Loss of conductance between mesophyll symplasm and intercellular air spaces explains nonstomatal control of transpiration.

The conventional assumption is that stomatal conductance ([Formula: see text]) dominates the regulation of water and carbon dioxide fluxes between leaves and the atmosphere. Here, a nanoreporter of water status at the mesophyll cell surface and local xylem within intact maize leaves documents significant undersaturation of water vapor in the outside-xylem zone (OXZ) and a large loss of conductance of this zone ([Formula: see text]) at moderate xylem water stress, without stomatal closure or turgor loss. The ratio of the resistances [Formula: see text] serves as a predictive phenotype of undersaturation, nonstomatal regulation of transpiration, errors in standard gas exchange analysis, and an increase of intrinsic water use efficiency ([Formula: see text]). Cell-scale access to water status reveals symplasmic-apoplasmic disequilibrium and informs a biophysical model that can explain experimental observations quantitatively based on localization of variable conductance to the plasma membrane. This work opens paths of inquiry into the molecular basis and functional consequences of nonstomatal regulation of transpiration.