Combining timelapse imaging resolved leaf water potential with gas exchange provides insights into stomatal control by water.

Stomatal responses to changes in leaf water status are amongst the most important responses of guard cells for balancing carbon gain with water loss, yet we do not know if guard cells are hydraulically linked to the water status of the leaf across vascular land plants. We combined time-lapsing imaging of leaf tissue in the cuvette of a gas analyzer to provide a dynamic measurement of leaf water potential (Ψl) while simultaneously recording leaf gas exchange to test these questions. Using this method in a representative lycophyte Selaginella moellendorfii we show that stomata respond passively to changes in Ψl by closing as soon as Ψl declines and opening rapidly on rehydration, confirming a close hydraulic connection between guard cells and the leaf in lycophytes. In contrast, in a representative angiosperm Solanum americanum, wrong-way stomatal responses occurred when leaves experienced rapid changes in Ψl. In this species wrong-way responses no longer occurred once Ψl had declined to turgor loss point, at which point stomata will rapidly reopen on rehydration without a wrong-way response. These observations confirm a close hydraulic connection between the guard cell cytosol and leaf apoplast across vascular land plants and rule out a hypothesis that there is a rapid and transient rehydration of leaf tissue from embolized xylem on leaf excision, as an explanation for wrong-way stomatal opening. The ability to simultaneously record leaf gas exchange and Ψl provides a powerful tool for resolving outstanding questions related to the hydraulic regulation of stomatal aperture.