Rice B2 and B3 subgroup RAF kinases play central roles in both ABA and osmotic stress signaling

Protein kinases in the mitogen-activated protein kinase kinase kinase (MAPKKK) superfamily are known to be central actors in orchestrating cell differentiation, growth, and responses to extracellular stimuli in eukaryotes. However, the rice (Oryza sativa L.) MAPKKKs, especially the rapidly accelerated fibrosarcoma (RAF) subgroup ones, have rarely been studied yet. In this study, we generate high-order mutants of B1, B2, and B3 RAF subgroup MAPKKKs. The resulting raf-b2, raf-b3q, and raf-b2/3q mutants are hyposensitive to abscisic acid (ABA) but show dramatic sensitivity to various osmotic stresses. The rice B2 and B3 RAFs can phosphorylate stress-activated protein kinases (SAPKs), and in the rice raf-b2/3q, the ABA-triggered activation of SAPKs is largely impaired, and the expression of stress-response genes is decreased compared to that in the wild type. Our study reveals a crucial role of B2 and B3 subgroup RAFs in ABA signaling and osmotic stresses and sheds light on the functional divergence of the RAF-SAPK cascade in different plant species.