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

Flaveria trinervia C₄ phosphoenolpyruvate carboxylase (PEPc) has higher affinity for HCO₃⁻ than its closely related Flaveria pringlei C₃ PEPc, which drives higher modeled rates of C₄ photosynthesis under low CO₂ partial pressures.

Abstract

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 K_HCO3 (K_m for HCO₃⁻) to increase the carbon flux entering the C₄ cycle, particularly during conditions that limit CO₂ availability. However, the decrease in K_HCO3 has been hypothesized to cause an unavoidable increase in K_PEP (K_m for PEP). Therefore, the amino acid residue S774 in the C₄ enzyme, which has been shown to increase K_PEP, should lead to a decrease in K_HCO3. Several studies reported the effect S774 has on K_PEP; however, the influence of this amino acid substitution on K_HCO3 has not been tested. To test these hypotheses, membrane-inlet mass spectrometry (MIMS) was used to measure the K_HCO3 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 K_HCO3 and K_PEP 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 et al., 2007), seed development (Sangwan et al., 1992; O’Leary et al., 2011), 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 et al., 2002; 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 et al., 1975; 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 et al., 2007; Rosnow et al., 2014) and upstream promoter region (Schaffner and Sheen, 1992; Gowik et al., 2004). 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 et al., 2004). Work on PEPc peptide sequences from members of the Poaceae, Amaranthaceae, Asteraceae, and Cyperaceae families (Christin et al., 2007), as well as the Chenopodaceae family (Rosnow et al., 2014), 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 et al., 2007; Rosnow et al., 2014). 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 K_PEP (K_m 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 et al., 2002; Paulus et al., 2013). 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 et al., 2013).

As PEPc transitioned from C₃ to C₄ function, it has been suggested that certain amino acid substitutions were under positive selection to alter K_PEP and K_HCO3 (K_m for HCO₃⁻). It is hypothesized that certain mutations in the C₄ppc coding region resulted from strong selective pressures to obtain a lower K_HCO3 than that of the C₃ PEPc (Jacobs et al., 2008). The lower K_HCO3 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 K_PEP, with values typically reported between 100 µM and 590 µM (Svensson et al., 1997; Dong et al., 1998; Westhoff and Gowik, 2004; Lara et al., 2006; Rosnow et al., 2015), as compared with C₃ non-photosynthetic PEPc K_PEP values which range from 13 µM to 60 µM (Westhoff et al., 1997; Bläsing et al., 2002; Gowik et al., 2006; Lara et al., 2006; Rosnow et al., 2015). It was hypothesized that this increase in C₄K_PEP was an unavoidable consequence of the reduction of K_HCO3 since the two kinetic traits may be linked by certain amino acids (Jacobs et al., 2008; Gowik and Westhoff, 2011). Alternatively, since the PEP pools in a C₄ leaf are higher than in a C₃ leaf, the high K_PEP 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 et al. (1991) and Hermans and Westhoff (1992), and substituting the conserved C₄ serine for the conserved C₃ alanine in F. trinervia (S774A) significantly decreased the K_PEP of the C₄ PEPc (Bläsing et al., 2000; Engelmann et al., 2002). Since the S774A substitution affects the K_PEP of PEPc, it is possible that it may also affect the K_HCO3, making S774 one of the residues potentially linking K_PEP and K_HCO3. However, this serine residue was unimportant for the high K_PEP in the C₄ Chenopodaceae (Rosnow et al., 2014).

The only study to publish K_HCO3 of both a C₃ and C₄ PEPc showed that the K_HCO3 of five C₄ species representing the Poaceae and Amaranthaceae families was ~26 µM compared with preliminary evidence suggesting that the K_HCO3 of the C₃ PEPc from Flaveria cronquistii (Asteraceae family) was 80 µM (Bauwe, 1986). Other studies reported C₄ PEPc K_HCO3 values ranging from 14 µM to 180 µM (Janc et al., 1992; Gao and Woo, 1995; Parvathi et al., 2000; Boyd et al., 2015), where the C₃K_HCO3 of 80 µM falls within this reported range of C₄K_HCO3 values. However, comparing K_HCO3 for closely related C₃ and C₄ PEPc isoforms can provide a more accurate analysis of the change in C₃ to C₄K_HCO3 and whether there was a strong selective force on PEPc K_HCO3, but to date this has not been performed. Additionally, the previously reported K_HCO3 values were obtained by coupling PEPc activity to spectrophotometrically measured NADH oxidation rates. It is difficult to obtain accurate K_HCO3 values using this method because it does not directly measure changes in HCO₃⁻ concentration in the assay. This is problematic because measurements of K_HCO3 require accurate determinations of PEPc activity and HCO₃⁻ concentrations below the K_HCO3, 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 (C_i) concentrations, including concentrations well below the K_HCO3, without the complication of coupling PEPc activity to the NADH dehydrogenase reaction (Boyd et al., 2015).

In this study, we use MIMS to obtain K_HCO3 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 et al., 1984; Svensson et al., 1997). We found that the S774A substitution increases the C₄K_HCO3, whereas the A774S substitution did not affect the C₃K_HCO3, suggesting that additional amino acids besides S774 are involved in the C₄K_HCO3 trait. Since previous studies reported PEPc K_PEP changing in the presence of the allosteric activator glucose 6-phosphate (G6-P) and the inhibitor malate (Huber and Edwards, 1975; Wedding et al., 1990; Gupta et al., 1994; Bläsing et al., 2002), we tested whether these allosteric regulators also affected K_HCO3. We report that G6-P and malate have a minimal effect on the K_HCO3 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 K_HCO3 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 et al., 1984), and PEPc cDNA constructs used in Svensson et al. (1997) and Bläsing et al. (2000) 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 et al. (1997).

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 et al., 1997). 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 et al. (1997).

Fractions from the phenyl–Sepharose column were analyzed for PEPc activity by coupling the PEPc and NADH dehydrogenase reactions following the protocol of Boyd et al. (2015). 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 et al. (2015). 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 V_Pmax, K_HCO3, 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 et al. (2010). A CO₂ calibration was conducted before each HCO₃⁻ response curve as reported by Boyd et al. (2015). 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 et al. (2015) 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 C_i and total CO₂ in the cuvette, the HCO₃⁻ concentration could be deduced by subtracting the amount of CO₂ in the cuvette from the total C_i measured in the cuvette (Boyd et al., 2015).

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 (V_P) 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 C_i. Once the C_i 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 V_Pmax, K_HCO3, and Hill value (h) were obtained by using the Hill equation:

$$V_P=\frac{V_{Pmax} \times {\left[{\text{HCO}}_3^{-}\right]}^{\text{h}}}{{\left(K_{HCO3}\right)}^{\text{h}} + {\left[{\text{HCO}}_3^{-}\right]}^{\text{h}}}$$ (1)

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 K_HCO3

To measure the effect of G6-P on K_HCO3, G6-P was omitted from the assay described above to compare K_HCO3 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 K_HCO3 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 K_PEP values (2×K_PEP) in the absence of G6-P (Westhoff and Gowik, 2004).

Extraction source, pH, and calibration effects on PEPc K_HCO3

Previously, MIMS measurements of desalted PEPc extracts from S. viridis reported a K_HCO3 of 62.8 ± 5.0 µM (Boyd et al., 2015). Therefore, we tested whether differences in PEPc source (plant versus E. coli), pH, or MIMS calibrations caused the K_HCO3 reported here to differ from those of Boyd et al. (2015). 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 K_HCO3 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 K_HCO3 values obtained at pH 7.6, the pH used in this study, with K_HCO3 values obtained at pH 7.8 used by Boyd et al. (2015). These extracts were also used to compare K_HCO3 values obtained at pH 7.8 using either the current calibration method outlined above or the calibration method of Boyd et al. (2015).

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 K_HCO3 between PEPc isoforms. Two-way ANOVA (P<0.05) and Tukey HSD post-hoc tests were used to analyze statistically significant differences in K_HCO3 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 K_HCO3 assayed at pH 7.6 and 7.8 and for S. viridis PEPc K_HCO3 assayed at pH 7.8 using the two calibration methods.

Modeling the effect of K_HCO3 on C₄ photosynthesis

The modeled effect of K_HCO3 on the response of C₄ enzyme-limited photosynthetic CO₂ assimilation (A_c) to changing mesophyll CO₂ concentrations (C_m) 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 et al. (1994), von Caemmerer (2000), Tholen and Zhu (2011), and Ubierna et al. (2013), 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 (V_Pmax), the K_m for bicarbonate (K_HCO3), and the co-operativity of the PEPc active sites (h) from 25 °C MIMS measurements of PEPc activity (V_p) 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 K_HCO3 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 V_Pmax 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 K_HCO3 of the C₃, C₄, and chimeric PEPc isoforms. The K_HCO3 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 K_HCO3 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 V_Pmax (Supplementary Table S2). The reverse substitution, C₃-A774S made in the C₃ PEPc, did not significantly change the K_HCO3 (from 64.0 ± 2.4 µM to 61.5 ± 9.1 µM; Fig. 1) nor did it affect V_Pmax (Supplementary Table S2).

The impact of G6-P and malate on K_HCO3

The V_Pmax did not change by omitting G6-P from the assay, regardless of the PEPc isoform (Supplementary Table S2). Additionally, the K_HCO3 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 K_HCO3 of the different PEPc isoforms. The K_HCO3 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 K_HCO3 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 V_Pmax 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 K_HCO3 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, V_Pmax 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 K_HCO3 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 K_PEP of the C₃ and C₄ isoforms (Bläsing et al., 2002; Paulus et al., 2013), 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 K_HCO3 of different PEPc isoforms in the absence and presence of G6-P. (A) K_HCO3 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 K_HCO3 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 K_HCO3 by allosteric regulators and between different isoforms, but a Tukey HSD post-hoc test determined no significant allosteric effect on K_HCO3 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 K_PEP 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×K_PEP for each isoform.

Effects of MIMS calibrations, assay conditions, and extraction sources on K_HCO3

At pH 7.6, the C₄F. trinervia PEPc extracted from E. coli had a lower K_HCO3 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 K_HCO3 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 et al. (2015) MIMS calibration method at pH 7.8, the S. viridis K_HCO3 increased to 62.9 ± 8.7 µM (Fig. 5B).

Fig. 5.

The K_HCO3 measured under different assay conditions for PEPc isoforms extracted from F. trinervia, S. viridis, and E. coli. (A) The K_HCO3 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 K_HCO3 values measured from assays at pH 7.8 using either the current MIMS calibration or the Boyd et al. (2015) calibration. Significance between the K_HCO3 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 K_HCO3 of S. viridis PEPc assayed at pH 7.8 obtained by either the current calibration method or the Boyd et al. (2015) calibration method was determined by a Student’s t-test (P<0.05).

Modeling the effect of different K_HCO3 values on C₄ photosynthesis

The C₃ and C₄ PEPc K_HCO3 values from Fig. 1 were input into the C₄ photosynthesis model from von Caemmerer (2000) to determine how differences in K_HCO3 would impact modeled rates of C₄ photosynthesis. Varying K_HCO3 with a constant V_Pmax significantly changed the modeled rates of C₄ photosynthesis under CO₂ conditions below 20 Pa. The lower K_HCO3 of the C₄ PEPc resulted in higher modeled rates of C₄ photosynthesis at these low mesophyll CO₂ concentrations (C_m; Fig. 6). However, the difference between the C₃ and C₄K_HCO3 modeled no differences in net CO₂ assimilation above ~20 Pa C_m (Fig. 6).

Fig. 6.

Modeled rates of C₄ photosynthesis with C₃ and C₄K_HCO3. The K_HCO3 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 (A_net) at various mesophyll CO₂ concentrations (C_m). Symbols represent means ±SD of four independent K_HCO3 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 (V_pr), Rubisco carboxylation (V_Cmax), and maximum PEPc carboxylation per unit leaf area [V_Pmax(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 pK_a 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 et al., 2008; Gowik and Westhoff, 2011) have hypothesized that there was a strong selective pressure to reduce the K_HCO3 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 K_PEP and its allosteric regulation (Engelmann et al., 2002; Endo et al., 2008). Therefore, the aim of this research was to test the hypotheses that the K_HCO3 of the C₄ PEPc isoform from F. trinervia would be lower than the K_HCO3 of the C₃ PEPc isoform from F. pringlei and that changes to residue 774 will impact K_HCO3 and its allosteric regulation. Residue 774 was chosen because others have shown the C₄F. trinervia S774A substitution reduces K_PEP (Bläsing et al., 2000; Endo et al., 2008). Furthermore, residue 774 is near both the PEP- and HCO₃⁻-binding sites, and may also influence K_HCO3. We have analyzed the influence of this residue on K_HCO3 and showed that the C₄-S774A chimeric PEPc had a significantly higher K_HCO3 compared with the C₄ PEPc (Fig. 1). This fits with previous data that suggest that K_PEP and K_HCO3 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 K_HCO3 and a simultaneous increase to K_PEP (Gao and Woo, 1996). Alternatively, swapping Lys600 with either an arginine or threonine in F. trinervia led to increases in both K_PEP and K_HCO3, but this residue is one of the four conserved amino acids comprising the HCO₃⁻-binding site (Gao and Woo, 1995; Kai et al., 2003).

The chimeric C₃ PEPc of F. pringlei, C₃-A774S, had a minimal effect on K_HCO3 (Fig. 1). This same amino acid substitution was also shown not to influence the K_PEP of the C₃ PEPc with G6-P present in the assay. However, in the absence of G6-P, the same A774S substitution did increase K_PEP (Bläsing et al., 2000). 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 C₃K_PEP even closer to the established C₄K_PEP value (Engelmann et al., 2002). However, further research is needed to understand how changes in PEPc amino acid composition influence K_HCO3, particularly how specific amino acid changes influence the impact that allosteric regulators such as G6-P and malate have on K_HCO3.

The influence of G6-P and malate on K_HCO3

PEPc is regulated by post-translational modifications (PTMs) and interactions with allosteric effectors. Previous studies showed that glycine and G6-P activate PEPc (Svensson et al., 1997; Westhoff et al., 1997; Endo et al., 2008) 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 K_PEP, which can be altered by the binding of G6-P and malate to PEPc (Wedding et al., 1990; Bläsing et al., 2002; Rosnow et al., 2015). We show that neither G6-P nor malate appears to impact K_HCO3 and the co-operativity of HCO₃⁻ binding to the extent that they influence K_PEP and co-operative PEP binding (Figs 2, 3; Supplementary Tables S2, S3). Using the crystal structure (4BXC) deposited by Schlieper et al. (2014), 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 et al., 2013). 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 et al., 2003; Paulus et al., 2013).

Another possibility is that PEP binds before HCO₃⁻ (Janc et al., 1992), 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 K_HCO3. For example, G6-P has a greater activating effect on PEPc under limiting PEP concentrations at 0.5 mM (Gupta et al., 1994). In addition, multiple studies suggest that there is a regulatory PEP-binding site different from the PEPc active site (Rustin et al., 1988; Rodríguez-Sotres and Muñoz-Clares, 1990; Mújica-Jiménez et al., 1998; Yuan et al., 2006), 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 K_PEP 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 et al., 2002; Paulus et al., 2013). 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×K_PEP (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 et al., 1994). 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 C_i concentrations below the K_HCO3 of PEPc (Beckmann et al., 2009; Cousins et al., 2010). Previously, Boyd et al. (2015) reported a MIMS-measured K_HCO3 value of 62.8 µM for the S. viridis C₄ PEPc which is higher than our MIMS-measured K_HCO3 value of 26.6 µM for the F. trinervia C₄ PEPc extracted from E. coli. This difference in K_HCO3 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 K_HCO3 values at pH 7.6 (Fig. 5A). Bauwe (1986) also reported similar K_HCO3 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 et al., 1997) and had a significantly lower K_HCO3 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 et al. (2000) observed a decrease in K_HCO3 as PEPc changed from the unphosphorylated to the phosphorylated state, and that PEPc extracts from illuminated leaves had lower K_HCO3 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 K_HCO3 between the plant and E. coli extracts is due to changes in PTMs. Alternatively, Bauwe (1986) observed that unpurified C₄ PEPc had higher K_HCO3 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 K_HCO3 relative to the C₄ PEPc purified from E. coli. The potential differences in PTMs and enzyme purity do not completely explain why the K_HCO3 for the S. viridis PEPc reported here and by Boyd et al. (2015) differ; however, this discrepancy in K_HCO3 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 et al. (2015), increased the S. viridis PEPc K_HCO3 by ~21% (Fig. 5A, B). In addition to the pH of the assay buffer, the MIMS calibration method can alter the measured K_HCO3. 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 K_HCO3 of PEPc (Boyd et al., 2015), we have improved the MIMS calibration method to obtain more accurate K_HCO3 values to analyze kinetic differences between PEPc isoforms. The calibration method presented here differed from that of Boyd et al. (2015) 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 K_HCO3. Using the calibration method and pH of 7.8 from Boyd et al. (2015), the K_HCO3 values reported here (62.9 ± 8.7 µM) and by Boyd et al. (2015) (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 K_HCO3. 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 K_HCO3 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 K_HCO3 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 K_HCO3 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 et al., 1997; Bailey et al., 2000). Therefore, there is likely to be strong selective pressure to increase PEPc affinity for HCO₃⁻ in C₄ plants to increase the amount of C_i entering C₄ photosynthesis. Our results, combined with those of others (Bläsing et al., 2000; Engelmann et al., 2002; Endo et al., 2008), show that as C₄K_HCO3 dropped, there was a concurrent increase in C₄K_PEP. 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 K_PEP may not negatively impact C₄ photosynthesis rates to the extent that changes in K_HCO3 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 K_HCO3 impacts net CO₂ assimilation rates, particularly at low CO₂ availability, suggesting selective pressure to reduce the C₄K_HCO3 to optimize inorganic carbon flux through C₄ photosynthesis. Alternatively, the increase in K_PEP can be seen as strengthening the diurnal regulation of C₄ PEPc but residue S774 appears to link K_HCO3 and K_PEP, indicating that the increase in K_PEP could be a negative consequence of reducing K_HCO3. Testing different plant species will provide new insights into which amino acids control K_HCO3 and provide a better understanding of the structure and function relationship of the enzyme. Obtaining a better understanding of what controls K_HCO3 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 K_HCO3 across the diverse lineages of C₄ plants and finding relationships between certain amino acid residues and ranges of K_HCO3 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.

Glossary

Abbreviations

CA

carbonic anhydrase

C_i

inorganic carbon

C_m

mesophyll CO₂ concentration

G6-P

glucose 6-phosphate

h

Hill value

HCO₃⁻

bicarbonate

K_HCO3

K _m for HCO₃⁻

K_PEP

K _m for PEP

MIMS

membrane-inlet mass spectrometry

OAA

oxaloacetate

PEG

polyethylene glycol

PEP

phosphoenolpyruvate

PEPc

phosphoenolpyruvate carboxylase

PTM

post-translational modification

V_P

PEPc activity

V_Pmax

maximum rate of PEPc carboxylation

V_Pmax(plant)

maximum rate of PEPc carboxylation per unit leaf area.