The role of leaf width and conductances to CO₂ in determining water use efficiency in C₄ grasses

C₄ plants achieve higher photosynthesis (An) and intrinsic water use efficiency (iWUE) than C₃ plants, but processes underpinning the variability in An and iWUE across the three C₄ subtypes remain unclear, partly because we lack an integrated framework for quantifying the contribution of diffusional and biochemical limitations to C₄ photosynthesis. We exploited the natural diversity among C₄ grasses to develop an original mathematical approach for estimating eight key processes of C₄ photosynthesis and their relative limitations to An. We also developed a new formulation to estimate mesophyll conductance (gm) based on actual hydration rates of CO₂ by carbonic anhydrases. We found a positive relationship between gm and iWUE and an inverse correlation with gsw among C₄ grasses. We also revealed subtype-specific regulatory processes of iWUE that may be related to known anatomical traits characterising each C₄ subtype. Leaf width was an important determinant of iWUE and showed significant correlations with key limitations of An, especially among NADP-ME species. In conclusion, incorporating leaf width in breeding trials may unlock new opportunities for C₄ crops because the revealed negative relationship between leaf width and iWUE may translate into higher crop and canopy WUE.This research was funded by the Australian Research Council Centre of Excellence for Translational Photosynthesis (CE1401000015) awarded to OG and which funded FJC and RES. ABC was supported by the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences, Department of Energy (grant no. DE-SC0001685)