This study investigates the functional analysis of oxidative stress-activated mitogen-activated protein kinase (MAPK) cascade in plants. The authors found that hydrogen peroxide (H2O2) is a potent activator of MAPKs in Arabidopsis leaf cells. Using epitope tagging and protoplast transient expression assays, they demonstrated that H2O2 can activate a specific Arabidopsis MAPKK, 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. This observation provides a molecular link between oxidative stress and auxin signal transduction. Additionally, transgenic tobacco plants expressing 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. These findings suggest that manipulation of key regulators of an oxidative stress signaling pathway, such as ANP/NPK1, can provide a strategy for engineering multiple stress tolerance, which may benefit agriculture.This study investigates the functional analysis of oxidative stress-activated mitogen-activated protein kinase (MAPK) cascade in plants. The authors found that hydrogen peroxide (H2O2) is a potent activator of MAPKs in Arabidopsis leaf cells. Using epitope tagging and protoplast transient expression assays, they demonstrated that H2O2 can activate a specific Arabidopsis MAPKK, 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. This observation provides a molecular link between oxidative stress and auxin signal transduction. Additionally, transgenic tobacco plants expressing 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. These findings suggest that manipulation of key regulators of an oxidative stress signaling pathway, such as ANP/NPK1, can provide a strategy for engineering multiple stress tolerance, which may benefit agriculture.