ZmMPK5 phosphorylates ZmNAC49 to enhance oxidative stress tolerance in maize.

Mitogen-activated protein kinase (MPK) is a critical regulator of the antioxidant defence system in response to various stimuli. However, how MPK directly and exactly regulates antioxidant enzyme activities is still unclear. Here, we demonstrated that a NAC transcription factor ZmNAC49 mediated the regulation of antioxidant enzyme activities by ZmMPK5. ZmNAC49 expression is induced by oxidative stress. ZmNAC49 enhances oxidative stress tolerance in maize, and it also reduces superoxide anion generation and increases superoxide dismutase (SOD) activity. A detailed study showed that ZmMPK5 directly interacts with and phosphorylates ZmNAC49 in vitro and in vivo. ZmMPK5 directly phosphorylates Thr-26 in NAC subdomain A of ZmNAC49. Mutation at Thr-26 of ZmNAC49 does not affect the interaction with ZmMPK5 and its subcellular localisation. Further analysis found that ZmNAC49 activates the ZmSOD3 expression by directly binding to its promoter. ZmMPK5-mediated ZmNAC49 phosphorylation improves its ability to bind to the ZmSOD3 promoter. Thr-26 of ZmNAC49 is essential for its transcriptional activity. In addition, ZmSOD3 enhances oxidative stress tolerance in maize. Our results show that phosphorylation of Thr-26 in ZmNAC49 by ZmMPK5 increased its DNA-binding activity to the ZmSOD3 promoter, enhanced SOD activity and thereby improved oxidative stress tolerance in maize.

The overexpression of a maize mitogen-activated protein kinase gene (ZmMPK5) confers salt stress tolerance and induces defence responses in tobacco.

As sessile organisms, plants are exposed to potential dangers, including multiple biotic and abiotic stresses. The mitogen-activated protein kinase (MAPK) is a universal signalling pathways involved in these processes. A previous study showed that maize ZmMPK5 is induced by various stimuli at transcriptional and post-translational levels. In this study, ZmMPK5 was overexpressed in tobacco to further analyse its biological functions. Under salt and oxidative stresses, ZmMPK5-overexpressing lines displayed less severe damage and stronger growth phenotypes corresponding to a series of physiological changes. In addition, the transgenic lines accumulated less reactive oxygen species (ROS) and had higher levels of antioxidant enzyme activity and metabolites than wild-type (WT) plants following NaCl treatment. Quantitative RT-PCR revealed that the expression of ROS-related and stress-responsive genes was higher in transgenic plants than in WT plants. Furthermore, transgenic lines exhibited enhanced resistance to viral pathogens, and expressed constitutively higher transcript levels of pathogenesis-related genes, such as PR1a, PR4, PR5 and EREBP. Taken together, these results demonstrated that ZmMPK5 is involved in salt stress, oxidative stress and pathogen defence signalling pathways, and its function may be at least partly devoted to efficiently eliminating ROS accumulation under salt stress.

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 65 kDa 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 H2O2 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.