Summary Stable carbon isotope ratios (δ13C) of terrestrial plants are employed across a diverse range of applications in environmental and plant sciences; however, the kind of information that is desired from the δ13C signal often differs. At the extremes, it ranges between purely environmental and purely biological. Here, we review environmental drivers of variation in carbon isotope discrimination (Δ) in terrestrial plants, and the biological processes that can either damp or amplify the response. For C3 plants, where Δ is primarily controlled by the ratio of intercellular to ambient CO2 concentrations (ci/ca), coordination between stomatal conductance and photosynthesis and leaf area adjustment tends to constrain the potential environmentally driven range of Δ. For C4 plants, variation in bundle‐sheath leakiness to CO2 can either damp or amplify the effects of ci/ca on Δ. For plants with crassulacean acid metabolism (CAM), Δ varies over a relatively large range as a function of the proportion of daytime to night‐time CO2 fixation. This range can be substantially broadened by environmental effects on Δ when carbon uptake takes place primarily during the day. The effective use of Δ across its full range of applications will require a holistic view of the interplay between environmental control and physiological modulation of the environmental signal.

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.

The efficiency of C4 photosynthesis in Zea mays, Miscanthus x giganteus and Flaveria bidentis in response to light was determined using measurements of gas exchange, 13 CO2 photosynthetic discrimination, metabolite pools and spectroscopic assays, with models of C4 photosynthesis and leaf 13 CO2 discrimination. Spectroscopic and metabolite assays suggested constant energy partitioning between the C4 and C3 cycles across photosynthetically active radiation (PAR). Leakiness (f), modelled using C4 light-limited photosynthesis equations (fmod), matched values from the isotope method without simplifications (fis) and increased slightly from high to low PAR in all species. However, simplifications of bundle-sheath [CO2] and respiratory fractionation lead to large overestimations of f at low PAR with the isotope method. These species used different strategies to maintain similar f. For example, Z. mays had large rates of the C4 cycle and low bundle-sheath cells CO2 conductance (gbs). While F. bidentis had larger gbs but lower respiration rates and M. giganteus had less C4 cycle capacity but low gbs, which resulted in similar f. This demonstrates that low gbs is important for efficient C4 photosynthesis but it is not the only factor determining f. Additionally, these C4 species are able to optimize photosynthesis and minimize f over a range of PARs, including low light.

Leakiness (Φ), the proportion of carbon fixed by phosphoenolpyruvate carboxylation that leaks out of the bundle-sheath cells, determines C(4) photosynthetic efficiency. Large increases in Φ have been described at low irradiance. The underlying mechanisms for this increase remain uncertain, but changes in photorespiration or the energy partitioning between the C(4) and C(3) cycles have been suggested. Additionally, values of Φ at low light could be magnified from assumptions made when comparing measured photosynthetic discrimination against (13)C (Δ) with the theoretical formulation for Δ. For example, several simplifications are often made when modelling Δ to predict Φ including: (i) negligible fractionation during photorespiration and dark respiration; (ii) infinite mesophyll conductance; and (iii) CO(2) inside bundle-sheath cells (C(s)) is much larger than values in mesophyll cells (C(m)). Theoretical models for C(4) photosynthesis and C(4) Δ were combined to evaluate how these simplifications affect calculations of Δ and Φ at different light intensities. It was demonstrated that the effects of photorespiratory fractionations and mesophyll conductance were negligible at low light. Respiratory fractionation was relevant only when the magnitude of the fractionation factor was artificially increased during measurements. The largest error in estimating Φ occurred when assuming C(s) was much larger than C(m) at low light levels, when bundle-sheath conductance was large (g(s)), or at low O(2) concentrations. Under these conditions, the simplified equation for Δ overestimated Φ, and compromised comparisons between species with different g(s), and comparisons across O(2) concentrations.