Mesophyll conductance (g ) is an important factor limiting rates of C photosynthesis. However, its role in C photosynthesis is poorly understood because it has been historically difficult to estimate. We use two methods to derive the temperature responses of g in C species. The first (Δ O) combines measurements of gas exchange with models and measurements of O discrimination. The second method (in vitro V ) derives g by retrofitting models of C photosynthesis and C discrimination with gas exchange, kinetic constants and in vitro V measurements. The two methods produced similar g for Setaria viridis and Zea mays. Additionally, we present the first temperature response (10-40°C) of C g in S. viridis, Z. mays and Miscanthus × giganteus. Values for g at 25°C ranged from 2.90 to 7.85 μmol m s Pa . Our study demonstrated that: the two described methods are suitable to calculate g in C species; g values in C are similar to high-end values reported for C species; and g increases with temperature analogous to reports for C species and the response is species specific. These results improve our mechanistic understanding of C photosynthesis.

ORCID IDs: 0000-0003-4009-7700 (R.A.B.); 0000-0001-7184-5113 (A.G.).The photosynthetic assimilation of CO 2 in C 4 plants is potentially limited by the enzymatic rates of Rubisco, phosphoenolpyruvate carboxylase (PEPc), and carbonic anhydrase (CA). Therefore, the activity and kinetic properties of these enzymes are needed to accurately parameterize C 4 biochemical models of leaf CO 2 exchange in response to changes in CO 2 availability and temperature. There are currently no published temperature responses of both Rubisco carboxylation and oxygenation kinetics from a C 4 plant, nor are there known measurements of the temperature dependency of the PEPc Michaelis-Menten constant for its substrate HCO 3 2 , and there is little information on the temperature response of plant CA activity. Here, we used membrane inlet mass spectrometry to measure the temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc carboxylation kinetics, and the activity and first-order rate constant for the CA hydration reaction from 10°C to 40°C using crude leaf extracts from the C 4 plant Setaria viridis. The temperature dependencies of Rubisco, PEPc, and CA kinetic parameters are provided. These findings describe a new method for the investigation of PEPc kinetics, suggest an HCO 3 2 limitation imposed by CA, and show similarities between the Rubisco temperature responses of previously measured C 3 species and the C 4 plant S. viridis.

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Optimization of Rubisco kinetics could improve photosynthetic efficiency, ultimatly 2 1 resulting in increased crop yield. However, imprecise knowledge of the reaction mechanism and 2 2 the individual rate constants limit our ability to optimize the enzyme. Membrane inlet mass Rubisco temperature response and relavance of individual rate constants of the reaction 3 3 mechanisms to potential breakpoints.

Summary Use of a complete dynamic model of NADP‐malic enzyme C4 photosynthesis indicated that, during transitions from dark or shade to high light, induction of the C4 pathway was more rapid than that of C3, resulting in a predicted transient increase in bundle‐sheath CO2 leakiness (ϕ). Previously, ϕ has been measured at steady state; here we developed a new method, coupling a tunable diode laser absorption spectroscope with a gas‐exchange system to track ϕ in sorghum and maize through the nonsteady‐state condition of photosynthetic induction. In both species, ϕ showed a transient increase to > 0.35 before declining to a steady state of 0.2 by 1500 s after illumination. Average ϕ was 60% higher than at steady state over the first 600 s of induction and 30% higher over the first 1500 s. The transient increase in ϕ, which was consistent with model prediction, indicated that capacity to assimilate CO2 into the C3 cycle in the bundle sheath failed to keep pace with the rate of dicarboxylate delivery by the C4 cycle. Because nonsteady‐state light conditions are the norm in field canopies, the results suggest that ϕ in these major crops in the field is significantly higher and energy conversion efficiency lower than previous measured values under steady‐state conditions.