Abstract
Mitogen-activated protein kinases (MAPKs/MPKs) are pivotal regulators in many stress-signaling pathways in plants. The dual phosphorylation of the TXY motif by MAP kinase kinases (MKKs) is essential for activating MAPKs. Here, we reveal a mechanism for MAPK activation that bypasses the need for MKKs. We identified rice (Oryza sativa) calcium-dependent protein kinase 5 (OsCPK5) and OsCPK13 as positive regulators in salt stress tolerance. These kinases are essential for the full activation of OsMPK3 and OsMPK6 in response to elevated sodium levels, with both OsMPK3 and OsMPK6 also acting as positive regulators in rice salt tolerance. Biochemical analysis demonstrated that OsCPK5/13 directly interact with and activate OsMPK3/6 by phosphorylating the TXY motif in vitro and in vivo. Additionally, we have discovered that OsCPK5/13 relocate from the cell membrane to the nucleus in response to salt stress. This process relies on their N-terminal myristoylation and a calcium-dependent phosphorylation event within the N-terminus. Our results elucidate a MAPK activation pathway in rice that is independent of traditional MKK-mediated phosphorylation, highlighting the crucial roles of OsCPK5 and OsCPK13 in directly phosphorylating and activating OsMPK3/6, which are important for rice tolerance to salt stress.