2 DISTINCT SOURCES OF ELICITED REACTIVE OXYGEN SPECIES IN TOBACCO EPIDERMAL-CELLS

Reactive oxygen species (ROS) play a prominent role in early and later stages of the plant pathogenesis response, putatively acting as both cellular signaling molecules and direct antipathogen agents. A single- cell assay, based on the fluorescent probe dichlorofluorescein, was us ed to scrutinize the generation and movement of ROS in tobacco epiderm al tissue, ROS, generated within cells, quickly moved apoplastically a s H2O2 into neighboring cells. Two classes of rapidly elicited intrace llular ROS, originating from distinct sources, were distinguished. Cry ptogein, the fungal elicitor from Phytophthora cryptogea, induced ROS from a flavin-containing oxidase source. ROS accumulation could be inh ibited by a number of pharmacological agents, suggesting induction thr ough an active signal transduction pathway. The insensitivity of the i ncrease in ROS to the external addition of enzymes that dissipate ROS suggests that this oxidative increase is primarily intracellular. In c ontrast, amines and polyamines, compounds that form during wounding an d pathogenesis, induced ROS at an apoplastic site from peroxidase-or a mine oxidase-type enzyme(s). Salicylic acid, a putative inhibitor of c ellular catalases and peroxidases, did not induce cellular ROS, as mea sured by dichlorofluorescein fluorescence, The physiological relevance of ROS-generated signals was indicated by the rapid alteration of the epidermal cell glutathione pool and the cellular redox state. In addi tion, induction of ROS by all elicitors was correlated with subsequent cell death.