Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells

Elevations in cytoplasmic calcium ([Ca2+](cyt)) are an important component of early abscisic acid (ABA) signal transduction. To determine whether defi ned mutations in ABA signal transduction affect [Ca2+](cyt) signaling, the Ca2+-sensitive fluorescent dye fura 2 was loaded into the cytoplasm of Arab idopsis guard cells. Oscillations in [Ca2+](cyt) could be induced when the external calcium concentration was increased, showing viable Ca2+ homeostas is in these dye-loaded cells. ABA-induced [Ca2+](cyt) elevations in wild-ty pe stomata were either transient or sustained, with a mean increase of simi lar to 300 nM, Interestingly, ABA-induced [Ca2+](cyt) increases were signif icantly reduced but not abolished in guard cells of the ABA-insensitive pro tein phosphatase mutants abi1 and abi2. Plasma membrane slow anion currents were activated in wild-type, abi1, and abi2 guard cell protoplasts by incr easing [Ca2+](cyt), demonstrating that the impairment in ABA activation of anion currents in the abi1 and abi2 mutants was bypassed by increasing [Ca2 +](cyt). Furthermore, increases in external calcium alone (which elevate [C a2+](cyt)) resulted in stomatal closing to the same extent in the abil and abi2 mutants as in the wild type. Conversely, stomatal opening assays indic ated different interactions of abil and abi2, with Ca2+-dependent signal tr ansduction pathways controlling stomatal closing versus stomatal opening. T ogether, [Ca2+](cyt) recordings, anion current activation, and stomatal clo sing assays demonstrate that the abil and abi2 mutations impair early ABA s ignaling events in guard cells upstream or close to ABA-induced [Ca2+](cyt) elevations. These results further demonstrate that the mutations can be by passed during anion channel activation and stomatal closing by experimental elevation of [Ca2+](cyt).