Abscisic acid stimulation of phospholipase D in the barley aleurone is G-protein-mediated and localized to the plasma membrane

We have previously determined that phospholipase D (PLD) is activated by ab scisic acid (ABA), and this activation is required for the ABA response of the cereal aleurone cell. In this study, ABA-stimulated PLD activity was re constituted in vitro in microsomal membranes prepared from aleurone protopl asts. The transient nature (20 min) and degree (1.5- to 2-fold) of activati on in vitro were similar to that measured in vivo. Stimulation by ABA was o nly apparent in the membrane fraction and was associated with a fraction en riched in plasma membrane. These results suggest that an ABA receptor syste m and elements licking it to PLD activation are associated with the aleuron e plasma membrane. The activation of PLD in vitro by ABA was dependent on t he presence of GTP. Addition of GTP gammaS transiently stimulated PLD in an ABA-independent manner, whereas treatment with GDP betaS or pertussis toxi n blocked the PLD activation by ABA. Application of pertussis toxin to inta ct aleurone protoplasts inhibited the ability of ABA to activate PLD as wel l as antagonizing the ability of ABA to down-regulate gibberellic acid-stim ulated alpha -amylase production. All of these data support the hypothesis that ABA stimulation of PLD activity occurs at the plasma membrane and is m ediated by G-protein activity.