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

Crop species with the C₄ photosynthetic pathway are generally characterized by high productivity, especially in environmental conditions favouring photorespiration. In comparison with the ancestral C₃ pathway, the biochemical and anatomical modifications of the C₄ pathway allow spatial separation of primary carbon acquisition in mesophyll cells and subsequent assimilation in bundle-sheath cells. The CO ₂-concentrating C₄ cycle has to operate in close coordination with CO₂ reduction via the Calvin-Benson-Bassham (CBB) cycle in order to keep the C₄ 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 C₄ 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 (δ ¹³C) 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 C₄ 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 C₄ acid and associated decarboxylase could also aid in maintaining C₄/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.