Synergistic Activation of the Arabidopsis NADPH Oxidase AtrbohD by Ca²⁺ and Phosphorylation*

Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91^phox) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91^phox/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca²⁺. However, the significance of Ca²⁺ binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca²⁺ ionophore that induces Ca²⁺ influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca²⁺ binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca²⁺ binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.