 1992; Gowik ). Changes to the C4ppc promoter region led to strong, mesophyll-specific expression, resulting in high PEPc activity to drive the CO2-concentrating mechanism of C4 photosynthesis (Gowik ). Work on PEPc peptide sequences from members of the Poaceae, Amaranthaceae, Asteraceae, and Cyperaceae families (Christin ), as well as the Chenopodaceae family (Rosnow ), identified amino acid residues predicted to be under positive selection in these C4 lineages. Comparing PEPc sequences of species within and between families shows that C4 PEPc isoforms from different species possess different combinations of amino acid residues under positive selection (Christin ; Rosnow ). These findings suggest that there are multiple ways the C4 PEPc kinetic properties can arise in different C4 origins or that there is diversity in the PEPc kinetics between species.

An increase in PEPc activity in the leaf mesophyll cytosol in the intermediate C3/C4 species would be likely to lead to selection for changes in kinetic properties (Westhoff and Gowik, 2004). This was previously tested in a variety of C3/C4 species that displayed a progression in altered KPEP (Km for PEP) and decreased malate sensitivity (Westhoff and Gowik, 2004). C4 plants contain high levels of malate in the mesophyll cytosol, so there would be selection for amino acid substitutions that transition the malate-sensitive C3 PEPc to a less sensitive C4 PEPc (Bläsing ; Paulus ). This is supported by the Gly884 substitution in the Flaveria trinervia C4 PEPc to the Flaveria pringlei C3 PEPc arginine that caused the C4 PEPc to lose its resistance to malate (Paulus ).

As PEPc transitioned from C3 to C4 function, it has been suggested that certain amino acid substitutions were under positive selection to alter KPEP and KHCO3 (Km for HCO3−). It is hypothesized that certain mutations in the C4ppc coding region resulted from strong selective pressures to obtain a lower KHCO3 than that of the C3 PEPc (Jacobs ). The lower KHCO3 of the C4 PEPc may enhance the efficiency of C4 photosynthesis, especially when HCO3− availably is low due to reduced stomatal conductance. Alternatively, the C4 PEPc has been shown to have a higher KPEP, with values typically reported between 100 µM and 590 µM (Svensson ; Dong ; Westhoff and Gowik, 2004; Lara ; Rosnow ), as compared with C3 non-photosynthetic PEPc KPEP values which range from 13 µM to 60 µM (Westhoff ; Bläsing ; Gowik ; Lara ; Rosnow ). It was hypothesized that this increase in C4KPEP was an unavoidable consequence of the reduction of KHCO3 since the two kinetic traits may be linked by certain amino acids (Jacobs ; Gowik and Westhoff, 2011). Alternatively, since the PEP pools in a C4 leaf are higher than in a C3 leaf, the high KPEP of the C4 PEPc may ensure stronger diurnal regulation of PEPc (Budde and Chollet, 1986; Hatch, 1987).

Residue S774 in F. trinervia (S780 in maize) was shown to be under positive selection by Poetsch and Hermans and Westhoff (1992), and substituting the conserved C4 serine for the conserved C3 alanine in F. trinervia (S774A) significantly decreased the KPEP of the C4 PEPc (Bläsing ; Engelmann ). Since the S774A substitution affects the KPEP of PEPc, it is possible that it may also affect the KHCO3, making S774 one of the residues potentially linking KPEP and KHCO3. However, this serine residue was unimportant for the high KPEP in the C4 Chenopodaceae (Rosnow ).

The only study to publish KHCO3 of both a C3 and C4 PEPc showed that the KHCO3 of five C4 species representing the Poaceae and Amaranthaceae families was ~26 µM compared with preliminary evidence suggesting that the KHCO3 of the C3 PEPc from Flaveria cronquistii (Asteraceae family) was 80 µM (Bauwe, 1986). Other studies reported C4 PEPc KHCO3 values ranging from 14 µM to 180 µM (Janc ; Gao and Woo, 1995; Parvathi ; Boyd ), where the C3KHCO3 of 80 µM falls within this reported range of C4KHCO3 values. However, comparing KHCO3 for closely related C3 and C4 PEPc isoforms can provide a more accurate analysis of the change in C3 to C4KHCO3 and whether there was a strong selective force on PEPc KHCO3, but to date this has not been performed. Additionally, the previously reported KHCO3 values were obtained by coupling PEPc activity to spectrophotometrically measured NADH oxidation rates. It is difficult to obtain accurate KHCO3 values using this method because it does not directly measure changes in HCO3− concentration in the assay. This is problematic because measurements of KHCO3 require accurate determinations of PEPc activity and HCO3− concentrations below the KHCO3, which is in the micromolar range. To overcome this problem, membrane-inlet mass spectrometry (MIMS) can be used to measure HCO3− consumption by PEPc accurately and directly in real-time over a wide range of inorganic carbon (Ci) concentrations, including concentrations well below the KHCO3, without the complication of coupling PEPc activity to the NADH dehydrogenase reaction (Boyd ).

In this study, we use MIMS to obtain KHCO3 values for the photosynthetic PEPc from the C4 plant F. trinervia and the non-photosynthetic PEPc from the C3 plant F. pringlei that were overexpressed and purified from the PEPc-less PCR1 Escherichia coli strain (Sabe ; Svensson ). We found that the S774A substitution increases the C4KHCO3, whereas the A774S substitution did not affect the C3KHCO3, suggesting that additional amino acids besides S774 are involved in the C4KHCO3 trait. Since previous studies reported PEPc KPEP changing in the presence of the allosteric activator glucose 6-phosphate (G6-P) and the inhibitor malate (Huber and Edwards, 1975; Wedding ; Gupta ; Bläsing ), we tested whether these allosteric regulators also affected KHCO3. We report that G6-P and malate have a minimal effect on the KHCO3 of PEPc. We address how differences in calibration methods, assay conditions, and enzyme extractions can produce different in vitro kinetic values, and we report an improvement to the MIMS PEPc assay. Lastly, we demonstrate how the decrease in KHCO3 between the C3 and C4 PEPc isoforms increases the modeled rates of C4 photosynthesis at low CO2 concentrations.

Materials and methods

Generating PCR1 PEPc-overexpressing lines

The PEPc-less E. coli strain, PCR1 (Sabe ), and PEPc cDNA constructs used in Svensson and Bläsing were generously provided by Professor Peter Westhoff’s lab. The plasmid, pTrc99A, carrying the cDNA coding for either the C4F. trinervia PEPc, the C3F. pringlei PEPc, or Flaveria PEPc with either an alanine or serine substitution at residue 774, C4-S774A or C3-A774S, respectively, was transformed into the PCR1 E. coli strain. PCR1 transformants producing plant PEPc were selected following the method of Svensson .

Growth of PCR1 PEPc-overexpressing lines for PEPc extraction

A 4 ml growth culture (Luria–Bertani broth; 0.1% w/v dextrose; 100 µg ml−1 ampicillin) was inoculated with a glycerol stock of the PCR1 strain carrying a Flaveria PEPc construct and was incubated at 28 °C with shaking at 160 rpm overnight. The following morning, the 4 ml culture was centrifuged at 1538 g for 10 min at room temperature. The supernatant was discarded, and the bacterial pellets were resuspended and transferred to a large 500 ml growth culture which was incubated at 28 °C and shaken at 160 rpm. After 6 h of incubation, isopropyl-β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 100 µM to the 500 ml growth culture to induce PEPc production overnight.

PEPc extraction and purification from E. coli

The 500 ml growth culture was centrifuged at 2602 g for 10 min at room temperature and the bacterial pellets were resuspended in a total volume of 20 ml of ice-cold lysis buffer [50 mM Tris–HCl, pH 8.0; 0.5 M NaCl; 10 mM DTT; 1 mM EDTA, pH 8.0; 20 µl ml−1E. coli protease inhibitor (Sigma); 1 mg ml−1 lysozyme (Bioworld); 10% (v/v) glycerol; 20% (w/v) sucrose]. The resuspended cells were placed in ice for 30 min and then lysed via sonication (BioLogics Ultrasonic Homogenizer 300 V/T). The sonicated cells were transferred to centrifuge tubes and were spun at 30597 g for 30 min at 4 °C. The supernatant was collected and MgCl2 was added to the supernatant to a final concentration of 10 mM. Polyethylene glycol (50% PEG 8000) was added to the supernatant to a final concentration of 6% (v/v) before placing the supernatant on ice for 15 min with gentle mixing. The supernatant was again spun at 30597 g for 20 min at 4 °C. The protein pellets were discarded and 50% PEG 8000 was added to the final concentration of 12% (v/v). The supernatant was slowly stirred on ice for 15 min before centrifugation at 30597 g for 20 min at 4 °C.

The protein pellet was collected and resuspended in 6 ml of Buffer A [0.5 M (NH4)2SO4; 20 mM Tris–HCl, pH 7.5; 0.1 mM DTT; 1 mM EDTA, pH 8.0; 5% (v/v) glycerol] supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) (Svensson ). The protein sample was loaded onto a HIC phenyl–Sepharose column (1 cm×5.5 cm) pre-incubated with Buffer A at a flow rate of 1.5 ml min−1. The hydrophobic properties of PEPc were used for the partial purification of PEPc by following the protocol of Svensson .

Fractions from the phenyl–Sepharose column were analyzed for PEPc activity by coupling the PEPc and NADH dehydrogenase reactions following the protocol of Boyd . Fractions displaying the highest PEPc activity were pooled and desalted in Buffer B [100 mM HEPES-KOH, pH 7.6; 1 mM DTT; 1 mM EDTA, pH 8.0] and concentrated using Corning Spin-X UF columns (6 ml volume, 100 K molecular weight cut-off) according to the Corning procedure. Glycerol was added to a final concentration of 20% (v/v) before storage at –80 °C. Total protein content of the PEPc samples was measured by a modified Bradford assay (Bio-Rad Protein Assay Kit II) (Bradford, 1976) using the Bio-Rad procedure.

Extracting PEPc from F. trinervia and Setaria viridis

PEPc samples were extracted and desalted from leaves of F. trinervia and S. viridis following the procedure of Boyd . Once the desalted PEPc extracts were collected and moved through a Millex-GP 0.22 µm syringe filter (Millipore), the extracts were concentrated by spinning the samples at 2880 g for 20 min at 4 °C in an Amicon Ultra-4 Ultracel-100K centrifugal filter (Millipore). Glycerol was added to the concentrated PEPc samples to a final concentration of 20% (v/v) and stored at –80 °C.

Obtaining VPmax, KHCO3, and Hill values for the different PEPc isoforms

The HCO3−-dependent PEPc assays were run in a 600 µl cuvette attached to the inlet of a mass spectrometer as described by Cousins . A CO2 calibration was conducted before each HCO3− response curve as reported by Boyd . The calibration consisted of three 2 µl injections of 10 mM NaHCO3 into 0.1 N HCl and three 6 µl injections of 100 mM NaHCO3 into the PEPc reaction mixture [100 mM HEPES-KOH, pH 7.6; 10 mM MgCl2; 1 mM DTT; 50 µg ml−1 carbonic anhydrase (CA); 5 mM G6-P; 5 mM PEP]. The second calibration step differs from Boyd as their 100 mM NaHCO3 injections went into buffer lacking DTT, G6-P, and PEP. DTT, G6-P, and PEP were added to the second calibration to take into account the pH changes these compounds have on the assay buffer. As the CO2 calibrations take into account total Ci and total CO2 in the cuvette, the HCO3− concentration could be deduced by subtracting the amount of CO2 in the cuvette from the total Ci measured in the cuvette (Boyd ).

To measure PEPc HCO3− kinetics, seven NaHCO3 concentrations (50, 100, 200, 350, 500, 750, and 1000 µM) were used for the assays. The CO2 was removed from the assay buffer containing 100 mM HEPES-KOH, pH 7.6 and 10 mM MgCl2 by continuously bubbling the buffer with humidified N2 gas starting at least 1 h prior to initiating the assays. The assay buffer (600 µl) followed by 1 mM DTT, 50 µg ml−1 CA, 5 mM G6-P, 5 mM PEP, and various NaHCO3 concentrations were added to the reaction cuvette and held at a constant 25 °C with a temperature-controlled water bath.

A blank rate was obtained by measuring the change in the mass 44 (12C16O16O) signal during a 30 s period before initiating the reaction with the addition of 10–15 µg of total protein of the PEPc extract. The PEPc reaction was run for 5 min but the first 20 s of the PEPc reaction were discarded to allow for enzyme mixing and rate stabilization. The MIMS reports a mass 44 signal every 0.8 s, so a robust 10 s running average of the change in mass 44 was used as a single data point for PEPc activity (VP) at an averaged [HCO3−]. Data points from 10 s running averages were taken immediately following the 20 s mixing phase. For the larger NaHCO3 injections (350–1000 µM NaHCO3), 10 s running averages were taken until a drop in PEPc activity was observed to avoid data points where there might be end-product inhibition of the reaction. For the lower NaHCO3 concentrations (50–200 µM), 10 s running averages were taken until the reaction was depleted of Ci. Once the Ci was depleted from the 50, 100, and 200 µM NaHCO3 injections, as indicated by a zero slope for the mass 44 signal, a 30 s running average of the zero slope was taken to obtain a mass 44 zero. These mass 44 zeroes accounted for mechanical drift in the MIMS as the different zeroes were taken at various times throughout the HCO3− response curve.

The kinetic parameters VPmax, KHCO3, and Hill value (h) were obtained by using the Hill equation:

where the Hill equation was fit to the HCO3− response curve using Excel’s Solver function to produce the kinetic parameters listed above.

Measuring the impact of G6-P and malate on KHCO3

To measure the effect of G6-P on KHCO3, G6-P was omitted from the assay described above to compare KHCO3 values in the presence or absence of 5 mM G6-P. Alternatively, 2.5 mM malate (pH 7.6) was added to the assay described above to determine if malate affects PEPc KHCO3 values in the presence or absence of G6-P.

Measuring PEP effects on malate inhibition

The MIMS assay described above was used to determine if PEP concentration affects malate inhibition of PEPc, in the absence of G6-P. Five malate concentrations were used (0, 1, 2, 3, and 4 mM) to determine the percentage change in enzyme activity of the C3 and C4 PEPc isoforms in the presence of saturating NaHCO3 (1000 µM) and when PEP was saturating (5 mM) or non-saturating (150 µM and 750 µM for the C3 and C4 isoforms, respectively). The non-saturating PEP concentrations were determined to be twice the reported KPEP values (2×KPEP) in the absence of G6-P (Westhoff and Gowik, 2004).

Extraction source, pH, and calibration effects on PEPc KHCO3

Previously, MIMS measurements of desalted PEPc extracts from S. viridis reported a KHCO3 of 62.8 ± 5.0 µM (Boyd ). Therefore, we tested whether differences in PEPc source (plant versus E. coli), pH, or MIMS calibrations caused the KHCO3 reported here to differ from those of Boyd . The MIMS assay described above containing 5 mM G6-P and no malate was used to test whether PEPc samples extracted from leaves of F. trinervia and S. viridis produced different KHCO3 values from the C4 PEPc extracted from E. coli. For each assay, 5–10 µl of plant extract was added to initiate the PEPc reaction. PEPc extracts from S. viridis were used to compare KHCO3 values obtained at pH 7.6, the pH used in this study, with KHCO3 values obtained at pH 7.8 used by Boyd . These extracts were also used to compare KHCO3 values obtained at pH 7.8 using either the current calibration method outlined above or the calibration method of Boyd .

Statistical analysis of experimental data

Statistical analyses of the kinetic data were performed using RStudio version 1.1.447 (RStudio Team, 2016). Homogeneity of variance was checked using Levene tests, and normality was checked using residual quantile plots and residual versus fitted value plots. Non-normal data were log transformed but they reported the same statistical outcomes as non-transformed data, so for simplicity only non-transformed data analyses are presented. One-way ANOVA and Tukey HSD post-hoc tests were used to determine statistical significance (P<0.05) of KHCO3 between PEPc isoforms. Two-way ANOVA (P<0.05) and Tukey HSD post-hoc tests were used to analyze statistically significant differences in KHCO3 between isoforms and the impact of potential allosteric effectors. A two-way repeated measures ANOVA (P<0.05) was used to test if the change in PEPc activity in response to malate significantly differed between C3 and C4 PEPc isoforms at various PEP concentrations. One-way ANOVA and Tukey HSD post-hoc tests were used to determine significant differences between C4 PEPc extracted from E. coli and PEPc extracted from F. trinervia and S. viridis leaves. Student’s t-tests (P<0.05) were separately used to determine significant differences in S. viridis PEPc KHCO3 assayed at pH 7.6 and 7.8 and for S. viridis PEPc KHCO3 assayed at pH 7.8 using the two calibration methods.

Modeling the effect of KHCO3 on C4 photosynthesis

The modeled effect of KHCO3 on the response of C4 enzyme-limited photosynthetic CO2 assimilation (Ac) to changing mesophyll CO2 concentrations (Cm) was determined by solving the quadratic formula using the set of equations as described by von Caemmerer (2000). The equations and input variables were taken from von Caemmerer , von Caemmerer (2000), Tholen and Zhu (2011), and Ubierna , and are presented in Supplementary Table S1 at JXB online.

Results

Kinetics of the C3, C4, and chimeric PEPc isoforms

The Hill equation was used to determine the maximum rate of PEPc carboxylation (VPmax), the Km for bicarbonate (KHCO3), and the co-operativity of the PEPc active sites (h) from 25 °C MIMS measurements of PEPc activity (Vp) in response to changes in HCO3− concentrations (Supplementary Fig. S1). Measurements were made on C4, C3, and chimeric Flaveria PEPc isoforms expressed and partially purified from E. coli. The C4 PEPc had a significantly lower KHCO3 than the C3 PEPc, 26.6 ± 1.7 µM and 64.0 ± 2.4 µM, respectively (Fig. 1). Additionally, the C3 PEPc had a lower VPmax compared with the C4 PEPc, 5.1 ± 0.7 µmol mg protein−1 min−1 and 8.1 ± 0.7 µmol mg protein−1 min−1, respectively (Supplementary Table S2). Neither isoform displayed co-operativity towards HCO3− binding, with Hill values close to 1.0 under all assay conditions (Supplementary Table S2).

Fig. 1.

The KHCO3 of the C3, C4, and chimeric PEPc isoforms. The KHCO3 values were obtained from the MIMS assayed in 100 mM HEPES-KOH buffer (pH 7.6) that contained 10 mM MgCl2, 5 mM PEP, 50 µg ml−1 CA, 1 mM DTT, and 5 mM G6-P. Error bars represent the mean ±SD of four independent extractions from E. coli for each PEPc isoform. Significance was determined by one-way ANOVA and Tukey HSD post-hoc tests. Bars with different letters are significantly different (P<0.05).

The substitution of the conserved C4 serine at residue 774 (780 in maize) with the conserved C3 alanine (C4-S774A) significantly increased the KHCO3 by 45% from 26.6 ± 1.7 µM to 38.6 ± 5.5 µM (Fig. 1). However, the C4-S774A substitution had no effect on VPmax (Supplementary Table S2). The reverse substitution, C3-A774S made in the C3 PEPc, did not significantly change the KHCO3 (from 64.0 ± 2.4 µM to 61.5 ± 9.1 µM; Fig. 1) nor did it affect VPmax (Supplementary Table S2).

The impact of G6-P and malate on KHCO3

The VPmax did not change by omitting G6-P from the assay, regardless of the PEPc isoform (Supplementary Table S2). Additionally, the KHCO3 values of the PEPc isoforms were not significantly altered by the presence or absence of G6-P in the assay (Fig. 2).

Fig. 2.

The impact of G6-P on the KHCO3 of the different PEPc isoforms. The KHCO3 values of the different PEPc isoforms were obtained from MIMS PEPc assays where 5 mM G6-P was present (white bars) or absent (gray bars) in the assay buffer. White bars are data represented from Fig. 1. Error bars represent the mean ±SD of four independent extractions from E. coli for each PEPc isoform. A two-way ANOVA determined that G6-P had a non-significant effect on KHCO3 but there was a significant isoform effect, and a Tukey HSD post-hoc test was used to determined significance. PEPc isoforms with different letters are significantly different (P<0.005).

Under the current measurement conditions of pH 7.6, 5 mM PEP, and the absence of G6-P, the addition of 2.5 mM malate decreased the VPmax in the C4, C4-S774A chimeric, and the C3 PEPc by 57.5, 24.4, and 6.8%, respectively (Supplementary Tables S2, S3). However, the KHCO3 values of the PEPc isoforms were not significantly altered by the presence of malate in the assay (Fig. 3A). HCO3− response curves in the absence of G6-P were not obtained for the C3-A774S PEPc due to severe inhibition of the chimeric PEPc by malate (Supplementary Fig. S2). When 5 mM G6-P and 2.5 mM malate were both present in the PEPc assay, VPmax decreased in the C4, C4-S774A chimeric, C3, and C3-A774S chimeric PEPc by 44.4, 28.6, 2, and 14%, respectively (Supplementary Tables S2, S3). However, the KHCO3 values of the PEPc isoforms were not significantly changed with both G6-P and malate in the assay (Fig. 3B). Interestingly, decreasing the total PEP concentration in the assay from 5 mM to twice the reported KPEP of the C3 and C4 isoforms (Bläsing ; Paulus ), 150 µM and 750 µM PEP, respectively, caused the C3 PEPc to lose activity dramatically in the presence of malate, whereas the change in PEP concentration had a smaller effect on malate inhibition of C4 PEPc activity (Fig. 4).

Fig. 3.

The effect of 2.5 mM malate on the KHCO3 of different PEPc isoforms in the absence and presence of G6-P. (A) KHCO3 values of the different PEPc isoforms obtained from MIMS PEPc assays in the presence (white bars) or absence (gray bars) of 2.5 mM malate without G6-P. (B) The KHCO3 of the different PEPc isoforms with 5 mM G6-P, in the presence (white bars) or absence (gray bars) of 2.5 mM malate. Gray bars in both (A) and (B) represent data from previous figures. Error bars represent the mean ±SD of four independent extractions from E. coli for each PEPc isoform. A two-way ANOVA determined significant effects on KHCO3 by allosteric regulators and between different isoforms, but a Tukey HSD post-hoc test determined no significant allosteric effect on KHCO3 in both (A) and (B). A Tukey HSD post-hoc test was performed on the isoform effect, and different letters represent significant differences (P<0.005).

Fig. 4.

Malate resistance of the C3 PEPc is affected more by changing PEP concentrations than that of the C4 PEPc. At 5 mM PEP (filled circles and squares), the C3 PEPc (solid line) is more resistant to malate than the C4 PEPc (dashed line). When the PEP concentration was dropped to twice the reported KPEP for the C3 and C4 PEPc isoforms (open circles and squares), malate resistance of the C3 PEPc dropped drastically compared with the malate resistance for the C4 PEPc. Malate activity assays were performed in 100 mM HEPES-KOH (pH 7.6), 10 mM MgCl2, 1 mM DTT, 50 µg ml−1 CA, 2.5 mM malate (pH 7.6), 1 mM NaHCO3, and various PEP concentrations. Shapes and error bars represent the mean ±SD of four independent extractions from E. coli for both the C3 and C4 PEPc isoforms. A two-way repeated measures ANOVA (P<0.05) determined that there was a significant difference between the increased sensitivity to malate of the C3 PEPc versus the C4 PEPc when PEP concentration was decreased to 2×KPEP for each isoform.

Effects of MIMS calibrations, assay conditions, and extraction sources on KHCO3

At pH 7.6, the C4F. trinervia PEPc extracted from E. coli had a lower KHCO3 than desalted plant PEPc extracts from F. trinervia, 26.6 ± 1.7 µM and 35.2 ± 3.2 µM, respectively (Fig. 5A). Although not statistically significant, changing the pH of the assay buffer from 7.6 to 7.8 increased the KHCO3 of S. viridis PEPc by 21.3% from 30.0 ± 3.0 µM to 36.4 ± 5.3 µM (Fig. 5A, 5B). When using the Boyd MIMS calibration method at pH 7.8, the S. viridis KHCO3 increased to 62.9 ± 8.7 µM (Fig. 5B).

Fig. 5.

The KHCO3 measured under different assay conditions for PEPc isoforms extracted from F. trinervia, S. viridis, and E. coli. (A) The KHCO3 values of the C4 PEPc extracts from E. coli (black bar; value from Fig. 1) and desalted PEPc extracts from F. trinervia and S. viridis (gray bars) were obtained from the MIMS assayed in 100 mM HEPES-KOH buffer (pH 7.6) with 10 mM MgCl2, 5 mM PEP, 50 µg ml−1 CA, 1 mM DTT, and 5 mM G6-P. Bars represent the mean ±SD of four independent PEPc extractions. Significance was determined by one-way ANOVA and Tukey HSD tests. Bars with different letters are significantly different (P<0.05). (B) S. viridis PEPc KHCO3 values measured from assays at pH 7.8 using either the current MIMS calibration or the Boyd calibration. Significance between the KHCO3 of S. viridis PEPc assayed at (A) pH 7.6 and (B) 7.8 was determined by a Student’s t-test (P=0.08). Significance between the KHCO3 of S. viridis PEPc assayed at pH 7.8 obtained by either the current calibration method or the Boyd calibration method was determined by a Student’s t-test (P<0.05).

Modeling the effect of different KHCO3 values on C4 photosynthesis

The C3 and C4 PEPc KHCO3 values from Fig. 1 were input into the C4 photosynthesis model from von Caemmerer (2000) to determine how differences in KHCO3 would impact modeled rates of C4 photosynthesis. Varying KHCO3 with a constant VPmax significantly changed the modeled rates of C4 photosynthesis under CO2 conditions below 20 Pa. The lower KHCO3 of the C4 PEPc resulted in higher modeled rates of C4 photosynthesis at these low mesophyll CO2 concentrations (Cm; Fig. 6). However, the difference between the C3 and C4KHCO3 modeled no differences in net CO2 assimilation above ~20 Pa Cm (Fig. 6).

Fig. 6.

Modeled rates of C4 photosynthesis with C3 and C4KHCO3. The KHCO3 values for the C3 PEPc (filled circles) and C4 PEPc (open circles) were input into the C4 photosynthesis model from von Caemmerer (2000) to determine the modeled rate of CO2 assimilation (Anet) at various mesophyll CO2 concentrations (Cm). Symbols represent means ±SD of four independent KHCO3 values presented in Fig. 1 input into the model, where all other variables in the C4 model were held constant. The maximal rates of PEP regeneration (Vpr), Rubisco carboxylation (VCmax), and maximum PEPc carboxylation per unit leaf area [VPmax(plant)] were set to 80, 60, and 120 µmol m−2 s−1, respectively (von Caemmerer, 2000), and all other values are presented in Supplementgary Table S1. A pKa of 6.12 and assumed a mesophyll cytosol pH of 7.2 were used to convert µM HCO3− to µM CO2. Pa CO2 was obtained by using Henry’s constant for CO2 (0.034 mol l−1 atm−1) and assumed standard pressure (101325 Pa atm−1).

Discussion

Kinetic changes during the evolution of the C4 PEPc

We and others (Jacobs ; Gowik and Westhoff, 2011) have hypothesized that there was a strong selective pressure to reduce the KHCO3 of the C4 PEPc isoform. Additionally, previous studies have reported that changing the PEPc amino acid residue 774 in Flaveria spp. (780 in maize) influences KPEP and its allosteric regulation (Engelmann ; Endo ). Therefore, the aim of this research was to test the hypotheses that the KHCO3 of the C4 PEPc isoform from F. trinervia would be lower than the KHCO3 of the C3 PEPc isoform from F. pringlei and that changes to residue 774 will impact KHCO3 and its allosteric regulation. Residue 774 was chosen because others have shown the C4F. trinervia S774A substitution reduces KPEP (Bläsing ; Endo ). Furthermore, residue 774 is near both the PEP- and HCO3−-binding sites, and may also influence KHCO3. We have analyzed the influence of this residue on KHCO3 and showed that the C4-S774A chimeric PEPc had a significantly higher KHCO3 compared with the C4 PEPc (Fig. 1). This fits with previous data that suggest that KPEP and KHCO3 are inversely linked through specific amino acid residues near the two binding sites. For example, the K829G substitution in the F. trinervia C4 PEPc resulted in a small decrease in KHCO3 and a simultaneous increase to KPEP (Gao and Woo, 1996). Alternatively, swapping Lys600 with either an arginine or threonine in F. trinervia led to increases in both KPEP and KHCO3, but this residue is one of the four conserved amino acids comprising the HCO3−-binding site (Gao and Woo, 1995; Kai ).

The chimeric C3 PEPc of F. pringlei, C3-A774S, had a minimal effect on KHCO3 (Fig. 1). This same amino acid substitution was also shown not to influence the KPEP of the C3 PEPc with G6-P present in the assay. However, in the absence of G6-P, the same A774S substitution did increase KPEP (Bläsing ). Taken together, these results support the analysis that multiple amino acid residues, in addition to S774 (S780 in maize), influence PEPc kinetics. Indeed, swapping amino acid residues 296–437 from the C4 enzyme into the C3-A774S chimeric PEPc increased the C3KPEP even closer to the established C4KPEP value (Engelmann ). However, further research is needed to understand how changes in PEPc amino acid composition influence KHCO3, particularly how specific amino acid changes influence the impact that allosteric regulators such as G6-P and malate have on KHCO3.

The influence of G6-P and malate on KHCO3

PEPc is regulated by post-translational modifications (PTMs) and interactions with allosteric effectors. Previous studies showed that glycine and G6-P activate PEPc (Svensson ; Westhoff ; Endo ) while the end-products, aspartic acid and malate, inhibit PEPc (Huber and Edwards, 1975). Additionally, previous studies have shown that the PEP-binding sites of the C4 PEPc tetramer display positive co-operativity for KPEP, which can be altered by the binding of G6-P and malate to PEPc (Wedding ; Bläsing ; Rosnow ). We show that neither G6-P nor malate appears to impact KHCO3 and the co-operativity of HCO3− binding to the extent that they influence KPEP and co-operative PEP binding (Figs 2, 3; Supplementary Tables S2, S3). Using the crystal structure (4BXC) deposited by Schlieper , the HCO3−-binding site is further from the G6-P and aspartic acid/malate allosteric binding sites than the PEP-binding site is from the allosteric sites, so any structural changes to PEPc caused by allosteric binding may affect the PEP-binding site more than the HCO3−-binding site. Additionally, the S774A and A774S substitutions did not influence the allosteric regulation of PEPc to the extent that the R884G and G884R substitutions affected malate sensitivity of the F. pringlei and F. trinervia PEPc isoforms, respectively (Paulus ). This discrepancy may be due to residue 884 being closer to the residues of the aspartate/malate-binding sites compared with residue 774, whereas residue 774 is closer to the PEP- and HCO3−-binding sites (Kai ; Paulus ).

Another possibility is that PEP binds before HCO3− (Janc ), potentially conferring the primary allosteric regulation of PEPc to the binding of PEP. Alternatively, under our assay conditions, the high PEP concentration may have reduced the impact G6-P and malate had on KHCO3. For example, G6-P has a greater activating effect on PEPc under limiting PEP concentrations at 0.5 mM (Gupta ). In addition, multiple studies suggest that there is a regulatory PEP-binding site different from the PEPc active site (Rustin ; Rodríguez-Sotres and Muñoz-Clares, 1990; Mújica-Jiménez ; Yuan ), and it is possible this regulatory PEP site may not be saturated under low PEP concentrations. Saturating this regulatory PEP-binding site might supersede G6-P activation and overcome malate inhibition of PEPc (Huber and Edwards, 1975). The assay conditions used in this study contained saturating (5 mM) levels of PEP, which were well above the KPEP of both PEPc isoforms, since limiting PEP would complicate the response to changes in HCO3− concentrations. The C3 PEPc was more resistant to malate inhibition than the C4 PEPc under these saturating PEP conditions, which is in contrast to previous reports (Bläsing ; Paulus ). However, we found that the C3 PEPc was more sensitive to malate than the C4 PEPc when the PEP concentration in the assay was reduced to 2×KPEP (Fig. 4). This suggests that the PEP regulatory site for the C4 PEPc may be less sensitive than the C3 PEPc to changes in free PEP availability. Alternatively, the C4 PEP regulatory site may not have as much influence on malate tolerance as the C3 PEP regulatory site under the current assay conditions.

We were unable to obtain kinetic data for the C3-A774S chimeric PEPc due to drastic inhibition of the enzyme by malate when G6-P was absent from the assay (Supplementary Fig. S2). This result was unexpected since the addition of 2.5 mM malate had a small effect on the activity of the C3 PEPc (Supplementary Tables S2, S3). However, since PEP and malate interact differently with the C3 and C4 PEPc isoforms, it is possible that the A774S substitution in the C3 PEPc modified these interactions to allow potent inhibition of the C3-A774S chimeric PEPc. Further analysis is needed to test the extent of malate inhibition on the C3-A774S PEPc and other chimeric PEPc isoforms under various assay conditions. It is worth noting that malate has a stronger inhibitory effect on PEPc at pH 7.0 than at pH 8.0 (Huber and Edwards, 1975; Gupta ). As discussed below, pH and other assay conditions used to measure PEPc activity can influence the absolute values of the kinetic parameters.

Assay conditions, extraction method, and source can affect PEPc kinetics

MIMS can directly measure dissolved CO2 even at very low Ci concentrations below the KHCO3 of PEPc (Beckmann ; Cousins ). Previously, Boyd reported a MIMS-measured KHCO3 value of 62.8 µM for the S. viridis C4 PEPc which is higher than our MIMS-measured KHCO3 value of 26.6 µM for the F. trinervia C4 PEPc extracted from E. coli. This difference in KHCO3 between the S. viridis and F. trinervia C4 PEPc may be due to any combination of species differences in enzyme kinetics, enzyme purity, pH of the assay, and MIMS calibrations. Plant PEPc extracted from F. trinervia, a dicot in the Asteraceae family, and S. viridis, a monocot in the Poaceae family, had similar KHCO3 values at pH 7.6 (Fig. 5A). Bauwe (1986) also reported similar KHCO3 values for different C4 PEPc isoforms extracted from multiple grasses and Glomphrena globosa, a dicot from the Amaranthaceae family.

The F. trinervia C4 PEPc partially purified from E. coli was reported to be unphosphorylated at the N-terminal serine residue (Svensson ) and had a significantly lower KHCO3 than the desalted plant PEPc extracts taken from F. trinervia leaves during the day (Fig. 5). This suggests that potential differences in post-translational modifications might influence the kinetic properties of PEPc. Parvathi observed a decrease in KHCO3 as PEPc changed from the unphosphorylated to the phosphorylated state, and that PEPc extracts from illuminated leaves had lower KHCO3 values than PEPc extracted in the dark. In the current study, the phosphorylation status of the PEPc extracts was not tested, so it cannot be confirmed that the difference in KHCO3 between the plant and E. coli extracts is due to changes in PTMs. Alternatively, Bauwe (1986) observed that unpurified C4 PEPc had higher KHCO3 values than purified C4 PEPc extracts. It is possible that the impurity of our desalted plant PEPc extracts from F. trinervia contributed to the increased KHCO3 relative to the C4 PEPc purified from E. coli. The potential differences in PTMs and enzyme purity do not completely explain why the KHCO3 for the S. viridis PEPc reported here and by Boyd differ; however, this discrepancy in KHCO3 can be explained by differences in assay conditions and MIMS calibrations.

Raising the pH of the PEPc assay from 7.6 to 7.8, the pH used by Boyd , increased the S. viridis PEPc KHCO3 by ~21% (Fig. 5A, B). In addition to the pH of the assay buffer, the MIMS calibration method can alter the measured KHCO3. This is because two MIMS calibrations are required to convert a voltage signal of mass 44 to a micromolar concentration of CO2 and to determine the HCO3− concentration in the reaction cuvette. Since the development of a novel MIMS technique to measure KHCO3 of PEPc (Boyd ), we have improved the MIMS calibration method to obtain more accurate KHCO3 values to analyze kinetic differences between PEPc isoforms. The calibration method presented here differed from that of Boyd because all reaction components except the enzyme extract were included in the calibration. This would account for slight pH changes to the assay when adding DTT, G6-P, or PEP, which is important for determining the CO2:HCO3− ratio. If there is a slight reduction in pH from adding assay components that is not accounted for during the calibrations, then the CO2:HCO3− ratio can be slightly overestimated, leading to higher estimations of KHCO3. Using the calibration method and pH of 7.8 from Boyd , the KHCO3 values reported here (62.9 ± 8.7 µM) and by Boyd (62.8 ± 5.0 µM) were nearly identical (Fig. 5B), suggesting that assay conditions such as pH and differences in MIMS calibration methods can affect the estimated KHCO3. These findings also highlight the important consideration of how well in vitro assay conditions reflect the in vivo conditions where PEPc operates. So far, in vivo PEPc kinetics can only be obtained by models using gas exchange (von Caemmerer, 2000). Further research is needed to compare in vitro and in vivo PEPc kinetics, since accurate PEPc kinetics are needed to model C4 photosynthesis.

K HCO3 affects modeled rates of C4 photosynthesis

The C4 photosynthesis model developed by von Caemmerer (2000) was used to test if differences in KHCO3 between the C3 and C4 PEPc isoforms were enough to influence rates of net CO2 assimilation during C4 photosynthesis. The C4 model predicts that a lower KHCO3 may not affect photosynthetic rates under high CO2 partial pressures (Fig. 6). This is expected since C4 photosynthesis rates are typically not limited by PEPc under these conditions (von Caemmerer, 2000). However, a large KHCO3 may limit rates of C4 photosynthesis under low CO2 partial pressures (Fig. 6), for example when reduced stomatal conductance limits CO2 movement into the leaf. Flux control analysis found that PEPc has substantial control of C4 photosynthesis under low CO2 partial pressures (Dever ; Bailey ). Therefore, there is likely to be strong selective pressure to increase PEPc affinity for HCO3− in C4 plants to increase the amount of Ci entering C4 photosynthesis. Our results, combined with those of others (Bläsing ; Engelmann ; Endo ), show that as C4KHCO3 dropped, there was a concurrent increase in C4KPEP. Due to the higher PEP levels observed in leaves of C4 plants compared with C3 plants (Leegood and von Caemmerer, 1994), it can be argued that there was stronger selective pressure to increase PEPc affinity for HCO3− than to maintain high affinities for PEP since an increase in KPEP may not negatively impact C4 photosynthesis rates to the extent that changes in KHCO3 can under lower CO2 partial pressures (Fig. 6).

Conclusion

The direct comparison of closely related C3 and C4 PEPc isoforms from Flaveria demonstrates that the photosynthetic C4 PEPc isoform has a significantly higher affinity for HCO3− than its closely related C3 PEPc isoform. This reduced KHCO3 impacts net CO2 assimilation rates, particularly at low CO2 availability, suggesting selective pressure to reduce the C4KHCO3 to optimize inorganic carbon flux through C4 photosynthesis. Alternatively, the increase in KPEP can be seen as strengthening the diurnal regulation of C4 PEPc but residue S774 appears to link KHCO3 and KPEP, indicating that the increase in KPEP could be a negative consequence of reducing KHCO3. Testing different plant species will provide new insights into which amino acids control KHCO3 and provide a better understanding of the structure and function relationship of the enzyme. Obtaining a better understanding of what controls KHCO3 will also lead to enhancing C4 photosynthesis, particularly at low CO2 partial pressures when stomata are partially closed. This raises interesting questions of whether there is a range in KHCO3 across the diverse lineages of C4 plants and finding relationships between certain amino acid residues and ranges of KHCO3 values can be beneficial for promoting strategies to optimize C4 photosynthesis in crop species for drought conditions.

Supplementary data

Supplementary data are available at JXB online.

Table S1. Variable descriptions and values used to model C4 photosynthesis.

Table S2. Kinetic properties of PEPc isoforms from F. trinervia and F. pringlei.

Table S3. Kinetic properties of PEPc isoforms in the presence of 2.5 mM malate.

Fig. S1. Representative MIMS responses of the C3 and C4 PEPc activities with changing HCO3− concentrations.

Fig. S2. HCO3− response curves for the C3, C3-A774S, C4, and C4-S774A PEPc isoforms in the presence of 2.5 mM malate.

Click here for additional data file.