Enhanced Ca binding to EF-hands through phosphorylation of conserved serine residues activates MpRBOHB and chitin-triggered ROS production.

NADPH oxidases/RBOHs catalyze apoplastic ROS production and act as key signaling nodes, integrating multiple signal transduction pathways regulating plant development and stress responses. Although RBOHs have been suggested to be activated by Ca binding and phosphorylation by various protein kinases, a mechanism linking Ca binding and phosphorylation in the activity regulation remained elusive. Chitin-triggered ROS production required cytosolic Ca elevation and Ca binding to MpRBOHB in a liverwort Marchantia polymorpha. Heterologous expression analysis of truncated variants revealed that a segment of the N-terminal cytosolic region highly conserved among land plant RBOHs encompassing the two EF-hand motifs is essential for the activation of MpRBOHB. Within the conserved regulatory domain, we have identified two Ser residues whose phosphorylation is critical for the activation in planta. Isothermal titration calorimetry analyses revealed that phosphorylation of the two Ser residues increased the Ca binding affinity of MpRBOHB, while Ca binding is indispensable for the activation, even if the two Ser residues are phosphorylated. Our findings shed light on a mechanism through which phosphorylation potentiates the Ca -dependent activation of MpRBOHB, emphasizing the pivotal role of Ca binding in mediating the Ca and phosphorylation-driven activation of MpRBOHB, which is likely to represent a fundamental mechanism conserved among land plant RBOHs.