Characterization of ion channel modulator effects on ABA- and malate-induced stomatal movements: strong regulation by kinase and phosphatase inhibitors, and relative insensitivity to mastoparans

In the present study abscisic acid-induced stomatal closing, and malate effects on stomatal apertures were analysed in the presence of guard cell ion channel regulators. A recent study has suggested that abscisic acid (ABA) activation of protein kinases and/or inhibition of protein phosphatases may be central to activation of guard cell slow anion channels and mediation of stomatal closing in Vicia faba (Schmidt et al., 1995). These findings were confirmed and extended in the present study showing that both in Vicia faba and in Commelina communis ABA-induced stomatal closings were abolished by kinase inhibitors and enhanced by the protein phosphatase inhibitor okadaic acid. Further detailed studies demonstrate that very high 40 mM extracellular malate concentrations are required to close stomata only partially and that okadaic acid also enhances malate-induced stomatal closing. In addition, when stomata are widely opened, even at 40 mM malate concentrations, no malate effect on stomatal apertures was observed. This finding may be explained by a complete inactivation of guard cell anion channels when stomatal apertures are opened very widely and suggests that extracellular malate cannot function as a primary CO2 signal in stomatal regulation. The G-protein regulators mastoparan and mas7 as well as neomycin showed no significant effects on light-induced stomatal opening and ABA-induced stomatal closing. Findings reported here correlate closely to recent findings on slow anion channel regulation in guard cells and support the hypothesis that activation of these anion channels by phosphorylation events and complete inactivation by dephosphorylation events is a rate-limiting component in guard cell signal transduction. Furthermore, the presented data support a model in which ABA-activation of protein kineses and/or inhibition of okadaic acid-sensitive protein phosphatases is central to ABA regulation of stomatal movements in Vicia faba and Commelina communis.