Activation by fatty acids of the production of active oxygen species by tobacco cells

Among the different transduction steps leading from hypoosmotic stress to oxidative burst in suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells, phospholipase activation was evidenced. Using thin layer chromatography and phospholipase inhibitors the involved lipase was strongly suggested to be a phospholipase A2 (EC 3.1.1.4). Fatty acids like arachidonate and linolenate stimulated the oxidative response and prevented its inhibition by a phospholipase inhibitor, confirming the physiological relevance of the phospholipase action. A production of active oxygen species by plasma membrane vesicles was demonstrated, using two different probes. The producing system characterized in vitro was NADPH-dependent, strongly depressed by iodonium diphenyl and activated by fatty acids like the oxidative response assayed in vivo. Several other anionic amphiphiles like SDS were able to mimic the activation of the oxidative response by fatty acids, both in vivo and in vitro, suggesting that negative charges may be involved in the action mode of fatty acids. Inversely, a cationic detergent was an efficient inhibitor of the hypoosmotically induced oxidative burst and the inhibition was fully reversed by SDS. The possible identification of the active oxygen synthase involved in hypoosmotic signalling with a plasma membrane-located NADPH oxidase is discussed.