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.