Activation of phospholipase D and the possible mechanism of activation in wound-induced lipid hydrolysis in castor bean leaves

Hydrolysis of membrane lipids has been suggested to provide messengers mediating defense gene expression in the wound signaling process. It is, however, unknown which lipolytic enzyme is involved in the signaling pathway. This study investigated the temporal and spatial activation of phospholipase D (PLD; EC 3.1.4.4) and the possible activation mechanism in response to wounding in castor bean (Ricinus communis L.) leaves. Wounding triggered a rapid activation of PLD-mediated phospholipid hydrolysis, as indicated by the in vivo increase in phosphatidic acid and free choline, at not only the site of wounding but also the undamaged area of wounded leaves. RNA blotting analysis indicated that PLD gene expression was not involved in the early phase of wounding-activation of PLD. Measurements of PLD by activity assay and immunoblotting suggest that the wounding-activation of PLD at unwounded cells results from intracellular translocation of PLD from cytosol to membranes, A similar translocation pattern of PLD was also obtained as a function of increased free calcium at physiological concentrations in a homogenization buffer, Based on the above results, it is proposed that wounding induces activation of PLD leading to phospholipid hydrolysis, and that the activation results from translocation of PLD to membranes, which is mediated by an increase in cytoplasmic calcium upon wounding.