MAP65-1a positively regulates H₂O₂ amplification and enhances brassinosteroid-induced antioxidant defence in maize

Brassinosteroid (BR)-induced antioxidant defence has been shown to enhance stress tolerance. In this study, the role of the maize 65kDa microtubule-associated protein (MAP65), ZmMAP65-1a, in BR-induced antioxidant defence was investigated. Treatment with BR increased the expression of ZmMAP65-1a in maize (Zea mays) leaves and mesophyll protoplasts. Transient expression and RNA interference silencing of ZmMAP65-1a in mesophyll protoplasts further revealed that ZmMAP65-1a is required for the BR-induced increase in expression and activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX). Both exogenous and BR-induced endogenous H₂O₂ increased the expression of ZmMAP65-1a. Conversely, transient expression of ZmMAP65-1a in maize mesophyll protoplasts enhanced BR-induced H ₂O₂ accumulation, while transient silencing of ZmMAP65-1a blocked the BR-induced expression of NADPH oxidase genes and inhibited BR-induced H₂O₂ accumulation. Inhibiting the activity and gene expression of ZmMPK5 significantly prevented the BR-induced expression of ZmMAP65-1a. Likewise, transient expression of ZmMPK5 enhanced BR-induced activities of the antioxidant defence enzymes SOD and APX in a ZmMAP65-1a-dependent manner. ZmMPK5 directly interacted with ZmMAP65-1a in vivo and phosphorylated ZmMAP65-1a in vitro. These results suggest that BR-induced antioxidant defence in maize operates through the interaction of ZmMPK5 with ZmMAP65-1a. Furthermore, ZmMAP65-1a functions in H₂O₂ self-propagation via regulation of the expression of NADPH oxidase genes in BR signalling.