The origin of nitric oxide (,NO) in plants is unclear and an 'NO synthase (NOS)-like enzyme and nitrate reductase (NR) are claimed as potential sources. Here we used wild-type and NR-defective double mutant plants to investigate 'NO production in Arabidopsis thaliana in response to Pseudomonas syringae pv maculicola. NOS activity increased substantially in leaves inoculated with P. syringae. However, electron paramagnetic resonance experiments showed a much higher 'NO formation that was dependent on nitrite and mitochondrial electron transport rather than on arginine or nitrate. Overall, these results indicate that NOS, NR and a mitochondrial-dependent nitrite-reducing activity cooperate to produce 'NO during A. thaliana-P. syringae interaction. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.5791738143820