ZmMPK5 is required for the NADPH oxidase-mediated self-propagation of apoplastic H₂O₂ in brassinosteroid-induced antioxidant defence in leaves of maize

Brassinosteroids (BRs) have been shown to induce hydrogen peroxide (H ₂O₂) accumulation, and BR-induced H₂O ₂ up-regulates antioxidant defence systems in plants. However, the mechanisms by which BR-induced H₂O₂ regulates antioxidant defence systems in plants remain to be determined. In the present study, the role of ZmMPK5, a mitogen-activated protein kinase, in BR-induced anitioxidant defence and the relationship between the activation of ZmMPK5 and H ₂O₂ production in BR signalling were investigated in leaves of maize (Zea mays) plants. BR treatment activated ZmMPK5, induced apoplastic and chloroplastic H₂O₂ accumulation, and enhanced the total activities of antioxidant enzymes. Such enhancements were blocked by pre-treatment with mitogen-activated protein kinase kinase (MAPKK) inhibitors and H₂O₂ inhibitors or scavengers. Pre-treatment with MAPKK inhibitors substantially arrested the BR-induced apoplastic H₂O₂ production after 6h of BR treatment, but did not affect the levels of apoplastic H₂O₂ within 1h of BR treatment. BR-induced gene expression of NADPH oxidase was also blocked by pre-treatment with MAPKK inhibitors and an apoplastic H₂O₂ inhibitor or scavenger after 120min of BR treatment, but was not affected within 30min of BR treatment. These results suggest that the BR-induced initial apoplastic H₂O₂ production activates ZmMPK5, which is involved in self-propagation of apoplastic H₂O₂ via regulation of NADPH oxidase gene expression in BR-induced antioxidant defence systems.