The Calcineurin B-Like Calcium Sensors CBL1 and CBL9 Together with Their Interacting Protein Kinase CIPK26 Regulate the Arabidopsis NADPH Oxidase RBOHF

This work provides evidence for the regulation of reactive oxygen species formation by a Ca-2-signaling network in Arabidopsis. In vivo and in vitro data indicate that the kinase CIPK26 upon interaction with the calcium sensors CBL1 or CBL9 enhances the activity of the NADPH oxidase RBOHF via phosphorylation.Stimulus-specific accumulation of second messengers like reactive oxygen species (ROS) and Ca-2 are central to many signaling and regulation processes in plants. However, mechanisms that govern the reciprocal interrelation of Ca-2 and ROS signaling are only beginning to emerge. NADPH oxidases of the respiratory burst oxidase homolog (RBOH) family are critical components contributing to the generation of ROS while Calcineurin B-like (CBL) Ca-2 sensor proteins together with their interacting kinases (CIPKs) have been shown to function in many Ca-2- signaling processes. In this study, we identify direct functional interactions between both signaling systems. We report that the CBL-interacting protein kinase CIPK26 specifically interacts with the N-terminal domain of RBOHF in yeast two-hybrid analyses and with the full-length RBOHF protein in plant cells. In addition, CIPK26 phosphorylates RBOHF in vitro and co-expression of either CBL1 or CBL9 with CIPK26 strongly enhances ROS production by RBOHF in HEK293T cells. Together, these findings identify a direct interconnection between CBLCIPK-mediated Ca-2 signaling and ROS signaling in plants and provide evidence for a synergistic activation of the NADPH oxidase RBOHF by direct Ca-2-binding to its EF-hands and Ca-2-induced phosphorylation by CBL1/9CIPK26 complexes.