Hydrogen Peroxide Stimulates Salicylic Acid Biosynthesis in Tobacco

Hydrogen peroxide induced the accumulation of free benzoic acid (BA) and salicylic acid (SA) in tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. Six hours after infiltration with 300 mM H2O2, the levels of BA and SA in leaves increased 5-fold over the levels detected in control leaves. The accumulation of BA and SA was preceded by the rapid activation of benzoic acid 2-hydroxylase (BA2H) in the H2O2-infiltrated tissues. This enzyme catalyzes the formation of SA from BA. Enzyme activation could be reproduced in vitro by addition of H2O2 or cumene hydroperoxide to the assay mixture. H2O2 was most effective in vitro when applied at 6 mM. In vitro activation of BA2H by peroxides was inhibited by the catalase inhibitor 3-amino-1,2,4-triazole. We suggest that H2O2 activates SA biosynthesis via two mechanisms. First, H2O2 stimulates BA2H activity directly or via the formation of its substrate, molecular oxygen, in a catalase-mediated reaction. Second, higher BA levels induce the accumulation of BA2H protein in the cells and provide more substrate for this enzyme.