Early wound-related changes in the leaf apoplast proteome of Medicago, truncatula have been characterized by 2-DE and MALDI-TOF/TOF and the differential expression of 28/110 extracellular proteins could be reproducibly observed 6 h after wounding. Wounding induced an initial (0-30 min) burst of O-2(-), followed by a later (3-6 h) production Of O-2(-) and H2O2. The infiltration of 5 mu M DPI <= 3 min after wounding inhibited both phases of the oxidative burst and suppressed wound-regulated changes in 9/28 extracellular proteins. DPI infiltrated 15 min after wounding only partially inhibited early O-2(-) production and was ineffective in suppressing wound-related changes in these proteins. This strongly suggests that in wounded Medicago, rapid O-2(-) is required for mobilizing the downstream (3-6 h), differential expression of several extracellular proteins. Further studies with DPI and exogenous sources of ROS supported the regulation of these proteins within early, wound-related ROS-signaling events. The study forms the basis for associating wound-related changes in the apoplast proteome with ROS-dependent and ROS-independent pathways. Proteins mobilized within the ROS-dependent pathway were largely ionically bound to cell walls and included SODs, peroxidases and germin-like proteins, suggesting their involvement within wound-activated, ROS regulatory loops.