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

C₄ plants achieve higher photosynthesis (A_n) and intrinsic water use efficiency (iWUE) than C₃ plants, but processes underpinning the variability in A_n 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 A_n. We also developed a new formulation to estimate mesophyll conductance (g_m) based on actual hydration rates of CO₂ by carbonic anhydrases. We found a positive relationship between g_m and iWUE and an inverse correlation with g_sw 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 A_n, 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.