Temperature response of mesophyll conductance in three C 4 species calculated with two methods: 18 O discrimination and in vitro V pmax

Summary Mesophyll conductance ( g m ) is an important factor limiting rates of C 3 photosynthesis. However, its role in C 4 photosynthesis is poorly understood because it has been historically difficult to estimate. We use two methods to derive the temperature responses of g m in C 4 species. The first (Δ 18 O) combines measurements of gas exchange with models and measurements of 18 O discrimination. The second method ( in vitro V pmax ) derives g m by retrofitting models of C 4 photosynthesis and 13 C discrimination with gas exchange, kinetic constants and in vitro V pmax measurements. The two methods produced similar g m for Setaria viridis and Zea mays . Additionally, we present the first temperature response (10–40°C) of C 4 g m in S. viridis , Z. mays and Miscanthus  ×  giganteus . Values for g m at 25°C ranged from 2.90 to 7.85 μmol m −2  s −1  Pa −1 . Our study demonstrated that: the two described methods are suitable to calculate g m in C 4 species; g m values in C 4 are similar to high‐end values reported for C 3 species; and g m increases with temperature analogous to reports for C 3 species and the response is species specific. These results improve our mechanistic understanding of C 4 photosynthesis.