RT Journal Article SR Electronic T1 Where in the leaf is intercellular CO2 (Ci)? Considerations and recommendations for assessing gaseous diffusion in leaves JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.05.05.079053 DO 10.1101/2020.05.05.079053 A1 Joseph R. Stinziano A1 Jun Tominaga A1 David T. Hanson YR 2020 UL http://biorxiv.org/content/early/2020/05/07/2020.05.05.079053.abstract AB The assumptions that water vapor exchange occurs exclusively through stomata, that the intercellular airspace is fully saturated with water vapor, and that CO2 gradients are negligible between stomata and the intercellular airspace have enabled significant advancements in photosynthetic gas exchange research for nearly 60 years via calculation of intercellular CO2 (Ci). However, available evidence suggests that these assumptions may be overused. Here we review the literature surrounding evidence for and against the assumptions made by Moss & Rawlins (1963). We reinterpret data from the literature by propagating different rates of cuticular water loss, CO2 gradients, and unsaturation through the data. We find that in general, when cuticle conductance is less than 1% of stomatal conductance, the assumption that water vapor exchange occurs exclusively through stomata has a marginal effect on gas exchange calculations, but this is not true when cuticle conductance exceeds 5% of stomatal conductance. Our analyses further suggest that CO2 and water vapor gradients have stronger impacts at higher stomatal conductance, while cuticle conductance has a greater impact at lower stomatal conductance. Therefore, we recommend directly measuring Ci whenever possible, measuring apoplastic water potentials to estimate humidity inside the leaf, and exercising caution when interpreting data under conditions of high temperature and/or low stomatal conductance, and when a species is known to have high cuticular conductance.Highlight Leaf water vapor and CO2 exchange have been successfully used to model photosynthetic biochemistry. We review critical assumptions in these models and make recommendations about which need to be re-assessed.