Distinct Cellular Locations of Carbonic Anhydrases Mediate Carbon Dioxide Control of Stomatal Movements

Elevated carbon dioxide (CO2) in leaves closes stomatal apertures. Research has shown key functions of the beta-carbonic anhydrases (beta CA1 and beta CA4) in rapid CO2-induced stomatal movements by catalytic transmission of the CO2 signal in guard cells. However, the underlying mechanisms remain unclear, because initial studies indicate that these Arabidopsis (Arabidopsis thaliana) beta CAs are targeted to distinct intracellular compartments upon expression in tobacco (Nicotiana benthamiana) cells. Which cellular location of these enzymes plays a key role in native guard cells in CO2-regulated stomatal movements remains unknown. Here, we express fluorescently tagged CAs in guard cells of ca1ca4 double-mutant plants and show that the specific locations of beta CA4 at the plasma membrane and beta CA1 in native guard cell chloroplasts each can mediate rapid CO2 control of stomatal movements. Localization and complementation analyses using a mammalian alpha CAII-yellow fluorescent protein in guard cells further show that cytoplasmic localization is also sufficient to restore CO2 regulation of stomatal conductance. Mathematical modeling of cellular CO2 catalysis suggests that the dynamics of the intracellular HCO3- concentration change in guard cells can be driven by plasma membrane and cytoplasmic localizations of CAs but not as clearly by chloroplast targeting. Moreover, modeling supports the notion that the intracellular HCO3- concentration dynamics in guard cells are a key mechanism in mediating CO2-regulated stomatal movements but that an additional chloroplast role of CAs exists that has yet to be identified.