Although plants accumulate L-proline (Pro) in response to several abiotic stresses, external Pro supply results toxic and this toxicity is apparently mediated by accumulation of reactive oxygen species (ROS). Pro catabolism takes place in mitochondria involving two consecutive steps catalyzed by ProDH and P5CDH. We have observed that Pro catabolism is altered in Arabidopsis thaliana tissues developing the Hypersensitive Response (HR) triggered by avirulent races of Pseudomonas syringae. Induction of ProDH and repression of P5CDH occur in these tissues. We here evaluated if ProDH and P5CDH genes are regulated by SA/JA/ROS and analyzed the requirement of ProDH for HR development. We found that SRO5, a salt- and ROS-sensitive gene which down regulates P5CDH trough a SRO5-P5CDH natural siRNA, was activated in HR. SA and JA stimulate similar ProDH, P5CDH and SRO5 transcriptional changes than those induced by the avirulent pathogen. To analyze if ProDH affects ROS generation, siRNAProDH lines were constructed. ROS induced by the avirulent pathogen or by external Pro treatment were diminished in these transgenic plants. Our results suggest that ProDH-mediated Pro degradation may affect HR development by modulating ROS generation.