PYR/PYL/RCAR Abscisic Acid Receptors Regulate K+ and Cl− Channels through Reactive Oxygen Species-Mediated Activation of Ca2+ Channels at the Plasma Membrane of Intact Arabidopsis Guard Cells

The discovery of the START family of abscisic acid (ABA) receptors places these proteins at the front of a protein kinase/phosphatase signal cascade that promotes stomatal closure. The connection of these receptors to Ca(2+) signals evoked by ABA has proven more difficult to resolve, although it has been implicated by studies of the pyrbactin-insensitive pyr1/pyl1/pyl2/pyl4 quadruple mutant. One difficulty is that flux through plasma membrane Ca(2+) channels and Ca(2+) release from endomembrane stores coordinately elevate cytosolic free Ca(2+) concentration ([Ca(2+)]i) in guard cells, and both processes are facilitated by ABA. Here, we describe a method for recording Ca(2+) channels at the plasma membrane of intact guard cells of Arabidopsis (Arabidopsis thaliana). We have used this method to resolve the loss of ABA-evoked Ca(2+) channel activity at the plasma membrane in the pyr1/pyl1/pyl2/pyl4 mutant and show the consequent suppression of [Ca(2+)]i increases in vivo. The basal activity of Ca(2+) channels was not affected in the mutant; raising the concentration of Ca(2+) outside was sufficient to promote Ca(2+) entry, to inactivate current carried by inward-rectifying K(+) channels and to activate current carried by the anion channels, both of which are sensitive to [Ca(2+)]i elevations. However, the ABA-dependent increase in reactive oxygen species (ROS) was impaired. Adding the ROS hydrogen peroxide was sufficient to activate the Ca(2+) channels and trigger stomatal closure in the mutant. These results offer direct evidence of PYR/PYL/RCAR receptor coupling to the activation by ABA of plasma membrane Ca(2+) channels through ROS, thus affecting [Ca(2+)]i and its regulation of stomatal closure.