High temperature promotes guard cell expansion, which opens stomatal pores to facilitate leaf cooling. How the hightemperature signal is perceived and transmitted to regulate stomatal aperture is, however, unknown. Here, we used a reverse-genetics approach to understand high temperature-mediated stomatal opening in Arabidopsis (Arabidopsis thaliana). Our findings reveal that high temperature-induced guard cell movement requires components involved in blue lightmediated stomatal opening, suggesting cross talk between light and temperature signaling pathways. The molecular players involved include phototropin photoreceptors, plasma membrane H 1-ATPases, and multiple members of the 14-3-3 protein family. We further show that phototropin-deficient mutants display impaired rosette evapotranspiration and leaf cooling at high temperatures. Blocking the interaction of 14-3-3 proteins with their client proteins severely impairs high temperatureinduced stomatal opening but has no effect on the induction of heat-sensitive guard cell transcripts, supporting the existence of an additional intracellular high-temperature response pathway in plants.

Our current understanding of guard cell signaling pathways is derived from studies in a small number of model species. The ability to study stomatal responses in isolated epidermis has been an important factor in elucidating the mechanisms by which the stomata of these species respond to environmental stresses. However, such approaches have rarely been applied to study guard cell signaling in the stomata of graminaceous species (including many of the world’s major crops), in which the guard cells have a markedly different morphology to those in other plants. Our understanding of guard cell signaling in these important species is therefore much more limited. Here, we describe a procedure for the isolation of abaxial epidermal peels from barley, wheat and Brachypodium distachyon. We show that isolated epidermis from these species contains viable guard cells that exhibit typical responses to abscisic acid (ABA) and CO2, as determined by measurements of stomatal apertures. We use the epidermal peel assay technique to investigate in more detail interactions between different environmental factors in barley guard cells, and demonstrate that stomatal closure in response to external CO2 is inhibited at higher temperatures, whilst sensitivity to ABA is enhanced at 30°C compared to 20 and 40°C.