Bundle-sheath leakiness in C4 photosynthesis: a careful balancing act between CO2 concentration and assimilation

Crop species with the C-4 photosynthetic pathway are generally characterized by high productivity, especially in environmental conditions favouring photorespiration. In comparison with the ancestral C-3 pathway, the biochemical and anatomical modifications of the C-4 pathway allow spatial separation of primary carbon acquisition in mesophyll cells and subsequent assimilation in bundle-sheath cells. The CO2-concentrating C-4 cycle has to operate in close coordination with CO2 reduction via the Calvin-Benson-Bassham (CBB) cycle in order to keep the C-4 pathway energetically efficient. The gradient in CO2 concentration between bundle-sheath and mesophyll cells facilitates diffusive leakage of CO2. This rate of bundle-sheath CO2 leakage relative to the rate of phosphoenolpyruvate carboxylation (termed leakiness) has been used to probe the balance between C-4 carbon acquisition and subsequent reduction as a result of environmental perturbations. When doing so, the correct choice of equations to derive leakiness from stable carbon isotope discrimination (Delta C-13) during gas exchange is critical to avoid biased results. Leakiness responses to photon flux density, either short-term (during measurements) or long-term (during growth and development), can have important implications for C-4 performance in understorey light conditions. However, recent reports show leakiness to be subject to considerable acclimation. Additionally, the recent discovery of two decarboxylating C-4 cycles operating in parallel in Zea mays suggests that flexibility in the transported C-4 acid and associated decarboxylase could also aid in maintaining C-4/CBB balance in a changing environment. In this paper, we review improvements in methodology to estimate leakiness, synthesize reports on bundle-sheath leakiness, discuss different interpretations, and highlight areas where future research is necessary.