Plants subjected to wounding stress produce secondary metabolites. Several of these metabolites prevent chronic diseases and can be used as colorants, flavors, and as antimicrobials. This wound-induced production of plant secondary metabolites is mediated by signaling-molecules such as reactive oxygen species (ROS), ethylene (ET) and jasmonic acid (JA). However, their specific role and interactions that modulate the wound-respond in plants is not fully understood. In the present study, a subtractive cDNA library was generated, to better understand the global response of plants to wounding stress. Carrot (Daucus carota) was used as a model system for this study. A total of 335 unique expressed sequence tags (ESTs) sequences were obtained. ESTs sequences with a putative identity showed involvement in stress-signaling pathways as well as on the primary and secondary metabolism. Inhibitors of ROS biosynthesis, ET action, and JA biosynthesis alone and in combination were applied to wounded-carrots in order to determine, based on relative gene expression data, the regulatory role of ET, JA, and ROS on the wound-response in plants. Our results demonstrate that ROS play a key role as signaling-molecules for the wound-induced activation of the primary and secondary metabolism whereas ET and JA are essential to modulate ROS levels.