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Breeding economically important C 4 crops for enhanced whole-plant water-use efficiency (WUE plant ) is needed for sustainable agriculture. WUE plant is a complex trait and an efficient phenotyping method that reports on components of WUE plant , such as intrinsic water-use efficiency (WUE i , the rate of leaf CO 2 assimilation relative to water loss via stomatal conductance), is needed. In C 4 plants, theoretical models suggest that leaf carbon isotope composition (δ 13 C), when the efficiency of the CO 2 -concentrating mechanism (leakiness, ϕ) remains constant, can be used to screen for WUE i . The limited information about how ϕ responds to water limitations confines the application of δ 13 C for WUE i screening of C 4 crops. The current research aimed to test the response of ϕ to short- or long-term moderate water limitations, and the relationship of δ 13 C with WUE i and WUE plant , by addressing potential mesophyll CO 2 conductance (g m ) and biochemical limitations in the C 4 plant Sorghum bicolor. We demonstrate that g m and ϕ are not responsive to short- or long-term water limitations. Additionally, δ 13 C was not correlated with gas-exchange estimates of WUE i under short- and long-term water limitations, but showed a significant negative relationship with WUE plant . The observed association between the δ 13 C and WUE plant suggests an intrinsic link of δ 13 C with WUE i in this C 4 plant, and can potentially be used as a screening tool for WUE plant in sorghum., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

The conductance of carbon dioxide (CO 2 ) from the substomatal cavities to the initial sites of CO 2 fixation (g m ) can significantly reduce the availability of CO 2 for photosynthesis. There have been many recent reviews on: (i) the importance of g m for accurately modelling net rates of CO 2 assimilation, (ii) on how leaf biochemical and anatomical factors influence g m , (iii) the technical limitation of estimating g m , which cannot be directly measured, and (iv) how g m responds to long- and short-term changes in growth and measurement environmental conditions. Therefore, this review will highlight these previous publications but will attempt not to repeat what has already been published. We will instead initially focus on the recent developments on the two-resistance model of g m that describe the potential of photorespiratory and respiratory CO 2 released within the mitochondria to diffuse directly into both the chloroplast and the cytosol. Subsequently, we summarize recent developments in the three-dimensional (3-D) reaction-diffusion models and 3-D image analysis that are providing new insights into how the complex structure and organization of the leaf influences g m . Finally, because most of the reviews and literature on g m have traditionally focused on C 3 plants we review in the final sections some of the recent developments, current understanding and measurement techniques of g m in C 4 and crassulacean acid metabolism (CAM) plants. These plants have both specialized leaf anatomy and either a spatially or temporally separated CO 2 concentrating mechanisms (C 4 and CAM, respectively) that influence how we interpret and estimate g m compared with a C 3 plants., (© 2020 The Authors The Plant Journal © 2020 John Wiley & Sons Ltd.)