Two 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 used to scrutinize the generation and movement of ROS in tobacco epidermal tissue. ROS, generated within cells, quickly moved apoplastically as H2O2 into neighboring cells. Two classes of rapidly elicited intracellular ROS, originating from distinct sources, were distinguished. Cryptogein, the fungal elicitor from Phytophthora cryptogea, induced ROS from a flavin-containing oxidase source. ROS accumulation could be inhibited by a number of pharmacological agents, suggesting induction through an active signal transduction pathway. The insensitivity of the increase in ROS to the external addition of enzymes that dissipate ROS suggests that this oxidative increase is primarily intracellular. In contrast, amines and polyamines, compounds that form during wounding and pathogenesis, induced ROS at an apoplastic site from peroxidase- or amine oxidase-type enzyme(s). Salicylic acid, a putative inhibitor of cellular catalases and peroxidases, did not induce cellular ROS, as measured 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 addition, induction of ROS by all elicitors was correlated with subsequent cell death.