Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants

Abstract

Despite the recognition of H₂O₂ as a central signaling molecule in stress and wounding responses, pathogen defense, and regulation of cell cycle and cell death, little is known about how the H₂O₂ signal is perceived and transduced in plant cells. We report here that H₂O₂ is a potent activator of mitogen-activated protein kinases (MAPKs) in Arabidopsis leaf cells. Using epitope tagging and a protoplast transient expression assay, we show that H₂O₂ can activate a specific Arabidopsis mitogen-activated protein kinase kinase kinase, ANP1, which initiates a phosphorylation cascade involving two stress MAPKs, AtMPK3 and AtMPK6. Constitutively active ANP1 mimics the H₂O₂ effect and initiates the MAPK cascade that induces specific stress-responsive genes, but it blocks the action of auxin, a plant mitogen and growth hormone. The latter observation provides a molecular link between oxidative stress and auxin signal transduction. Finally, we show that transgenic tobacco plants that express a constitutively active tobacco ANP1 orthologue, NPK1, display enhanced tolerance to multiple environmental stress conditions without activating previously described drought, cold, and abscisic acid signaling pathways. Thus, manipulation of key regulators of an oxidative stress signaling pathway, such as ANP1/NPK1, provides a strategy for engineering multiple stress tolerance that may greatly benefit agriculture.