PURIFICATION AND CHARACTERIZATION OF ELICITOR-INDUCED LIPOXYGENASE IN TOBACCO CELLS

Lipoxygenase activity was induced in a tobacco cell suspension culture by treatment with glycopeptide elicitors prepared from the cell walls of Phytophthora parasitica var. nicotianae, and in tobacco seedlings infected by this fungal pathogen. Upon purification and characterization, the enzyme appeared to have a molecular weight of 96 000, a pI of 5.1 and a K(m) of 20.9 muM with linoleic acid as substrate. According to its acidic optimum pH, it belongs to type-2 lipoxygenases. Using linoleic, linolenic and arachidonic acids as substrates, the products formed in vitro by lipoxygenase were characterized. 9- and 5-hydroperoxides were the main products obtained from the C18 and C20 fatty acids, respectively, thereby indicating that a 5-lipoxygenase accounts for most of the elicitor-induced activity, since the main site of insertion of molecular oxygen is on C-5 of arachidonic acid. Small amounts of 13-hydroperoxides were also formed from the C18 fatty acids. In vitro, the strongest inhibitors of tobacco lipoxygenase were n-propylgallate and nordihydroguaiaretic acid. The possible involvement of this enzyme in signaling phenomena leading to defense induction in plants via jasmonic acid and other fatty acid-derived products is discussed.