Plant Physiology Preview. Published on September 15, 2015, as DOI:10.1104/pp.15.00586
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Temperature response of CA, PEPc, and Rubisco
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Asaph B. Cousins
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School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA
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1-509-335-7218
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[email protected]
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Biochemistry and Metabolism
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Temperature response of C4 photosynthesis: Biochemical analysis of Rubisco,
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Phosphoenolpyruvate Carboxylase and Carbonic Anhydrase in Setaria viridis.
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Ryan A. Boyd, Anthony Gandin, and Asaph B. Cousins
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School of Biological Sciences, Molecular Plant Sciences, Washington State University, Pullman,
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WA 99164-4236, USA
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The temperature response of carbonic anhydrase, phosphoenolpyruvate carboxylase, and
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Rubisco from the C4 plant Setaria viridis; implications for model C4 photosynthesis.
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Financial Source: This research was support by the Division of Chemical Sciences, Geosciences
and Biosciences, Office of Basic Energy Sciences, Photosynthetic Systems through DE-FG0209ER16062 and with instrumentation from an NSF Major Research Instrumentation grant
#0923562.
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Present addresses of authors:
Anthony Gandin
Université de Lorraine
Faculté des Sciences et Techniques
Ecologie et Ecophysiologie Forestières
F54506 Vandoeuvre Les Nancy, France
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Corresponding Author:
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Asaph B. Cousins
1-509-335-7218
[email protected]
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ABSTRACT
The photosynthetic assimilation of CO2 in C4 plants is potentially limited by the
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enzymatic rates of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco),
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phosphoenolpyruvate carboxylase (PEPc), and carbonic anhydrase (CA). Therefore, the activity
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and kinetic properties of these enzymes are needed to accurately parameterize C4 biochemical
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models of leaf CO2 exchange in response to changes in CO2 availability and temperature. There
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are currently no published temperature responses of both Rubisco carboxylation and oxygenation
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kinetics from a C4 plant, nor are there known measurements of the temperature dependency of
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the PEPc Michaelis-Menten constant for its substrate HCO3-, and there is little information on
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the temperature response of plant CA activity. Here we used membrane inlet mass spectrometry
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to measure the temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc
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carboxylation kinetics, and the activity and 1st order rate constant for the CA hydration reaction
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from 10 to 40 ᵒC using crude leaf extracts from the C4 plant Setaria viridis. The temperature
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dependencies of Rubisco, PEPc, and CA kinetic parameters are provided. These findings
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describe a new method for investigation of PEPc kinetics, suggest a HCO3- limitation imposed
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by CA, and show similarities between the Rubisco temperature response of previously measured
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C3 species and the C4 plant S. viridis.
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INTRODUCTION
Biochemical models of photosynthesis are often used to predict the effect of
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environmental conditions on net rates of leaf CO2 assimilation (Farquhar et al., 1980; von
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Caemmerer, 2000; von Caemmerer, 2013; Walker et al., 2013). With climate change, there is
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increased interest on modeling and understanding the effects of changes in temperature and CO2
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concentration on photosynthesis. The biochemical models of photosynthesis are primarily driven
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by the kinetic properties of the enzyme ribulose-1,5-bisphosphate (RuBP)
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carboxylase/oxygenase (Rubisco), the primary carboxylating enzyme of the C3 photosynthetic
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pathway catalyzing the reaction of RuBP with either CO2 or O2. However, the CO2 concentrating
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mechanism in C4 photosynthesis utilizes carbonic anhydrase (CA) to help maintain the chemical
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equilibrium of CO2 with HCO3- and phosphoenolpyruvate (PEP) carboxylase (PEPc) to catalyze
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the carboxylation of PEP with HCO3-. These reactions ultimately provide the elevated levels of
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CO2 to the compartmentalized Rubisco (Edwards and Walker, 1983). In C4 plants, it has been
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demonstrated that PEPc, Rubisco, and CA can limit rates of CO2 assimilation and influence the
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efficiency of the CO2 concentrating mechanism (von Caemmerer, 2000; von Caemmerer et al.,
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2004; Studer et al., 2014). Therefore, accurate modeling of leaf photosynthesis in C4 plants in
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response to future climatic conditions will require temperature parameterizations of Rubisco,
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PEPc, and CA kinetics from C4 species.
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Modeling C4 photosynthesis relies on parameterization of both PEPc and Rubisco
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kinetics making it more complex than for C3 photosynthesis (Berry and Farquhar, 1978; von
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Caemmerer, 2000). However, the activity of CA is not included in these models as it is assumed
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non-limiting under most conditions (Berry and Farquhar, 1978; von Caemmerer, 2000). This
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assumption is implemented by modeling PEPc kinetics as a function of CO2 partial pressure
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(pCO2) and not HCO3- concentration, assuming CO2 and HCO3- are in chemical equilibrium.
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However, there are questions regarding the amount of CA activity needed to sustain rates of C4
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photosynthesis and if CO2 and HCO3- are in equilibrium (von Caemmerer et al., 2004; Studer et
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al., 2014).
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The most common steady-state biochemical models of photosynthesis are derived from
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the Michaelis-Menten models of enzyme activity (von Caemmerer, 2000), which are driven by
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the maximum rate of reaction (Vmax) and the Michaelis-Menten constant (Km). Both of these
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parameters need to be further described by their temperature response to be used to model
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photosynthesis in response to temperature. However, the temperature response of plant CA
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activity has not been completed above 17 ᵒC and there is no known measured temperature
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response of Km HCO3- for PEPc (KP). Alternatively, Rubisco has been well studied and there are
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consistent differences in kinetic values between C3 and C4 species at 25 ᵒC (von Caemmerer and
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Quick, 2000; Kubien et al., 2008) but the temperature responses, including both carboxylation
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and oxygenation reactions, have only been performed in C3 species (Badger and Collatz, 1977;
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Jordan and Ogren, 1984; Bernacchi et al., 2001; Bernacchi et al., 2002; Walker et al., 2013).
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Here we present the temperature dependency of Rubisco carboxylation and oxygenation
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reactions, PEPc kinetics for HCO3-, and CA hydration from 10 to 40 ᵒC from the C4 species
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Setaria viridis (A-010) measured using membrane inlet mass spectrometry. Generally, the 25 ᵒC
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values of the Rubisco parameters were similar to previous measurements of C4 species. The
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temperature response of the maximum rate of Rubisco carboxylation (Vcmax) was high compared
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to most previous measurements from both C3 and C4 species, and the temperature response of the
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Michaelis constant for oxygenation (KO) was low compared to most previously measured
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species. Taken together, the modeled temperature response of Rubisco activity S. viridis were
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similar to the previously reported temperature responses of some C3 species. Additionally, the
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temperature response of the maximum rate of PEPc carboxylation (Vpmax) was similar to previous
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measurements. However, the temperature response of the Michaelis constant for HCO3- (KP) was
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lower than what has been predicted (Chen et al., 1994). For CA, deactivation of the hydration
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activity was observed above 25 ᵒC. Additionally, models of CA and PEPc show CA activity
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limits HCO3- availability to PEPc above 15 ᵒC, suggesting that CA limits PEP carboxylation
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rates in S. viridis when compared to the assumption that CO2 and HCO3- are in full chemical
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equilibrium.
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RESULTS
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Rubisco Temperature Response
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The maximum rate of Rubisco carboxylation (Vcmax) and oxygenation (Vomax), the
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Michaelis-Menten constants for carboxylation (KC) and oxygenation (KO), and the specificity of
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the enzyme for CO2 over O2 (SC/O) were measured simultaneously using membrane inlet mass
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spectrometry from 10 to 40 °C on crude leaf extracts of S. viridis (Table 1). The maximum
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turnover rate for carboxylation (kcatCO2) and oxygenation (kcatO2) were determined at 25 ᵒC using
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combined spectrophotometer and radiolabeled binding of the enzyme. At 25 ᵒC the values of
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kcatCO2, kcatO2, KC, and KO for S. viridis are within ranges previously measured for other C4 species
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(Yeoh et al., 1980; Sage and Seemann, 1993; Kubien et al., 2008). At 25 ᵒC the kcatCO2 was 5.44
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± 0.44 mol CO2 mol site-1 s-1, and the KC and KO were 94.7 ± 15.1 Pa and 28.9 ± 5.4 kPa,
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respectively. The measured Rubisco specificity for CO2 over O2 (SC/O) in S. viridis at 25 °C was
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1610 ± 66 Pa Pa-1. The ratio of the maximum carboxylation rate to the maximum oxygenation
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rate (Vcmax/Vomax) at 25 °C was 5.54 ± 0.73 and the ratio of the Michaelis-Menten constant for
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oxygenation to carboxylation (KO/KC) had a 25 °C value of 0.31 ± 0.04 kPa Pa-1.
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The Vcmax increased exponentially from 10 to 40 °C with an energy of activation (Ea) of
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78.0 ± 4.1 kJ mol-1(Fig. 1). The temperature response of Vomax was lower compared to Vcmax with
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an Ea equal to 55.3 ± 6.2 kJ mol-1 (Fig. 1). The KC and KO had Ea values of 64.2 ± 4.5 and 10.5 ±
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4.8 kJ mol-1, respectively (Fig. 1). The Vcmax/Vomax increased from 10 to 40 °C (Fig. 2) but the
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KO/KC decreased with temperature (Fig. 2). The ratio of catalytic rate of carboxylation to the
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Michaelis-Menten constant for CO2 (kcatCO2/KC) and oxygenation reactions (kcatO2/KO) increased
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with temperature (Fig. 2). The SC/O decreased with temperature from 10 to 40 °C with an Ea
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value of -31.1 ± 2.9 kJ mol-1 (Fig. 3).
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Rates of Rubisco carboxylation (vc) and oxygenation (vo) were modeled at pCO2 of 400
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Pa and pO2 of 35 kPa in response to temperature for kinetic parameters from S. viridis and
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previously published values normalized to S. viridis values at 25 ᵒC (Fig. 4). The temperature
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response of vc in S. viridis (Fig. 4) was similar to the in vitro measured A. glabriuscula (Badger
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and Collatz, 1977) and S. oleracea (Jordan and Ogren, 1984), but larger than the in vivo
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measured A. thaliana (Walker et al., 2013) and N. tabacum (Bernacchi et al., 2001; Bernacchi et
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al., 2002; Walker et al., 2013). The temperature response of vo in S. viridis was similar to
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previous measurements (Fig. 4). Additionally, the temperature dependency of S. viridis was
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compared to non-normalized temperature responses of vc and vo from previous measurements
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(Fig. S1). Under the low pCO2 (25 Pa) and ambient pO2 (21 kPa) expected in mesophyll of a C3
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leaf, vc was lower in S. viridis (Fig. S1) compared to the C3 species (Jordan and Ogren, 1984;
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Bernacchi et al., 2001; Bernacchi et al., 2002; Walker et al., 2013) with the exception of A.
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glabriuscula (Badger and Collatz, 1977). The S. viridis vo response (Fig. S1) was similar in all
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reports with the exception of A. glabriuscula (Badger and Collatz, 1977) that had a lower value
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above 35 ᵒC. At high pCO2 the predicted rates of vc for S. viridis (Fig. S1) were not different than
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S. oleracea (Jordan and Ogren, 1984), N. tabacum (Bernacchi et al., 2001; Bernacchi et al.,
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2002), and below 25 ᵒC A. thaliana (Walker et al., 2013). At high pCO2 conditions, S. viridis had
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a higher predicted vo (Fig. S1) at all temperatures compared to the C3 species (Badger and
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Collatz, 1977; Jordan and Ogren, 1984; Walker et al., 2013) with the exception of N. tabacum
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(Bernacchi et al., 2001; Bernacchi et al., 2002) above 25 ᵒC.
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Phosphoenolpyruvate Carboxylase Temperature Response
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The kinetic parameters of PEPc were measured using membrane inlet mass spectrometry
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on crude leaf extracts of S. viridis from 10 to 40 °C (Table 1; Fig. S2). The Vpmax at 25 ᵒC was
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450 ± 16 µmol m-2 s-1 when measured on freshly collected leaf tissue. The Vpmax increased with
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temperature from 10 to 40 °C, with an apparent deactivation at 35 and 40 ᵒC (Fig. 5), noted by a
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deviation from linearity when plotted as a log transformation (plot not shown). The measured
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PEPc Michaelis-Mentent constant for HCO3- (KP) was 60.2 µM HCO3- at 25 °C. The KP was
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calculated as 16.0 ± 1.3 Pa CO2 assuming a pH of 7.2 and a pKa of 6.12 appropriate for the
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mesophyll cytosol at 25 °C (Jenkins et al., 1989). Values of KP increased exponentially from 10
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to 40 °C with an Ea of 36.3 ± 2.4 kJ mol-1 when modeled in pCO2 assuming full equilibrium with
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HCO3- (Fig. 5). The temperature dependency of leaf PEPc activity (vp) modeled at a mesophyll
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cytosol pCO2 of 12 Pa at all temperatures increased with temperature but plateaued between 35
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and 40 °C (Fig. 5).
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Carbonic Anhydrase Temperature Response
The rate constant for carbonic anhydrase (CA) hydration activity (kCA) was determined
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from crude leaf extracts using membrane inlet mass spectrometry to measure rates of 18O
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exchange between labeled 13C18O2 and H216O (Table 1; Fig. S3). At 25 °C freshly collected leaf
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tissue of S. viridis had a kCA of 124 ±6 µmol CO2 m-2 s-1 Pa-1. The kCA increased from 10 to 30 °C
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but plateaued from 30 to 40 °C (Fig. 6). When CA activity was modeled for an expected
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mesophyll pCO2 of 12 Pa, the hydration rate (vh) was 1488 µmol m-2 s-1 at 25 °C (Fig. 6). The
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temperature change in vh was identical to kCA because the mesophyll pCO2 was assumed to be 12
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Pa at all temperatures. Alternatively, the temperature response of the uncatalyzed rate of CO2
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hydration in the leaf increased exponentially with temperature and is described by the rate
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constant (kc) having an Ea of 95.0 ± 1.0 kJ mol-1 (Fig. 6). The Ea for the uncatalyzed rate is much
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higher than the catalyzed; however, the uncatalyzed rate is at least 105 times lower from 10 to 40
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ᵒC (Fig. 6).
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The supply of HCO3- provided to PEPc by the hydration activity of CA was investigated
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using the model of Hatch and Burnell (1990) and the temperature responses reported here (Fig.
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7). At 12 Pa CO2 and pH 7.2, CA activity limits PEPc carboxylation above 15 °C by more than 5
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% compared to assuming full chemical equilibrium between CO2 and HCO3- (Fig. 7).
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Additionally, the modeled HCO3- concentration is lower at all temperatures compared to the
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concentration assuming HCO3- is in full equilibrium with CO2 (Fig. 7). The modeled vp/vh
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increased with temperature and is lower when vp is modeled based on HCO3- availability
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provided by CA activity compared to assuming CO2 and HCO3- are in chemical equilibrium (Fig.
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7). It should be noted that, the modeled limitation of CA to PEPc depends on temperatures, pH,
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and ionic strength as all of these conditions influence the ratio of CO2 to HCO3-.
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DISCUSSION
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Rubisco Temperature Response
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The C4 biochemical model of photosynthesis use both carboxylase and oxygenase
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parameters of Rubisco (von Caemmerer, 2000), therefore it is important to know the temperature
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dependency of both reactions in order to model C4 photosynthesis in response to temperature.
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Rubisco kinetic parameters have been measured in numerous C3 and C4 species at 25 ᵒC (Yeoh
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et al., 1980; Sage and Seemann, 1993; Kubien et al., 2008; Galmés et al., 2014); however, there
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are few studies that measure both Rubisco carboxylation and oxygenation parameters from C4
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species (Kubien et al., 2008; Cousins et al., 2010). The S. viridis 25 ᵒC values of Rubisco
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parameters are similar to previous measurements of C4 species. Notably the KC value is in the
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higher end of measured C4 species but similar to S. italica (Jordan and Ogren, 1983) and lower
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than S. geniculata (Yeoh et al., 1980). The SC/O was low compared to Flaveria C4 species and Z.
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mays (Kubien et al., 2008; Cousins et al., 2010), but larger than a previous measure of S. italica
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(Jordan and Ogren, 1983). However, the values reported here fit the tenuous understanding that
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C4 species have higher KC, kcatCO2 values and lower SC/O values compared to C3 species (Yeoh et
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al., 1980; Sage, 2002; Kubien et al., 2008; Savir et al., 2010; Whitney et al., 2011).
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Currently, there are no known temperature dependencies of the full suite of Rubisco
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kinetics for a C4 enzyme. However, for C3 plants there are two in vitro temperature responses of
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both carboxylase and oxygenase parameters (Atriplex glabriuscula and Spinacea oleracea) and
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three in vivo measurements from two species Arabidopsis thaliana and Nicotiana tabacum
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(Badger and Collatz, 1977; Jordan and Ogren, 1984; Bernacchi et al., 2001; Bernacchi et al.,
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2002; Walker et al., 2013). These C3 temperature responses are typically applied to C4 Rubisco
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25 °C values with the assumption that the temperature response is similar between these two
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photosynthetic functional types (Berry and Farquhar, 1978). However, this assumption has not
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been tested for the Rubisco parameters used in the C4 model of photosynthesis and the previous
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comparisons of C3 and C4 Rubisco temperature responses have been limited to kcatCO2 and SC/O
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(Björkman and Pearcy, 1970; Jordan and Ogren, 1984; Sage, 2002; Galmés et al., 2015), and a
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recent investigation comparing kcatCO2, KC, and SC/O between C3, C4, and intermediate species
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within the Flaveria lineage (Perdomo et al., 2015). However, these studies lack comparisons of
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the oxygenation parameters kcatO2 and KO needed to accurately predict the temperature response
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of carboxylation and oxygenation. Here we report the temperature response of complete C4
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Rubisco kinetics from 10 to 40 ᵒC. In general, the temperature responses of Rubisco parameters
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in S. viridis were similar to reports from previous C3 species; however, variation exists in how
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individual kinetic parameters change with temperature in relation to one another.
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For example, SC/O has been reported to decrease with temperature but the reason for the
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decrease has been debated (Badger and Andrews, 1974; Jordan and Ogren, 1984; Walker et al.,
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2013). However, there are no consistent trends in the literature on the temperature response of
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Vcmax/Vomax, suggesting the temperature responses of Vcmax and Vomax are nearly identical (Badger
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and Andrews, 1974; Jordan and Ogren, 1984; Bernacchi et al., 2001; Walker et al., 2013).
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Additionally, there appears to be very little Vcmax/KC temperature dependency (Fig. 2). The
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decrease of KO/KC and increase of Vomax/KO with temperature are consistent between studies (Fig.
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2; Badger and Collatz, 1977; Jordan and Ogren, 1984; Bernacchi et al., 2001; Bernacchi et al.,
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2002; Walker et al., 2013). Taken together, it is likely that the insensitivity of KO to temperature
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drives the decrease in specificity in Rubisco, because between species and measurement methods
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the temperature response of Vcmax, Vomax, and KC are similar and have a higher temperature
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response compared to KO (Badger and Collatz, 1977; Jordan and Ogren, 1984; Bernacchi et al.,
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2001; Bernacchi et al., 2002; Walker et al., 2013).
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For comparing the predicted vc and vo, the values were plotted in units of mol CO2 mol-1
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site s-1 so that a comparison of the predicted trade off of kcatCO2 and KC between C3 and C4
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species could be analyzed with temperature. Because no kcatCO2 values in these units are available
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for three of the previous four temperature response publications the in vitro value of 3.3 mol CO2
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mol-1 site s-1 measured by Walker et al. (2013) for both A. thaliana and N. tabacum was applied
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to all species except S. viridis. At low pCO2 the previously reported C3 Rubisco have higher vc
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compared to the S. viridis enzyme, with the exception of A. glabriuscula because the KC for A.
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glabriuscula at 25 ᵒC is three times larger than for other C3 enzymes included in this analysis.
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However, at high pCO2 the S. viridis Rubisco has a slightly higher vc compared to the C3
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enzymes. The minimal difference of vc observed between C3 and S. viridis Rubisco at high pCO2
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is likely the result of the high KC measured in S. viridis.
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The absolute values of kinetic parameters measured at 25 ᵒC for S. viridis are similar to
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previously published values from C4 plants, which generally differ from C3 parameters (Yeoh et
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al., 1980; von Caemmerer and Quick, 2000; Kubien et al., 2008; Savir et al., 2010; Whitney et
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al., 2011). However, there is less support in the literature for differences in the temperature
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response between C3 and C4 Rubisco (Jordan and Ogren, 1984; Sage, 2002; Galmés et al., 2015;
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Perdomo et al., 2015). The Rubisco temperature response of S. viridis presented here are similar
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to the available temperature responses of C3 species, suggesting a generally conserved variation
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in kinetic parameters. While the temperature response of Rubisco is becoming increasingly well
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studied less work has focused on PEPc kinetics, especially the temperature response of KP.
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PEPc Temperature Response
The temperature response of Vpmax presented here (Fig. 5) was similar to measurements
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from other species (Buchanan‐Bollig et al., 1984; Wu and Wedding, 1987; Chen et al., 1994;
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Massad et al., 2007), which showed a change in the temperature response above 25 ᵒC. While
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this change in the temperature response has been previously observed, the magnitude varies
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between studies and species (Buchanan‐Bollig et al., 1984; Wu and Wedding, 1987; Chen et al.,
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1994; Massad et al., 2007). It is unclear if there are differences in the temperature response of
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Vpmax between species, because there are not sufficient comparisons made within a single study.
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The KP is important for modeling the response of PEPc to changes in HCO3- availability.
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However, the temperature response of KP has been left out of models of C4 photosynthesis (Berry
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and Farquhar, 1978; von Caemmerer, 2000) or a predicted temperature response has been used
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(Chen et al., 1994; Massad et al., 2007). This is in contrast to the incorporation of measured
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Rubisco temperature responses that have been used in the C4 model (Berry and Farquhar, 1978).
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The temperature response of KP measured here was lower than a predicted temperature response
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(Fig. 5; Chen et al., 1994); however, it should be noted that the temperature response from Chen
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et al. (1994) was not actually measured but selected to have a Q10 of 2.1. Because the
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temperature dependency of Vpmax is similar between this study and Massad et al. (2007), the
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effect of differences in KP temperature response can be seen on modeled vp in Figure 5 where
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there is a 80 µmol m-2 s-1 increase in the optimum occurring 5 °C higher. This shift in modeled vp
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suggests that the use of the measured KP presented here is important for estimates of Vpmax values
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based on leaf gas exchange.
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Carbonic Anhydrase Temperature Response
CA activity is considered necessary for C4 photosynthesis because it provides PEPc with
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HCO3- by facilitating the hydration of dissolved CO2 in the mesophyll cytosol (von Caemmerer
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et al., 2004). Additionally, the activity of CA relative to PEPc activity (vp/vh) can influence the
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isotopic CO2 exchange in C4 plants (Farquhar, 1983). However, the activity of CA is not
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included in models of C4 photosynthesis and is generally thought to have little effect on 13CO2
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isotope exchange because vp/vh is assumed low (Berry and Farquhar, 1978; Farquhar, 1983; von
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Caemmerer, 2000). However, in some C4 grass species leaf CA hydration activity (vh) is
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reported to be just sufficient to maintain rates of C4 photosynthesis at 25 °C (Hatch and Burnell,
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1990; Cousins et al., 2008). Therefore, under conditions which limit leaf CO2 availability the rate
334
of vh leaf may restrict C4 photosynthesis and influence leaf CO2 isotope exchange. This
335
limitation may be particularly important at temperatures outside of 25 °C; however, the
336
temperature response of vh leaf is poorly understood.
337
The measured 25 °C values of vh leaf and the first order rate constant for CA hydration
338
activity (kCA) in S. viridis are four-fold higher than previously published values for other C4
339
grasses (Cousins et al., 2008). Published CA activity varies widely between studies, species,
340
tissue collection methods, and growth conditions (Hatch and Burnell, 1990; Gillon and Yakir,
341
2001; Affek et al., 2006; Cousins et al., 2008). This variation is poorly understood; however, it
342
raises questions regarding CA response to changes in growth conditions, particularly those that
343
limit CO2 availability to the leaf such as drought and temperature.
344
The measured temperature response of kCA from 10 to 25 °C was similar to a Q10 of 1.9
345
reported for Z. mays made from 0 to 17 °C in Z. mays and intermediate to two isozymes of
346
human CA measured from 0 to 37 °C, but deviated at 30 °C and above (Fig. 6A; Sanyal and
347
Maren, 1981; Burnell and Hatch, 1988). The CA temperature response reported here plateaued
348
between 25 and 40 °C, suggesting a deactivation of CA activity in S. viridis at temperatures
349
above 25 ᵒC (Fig. 1). This apparent deactivation was not observed in previous studies (Sanyal
350
and Maren, 1981; Burnell and Hatch, 1988).
351
352
As expected for a chemical reaction, the uncatalyzed rate of CO2 hydration increases
exponentially (Fig. 1). The activation energy (Ea) reported here for the uncatalyzed rate constant
23
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353
of CO2 hydration at pH 8.0 was larger than previously measured for pH 7.2 (Sanyal and Maren,
354
1981). Following the assumptions of Badger and Price (1994) regarding the volume of the
355
mesophyll cytoplasm per unit leaf area, the amount of the uncatalyzed CO2 hydration estimated
356
in the mesophyll cytoplasm was 105 times lower than the vh leaf. The importance of the
357
uncatalyzed rate when modeling isotopic discrimination is unclear and should be investigated
358
further, but the estimated uncatalyzed rates presented here are insufficient to support rates of
359
photosynthesis at any temperature (Fig. 6).
360
Using the steady state model of Hatch and Burnell (1990), HCO3- concentration and vp
361
were modeled using the temperature response of kCA, Vpmax and KP measured for S. viridis
362
assuming a constant mesophyll pCO2 of 12 Pa. This model was compared to the assumption of
363
current gas exchange models that assume HCO3- is at chemical equilibrium with CO2 in the
364
mesophyll cytosol (Fig. 7). The results show that CA limits vp by more than 5 % above 15 ᵒC
365
when compared to the full equilibrium model due to reduced HCO3- availability to PEPc (Fig. 7).
366
These results are contradictory to the assumptions of gas exchange models, which assume CA
367
activity is high enough to maintain full chemical equilibrium between CO2 and HCO3-, and may
368
have important implications for comparing models and measurements of C4 photosynthesis. For
369
example, in wild type Z. mays plants presented by Studer et al. (2014) there is an 80 % reduction
370
in in vivo compared to in vitro Vpmax. The in vivo value is calculated from the initial slope of net
371
CO2 assimilation against intercellular CO2 concentration (A-Ci curve); therefore, the lower Vpmax
372
calculated from an A-Ci curve is possibly driven by inaccurate estimates of the conductance of
373
CO2 from the intercellular air space to the mesophyll cytosol (gm) and the proposed
374
disequilibrium between CO2 and HCO3- due to limiting CA activity. Both of these factors would
375
lower the initial slope of an A-Ci curve and therefore in vivo estimates of Vpmax.
24
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
376
The CA limitation in wild type Z. mays is initially hard to reconcile with evidence from
377
CA mutants showing little change in net CO2 assimilation, except for homozygous mutants at
378
sub-ambient CO2 (Studer et al., 2014). However, using the model of Hatch and Burnell (1990)
379
combined with the PEPc limited models of net CO2 assimilation (von Caemmerer, 2000) and the
380
in vitro dataset presented by Studer et al. (2014) the predicted ‘apparent’ in vivo Vpmax in the wild
381
type, heterozygous and homozygous plants are 34, 33 and 13 μmol m-2 s-1, respectively. These
382
values are similar to the gas exchange estimates of in vivo Vpmax presented in Studer et al. (2014)
383
reanalyzed here using just the linear portion of the A-Ci curves as 36 ± 2, 34 ± 2, and 16 ± 1 for
384
the wild type, heterozygous and homozygous plants, respectively. This demonstrates that in
385
vitro data of CA and PEPc activities used with the Hatch and Burnell (1990) model and the PEPc
386
limited models of net CO2 assimilation (von Caemmerer, 2000) can accurately predict sublet
387
changes in leaf gas exchange measured in plants with low CA.
388
Although the difference between in vivo and in vitro PEPc can generally be predicted in
389
the example presented above there are still unknown factors that potentially regulate in vivo
390
PEPc activity. Of critical importance for estimating in vivo Vpmax are the values of gm; however,
391
there are no robust means for measuring gm in C4 plants, which also limits the accuracy of
392
defining a CA limitation to gas exchange measurements. Therefore, more work is needed to
393
define the importance of CA in C4 photosynthesis, especially in linking leaf level gas exchange
394
to biochemical models in order to better define carbon flux through the C4 pathway.
395
396
397
398
CONCLUSION
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate
carboxylase (PEPc) and carbonic anhydrase (CA) are potentially rate-limiting steps in the
25
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
399
photosynthetic assimilation of atmospheric CO2 in C4 plants. Therefore, the activity and kinetic
400
properties of these enzymes are needed to accurately parameterize biochemical models of leaf
401
CO2 exchange in response to changes in CO2 and temperature. To address this issue the
402
temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc carboxylation
403
kinetics, and CA hydration reaction from the C4 plants Setaria viridis (A-010) were analyzed
404
using a membrane inlet mass spectrometer. These findings suggest that the C4 Rubisco of S.
405
viridis has a similar temperature response to previously measured C3 Rubisco, the KP of PEPc
406
increases with temperature, and although modeling shows that CA limits HCO3- availability to
407
PEPc in S. viridis it also supports previous findings that large changes in CA activity have
408
minimal effect on net CO2 assimilation. These results advance our understanding of the
409
temperature response of CO2 assimilation in C4 plants and help to better parameterize the models
410
of C4 photosynthesis. However, more work is needed including leaf gas exchange and isotopic
411
measurements to determine the importance and implications of CA limitation to C4
412
photosynthesis.
413
414
MATERIALS AND METHODS
415
Growth Conditions
416
Seeds of Setaria viridis (A-010) were planted in 7.5 liter pots with LC-1 Sunshine mix
417
(Sun Gro Horticulture, Agawam, MA, USA). Plants were grown in environmental growth
418
chambers (Enconair Ecological GC-16) with a temperature of 28/18 ᵒC day/night, photoperiod of
419
16/8 hours day/night, a light intensity of 1000 µmol quanta m-2 s-1 at canopy level using 50% 400
420
watt high pressure sodium and 50% 400 watt metal halide lighting. Relative humidity was not
421
controlled and varied from 30 to 85% during the life of the plants as measured by the growth
26
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
422
chamber. Plants were watered as needed and fertilized twice a week using Peters 20-20-20 (J.R.
423
Peters, Inc., Allentown, PA, USA), and supplemented with Sprint 330 iron chelate (BASF
424
Corporation, Durham, NC, USA) as needed. Plants were grown for six weeks after germination
425
and sampled periodically between three to six weeks.
426
427
Sample Preparation
428
Carbonic Anhydrase Extraction
429
Leaf discs were extracted on ice in a glass homogenizer in 1 mL of 50 mM 4-(2-
430
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; pH 7.8), 1% polyvinylpolypyrrolidone
431
(PVPP), 1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM dithiothreitol (DTT), 0.1%
432
Triton X-100, and 2% protease inhibitor cocktail (P9599, Sigma-Aldrich). Crude extracts were
433
centrifuged at 4 °C for 1 min at 17,000 g, and the supernatant was collected for immediate use in
434
the CA assay.
435
436
437
Phosphoenolpyruvate Carboxylase Extraction
The mid-veins of youngest fully expanded leaves were removed and the remaining tissue
438
was cut into small pieces prior to grinding to a fine powder in liquid nitrogen using mortar and
439
pestle. The frozen powder was transferred to an ice-cooled mortar containing extraction buffer,
440
and ground on ice using a pestle until fully homogenized. Approximately 2 g of leaf tissue was
441
ground in 4 mL of 100 mM HEPES (pH 7.8), 10 mM DTT, 25 mM MgCl2, 1 mM EDTA, 10
442
mM NaHCO3, 1% PVPP, 0.5% Triton, 33 µL g -1 leaf tissue of protease inhibitor cocktail
443
(Sigma, St Louis, MO, USA) and sand. The extract was spun at 17,000 g for 10 minutes at 4 ᵒC.
444
The supernatant was collected and desalted using Econo Pac 10 DG column (BIO-RAD,
27
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
445
Hercules, CA, USA), filtered through Millex –GP 33mm syringe driven filter unit (Millipore,
446
Darmstadt, Germany), and then centrifuged using Amicon Ultra Ultracel 30K centrifugal filters
447
(Millipore, Darmstadt, Germany) at 4 ᵒC for an hour, at 4000 rpm (3000 g). The layer
448
maintained above the filter unit was collected, brought to 20% glycerol, flash frozen in liquid
449
nitrogen, and stored at -80 ᵒC until measured. Three individual plants were used, with one
450
extraction per plant.
451
452
453
Rubisco Extraction
Rubisco extraction followed that of PEPc except that most Rubisco activity was found to
454
be in the green layer of the pellet and not the supernatant following the first centrifugation. The
455
supernatant was discarded and the upper portion of the pellet was collected avoiding sand and
456
PVPP. The pellet was resuspended in extraction buffer and spun again at 4 °C, for 9 seconds, up
457
to 17,000 g to remove large particulates. The supernatant was collected and run through Econo
458
Pac 10DG desalting columns (BIO-RAD, Hercules, CA, USA) and the dark green fraction was
459
collected and centrifuged at 4 oC, for 10 minutes, at 17,000 g. Although no pellet formed, a
460
gradient in color from light green at the top of the tube, to dark green at the bottom was noted.
461
Most Rubisco activity was found to be in the bottom half of the sample. The upper light green
462
portion of the sample was discarded to concentrate Rubisco. The sample was brought to 20 %
463
glycerol, flash frozen in liquid nitrogen, and stored at -80 °C until measured. After being thawed
464
for use, the extract was allowed to activate in 15 mM MgCl2 and 15 mM NaHCO3 for 10 min at
465
room temperature before being placed on ice for the duration of the measurement. Measurements
466
for a single O2 level lasted approximately one hour and single aliquot was used per O2 level. A
467
new aliquot was thawed for each of the four O2 levels.
28
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
468
469
470
Extraction for Spectrophotometry
Rubisco activity, and PEPc activity at 25 °C were measured on freshly collected leaf
471
disks homogenized on ice in 1 mL of 100 mM HEPES (pH 7.8), 1% PVPP, 1 mM EDTA, 10
472
mM DTT, 0.1% Triton, and 1% protease inhibitor cocktail using a glass homogenizer. The
473
extract was then centrifuged at 17,000 g for 1 min at 4 °C and the supernatant was collected. The
474
supernatant was activated in 15 mM MgCl2 and 15 mM NaHCO3 for 10 min at room temperature
475
and placed on ice.
476
477
Membrane Inlet Mass Spectrometry
478
Carbonic Anhydrases Measurements
479
Carbonic anhydrase (CA) activity was measured using a membrane inlet mass
480
spectrometer (MIMS) to measure the rates of 18O2 exchange from labeled 13C18O2 to H216O with
481
a total carbon concentration of 1 mM (Silverman, 1982; Badger and Price, 1989; Hatch and
482
Burnell, 1990). The hydration rates were calculated from the enhancement in the rate of 18O loss
483
over the uncatalyzed rate with the non-enzymatic first order rate constant for the hydration of
484
CO2 calculated for the assay pH 8.03 at 25 ᵒC using the equation from Jenkins et al. (1989). The
485
measured temperature response was applied to the 25 ᵒC rate constant correcting for the change
486
in assay pH at each temperature (Fig. S3). The CO2 concentration was calculated using the
487
temperature appropriate pKa assuming an ionic strength of 0.1 M (Harned and Bonner, 1945),
488
and the partial pressure of CO2 was calculated using the temperature appropriate Henry’s
489
constant (Sander, 2015). The temperature response was determined on three biological replicates
490
separately extracted and measured three times (technical replication) from frozen leaf tissue at 5
29
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
491
ᵒC increments from 10 to 40 ᵒC. Total leaf CA activity at 25 ᵒC was determined from four
492
biological replicates measured on fresh tissue.
493
494
PEPc and Rubisco Measurements
495
PEPc and Rubisco assays were conducted in a 600 uL temperature controlled cuvette
496
linked to a mass spectrometer as described by Cousins et al. (2010). CO2 and O2 calibrations
497
were made daily at the measurement temperature as described by Cousins et al. (2010), with the
498
exception that no O2 zero or membrane consumption was determined because it was assumed to
499
be accounted for in the blank rate made immediately prior to measurement of the enzymatic rate.
500
To determine the O2 concentration dissolved in water during equilibrium with air, the
501
temperature dependency of the Henry’s constant for O2 was taken into account given the
502
measurement temperature. Because calibrations account for concentration of CO2 and total
503
inorganic carbon, the concentration of HCO3- was calculated as the difference between total
504
inorganic carbon and CO2.
505
Measurements of PEPc bicarbonate kinetics were similar to the method presented by
506
Cousins et al. (2010), except that only one O2 level (~80 µM) was used with five HCO3-
507
concentrations ranging from 0 µM to 3000 µM. The assay buffer contained 200 mM HEPES (pH
508
7.8, measured at 25 ᵒC), 20 mM MgCl2, 8 µg ml-1 CA, 5 mM glucose 6-phophate, and 4 mM
509
phosphoenolpyruvate (PEP). For all measurement points, except the lowest HCO3-, 30 seconds
510
were sampled prior to the initiation of the reaction as the blank rate; following a 30 second
511
mixing period after the injection of PEP to initiate the reaction, the next 30 seconds were
512
sampled as the enzymatic rate (Fig. 1). The blank rate was subtracted from the enzymatic rate.
513
For the lowest HCO3- concentration, the slope was found to change rapidly over the course of 30
30
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
514
seconds; therefore, the slope was calculated every five seconds during the 30 second interval.
515
Three biological replicates were measured at 5 ᵒC increments from 10 to 40 ᵒC.
516
Measurements of Rubisco kinetics were made following the methods of Cousins et al.
517
(2010). Samples were measured at four O2 concentrations ranging from 40 to 1600 µM, and five
518
CO2 concentrations ranging from 10 to 200 µM at each O2 level, for a total of 20 data points per
519
measurement. Measurements were made at 5 ᵒC intervals from 10 to 40 ᵒC, and four replicates
520
were measured per temperature. The assay buffer contained 200 mM HEPES, 20 mM MgCl2, 0.1
521
mM α-HPMS, 8 µg ml-1 CA, 0.6 mM RuBP. A total of 20 – 100 µL of extract was added per
522
measurement depending on the Rubisco concentration of the extract. One measurement initiating
523
the reaction with RuBP was made at each O2 concentration to test for any non-RuBP dependent
524
consumption of CO2 and O2. Non-RuBP dependent consumption of CO2 and O2 was considered
525
negligible because it did not have a significant effect on the final calculations of Rubisco kinetic
526
parameters.
527
528
For PEPc measurements, vp and HCO3- concentration ([HCO3-]) were fit to the equation:
=
[
[
]
(1)
]
529
solving for Vpmax and KP. For Rubisco measurements vc and vo were normalized by dividing all
530
measured values by their average to give uniform weight during the fitting calculation. The
531
normalized vc and vo, and corresponding CO2 concentration ([CO2]) and O2 concentration ([O2])
532
were fit simultaneously to the following equations:
533
=[
534
=[
[
]
(
[
]
(
]
⁄
)
]
⁄
)
535
solving for the parameters KC and KO. This was repeated using the non-transformed vc and vo
536
values while holding KC and KO constant and solving for Vcmax and Vomax. The normalizing of
(2)
(3)
31
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
537
data before solving for KC and KO improved the fits for vo. The KC and KO data obtained this way
538
matches the methods of plotting the apparent KC against [O2] (von Caemmerer et al., 1994). All
539
model fits were performed in the software package Origin 8 (OriginLab, Northampton, MA,
540
USA) using the non-linear curve fit function NLfit.
541
542
Enzyme Quantification
543
Rubisco Quantification
544
Rubisco activity was determined by enzyme coupled spectrophotometry, monitoring the
545
conversion rate of nicotinamide adenine dinucleotide (NADH) to NAD+ at 340 nm (Evolution
546
300 UV-Vis; Thermo Fisher Scientific,Waltham, MA, USA). The 1 mL assay contained 100 mM
547
4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS; pH 8 NaOH), 20 mM MgCl2, 1
548
mM EDTA, 1 mM Adenosine-5’ triphosphate(ATP), 5 mM creatine phosphate (CP), 20 mM
549
NaHCO3, 0.2 mM NADH, 12.5 units ml-1 creatine phosphokinase (CPK), 250 units ml-1 CA,
550
22.5 units ml-1 3-phosphoglyceric phosphokinase (PGK), 20 units ml-1,G3PDH, 56 units ml-1
551
TIM, 20 units ml-1 Glycerol-3PDH, and 10 uL of fresh leaf extract. The reaction was initiated
552
with 0.5 mM RuBP. Rubisco content was determined from the stoichiometric binding of
553
radiolabelled [14C] carboxy-arabinitolbisphosphate (14CABP; Collatz et al., 1979; Walker et al.,
554
2013). For 14CABP binding assays, the extract was incubated in 1 mM 14CABP for 45 minutes at
555
room temperature and then passed through a chromatography column (737–4731; Bio-Rad,
556
Hercules, CA, USA) packed with Sephadex G-50 fine beads (GE Healthcare Biosciences,
557
Pittsburgh, PA, USA). Rubisco content was determined from the Rubisco-bound fractions using
558
a scintillation counter and the specific activity of the 14CABP (14CABP; Collatz et al., 1979;
559
Walker et al., 2013).
32
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
560
The extract used to measure Rubisco kinetics was too viscous to move through the
561
columns used for 14CABP site quantification. To normalize the temperature response of Vcmax
562
and Vomax, from the MIMS measurements each extract was measured at 25 ᵒC for Vcmax using
563
enzyme coupled spectrophotometry. The 25 ᵒC Vcmax value was used to normalize rates obtained
564
from 10 to 40 ᵒC using MIMS. There was no significant difference between the 25 ᵒC values
565
measured on the spectrophotometer compared to MIMS.
566
567
568
PEPc Quantification
A similar enzyme coupled spectrophotometric assay as described above for Rubisco was
569
conducted for PEPc to determine the leaf level Vpmax. The 1mL assay contained 100 mM 4-(2-
570
Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS; pH 8 NaOH), 20 mM MgCl2, 1 mM
571
EDTA, 5 mM glucose 6-phosphate (G6P), 1 mM NaHCO3, 0.2 mM NADH, 12 units malate
572
dehydrogenase (MDH), and 10 uL of freshly collected leaf extract. The reaction was initiated
573
with 4 mM PEP.
574
575
576
577
Modeling the Temperature Response
The temperature response of the kinetic parameters kh, KP, Vcmax, Vomax, KC, KO, and SC/O
were fit to the equation:
=
578
(
.
)/(
.
)
(4)
579
where k25 is the value of the parameter at 25 ᵒC, Ea is the energy of activation, and R is the molar
580
gas constant (Badger and Collatz, 1977). The fit was calculated by taking the natural log of the
581
data plotted against
582
equal to ln(
(
.
)
using the linear function in Origin 8, such that the intercept was
) and the slope was equal to
/(298.15 ). The temperature response of kCA
33
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
583
and Vpmax were modeled to include a heat of deactivation (Hd) and entropy factor (ΔS) using the
584
equation (Farquhar et al., 1980; Leuning, 1997):
585
586
=
(
.
)/(
.
(
)
.
)⁄(
(
)⁄(
.
)
)
(5)
This model fitting was performed using the non-linear curve fit function NLfit in Origin 8.
587
588
589
Modeling the supply of HCO3- by CA
The model of Hatch and Burnell (1990) was used to calculate the CA limited model (Fig.
590
7). The rate of PEP carboxylation and HCO3- concentration was calculated by solving for the
591
following equations:
(6)
=
592
=
593
[
]
[
594
=
595
=
=
596
(7)
]
(8)
−
[
]
[
(9)
]
[
(10)
]
597
where vd is the rate of HCO3- dehydration catalyzed by CA, kr is the uncatalyzed rate constant for
598
the reverse reaction, kf is the uncatalyzed rate constant for the forward reaction, and kh is the
599
Henry’s Law constant used to determine the concentration of CO2 for any given pCO2 at the
600
appropriate temperature. The pCO2 was held constant at 12 Pa. The uncatalyzed rates of the
601
forward and reverse reactions at 25 ᵒC and pH 7.2 were calculated using the following equations
602
from Jenkins et al. (1989):
603
= 6.22 × 10
604
= 2 × 10
⁄[
] + 3.8 × 10
+ 4.96 × 10 [
]
(11)
(12)
34
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
605
where the temperature response of kf and kr measured by Sanyal and Maren (1981) for pH 7.2
606
was applied.
607
608
ACKNOWLEDGEMENTS
609
The authors would like to thank Murray Badger and Connor Bollinger-Smith for helping to
610
establish our MIMS protocol for Rubisco, Chuck Cody for maintaining plant growth facilities,
611
Susanne von Caemmerer and past and current members of Cousins Lab for helpful and insightful
612
discussions. Generous support to RAB was provided by a fellowship from the Seattle chapter of
613
Achievement Rewards for College Scientists (ARCS) Foundation.
614
615
616
617
618
619
35
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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