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Phospholipase A2s (PLA2s) are enzymes that liberate lysophospholipids and free fatty acids (FFAs) from membrane phospholipids in response to hormones and other external stimuli. This report describes the cloning and functional characterization of a PLA2 cDNA from Arabidopsis thaliana, AtsPLA2-alpha, which represents one of four secretory PLA2 (sPLA2) genes in Arabidopsis. The encoded protein is 148-amino acid polypeptide and is predicted to contain a 20-amino acid signal peptide at its amino terminus. The predicted mature form (Mr=14,169) of AtsPLA2-alpha exhibited approximately 5 times the specific activity of its pre-processed form. Different from animal sPLA2s, AtsPLA2-alpha showed a significant preference for the acyl group linoleic acid over palmitic acid in phospholipid hydrolysis. Like some animal sPLA2s, however, it has a slight preference for phosphatidylethanolamine over phosphatidylcholine as the substrate. The specific activity of AtsPLA2-alpha continuously increased as the Ca2+ concentration was increased to 10 mM, and the optimal pH range was very broad and biphasic between 6 and 11. AtsPLA2-alpha transcript was detected at low levels in roots, stems, leaves, and flowers but not in siliques.

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 bear (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.