Activation of a rice ROS-producing enzyme OsrbohB by Ca²⁺ binding and phosphorylation.

Abstract

Reactive oxygen species (ROS) produced by plant NADPH oxidases, encoded by rboh (respiratory burst oxidase homolog) genes, have been shown to play important roles in stress signaling and development. However, regulatory mechanisms of ROS production by OsrbohB are still largely unknown. We recently showed that heterologous expression in HEK293T cells is useful to characterize the ROS-producing activity and regulatory mechanisms of plant rboh (Ogasawara et al. 2008). OsrbohB has been shown to play a crucial role in disease resistance in rice (Wong et al. 2007). OsrbohB expressed in HEK293T cells showed a small basal ROS-producing activity, which was activated by a Ca²⁺ ionophore, ionomycin. Site-directed mutagenesis revealed that binding of Ca²⁺ to the first EF hand motif is critical for activation by Ca²⁺. The ionomycin-triggered ROS production was dramatically enhanced by a protein phosphatase inhibitor, calyculin A, suggesting that the binding of Ca²⁺ and phosphorylation synergistically activate the ROS-producing activity of OsrbohB. Results using various site-directed mutants will be presented and possible regulatory mechanisms of the ROS-producing enzyme will be discussed.