Melatonin is required for H2O2- and NO-mediated defense signaling through MAPKKK3 and OXI1 in Arabidopsis thaliana

Melatonin influences plant innate immunity through the mitogen-activated protein kinase (MAPK) pathway. However, the most upstream MAPK component in melatonin signaling and the dependence of generation of a reactive oxygen species (ROS) burst on melatonin synthesis and signaling remain unclear. In this study, treatment of several mekk (alias mapkkk)-knockout Arabidopsis mutants with melatonin revealed that the MAPKKK3 and OXI1 (oxidative signal-inducible1) kinases are responsible for triggering melatonin-induced defense signaling pathways. In addition, melatonin induction upon infection with the avirulent pathogen Pseudomonas syringae DC3000 (avrRpt2) was independent of H2O2 and NO individually, but dependent on the combination of H2O2 and NO. Moreover, melatonin-mediated induction of the expression of defense-related genes, such as PR1 and ICS1, was not altered in the H2O2-deficient rbohD/F-knockout mutant cotreated with an NO scavenger, indicating that melatonin functions downstream of the ROS and NO burst. Collectively, the data indicate that melatonin-mediated induction of an innate immune response requires multiple signaling molecules and activation of MAPKKK3 and OXI1, followed by triggering of downstream MAPK cascades, such as MAPK3 and MAPK6.