The CaM1-associated CCaMK–MKK1/6 cascade positively affects lateral root growth via auxin signaling under salt stress in rice

Ca2+/calmodulin (CaM)-dependent protein kinases (CCaMKs) and mitogen-activated protein kinase kinases (MAPKKs) are two types of kinases that regulate salt stress response in plants. It remains unclear, however, how they cooperatively affect lateral root growth under salt stress. Here, two conserved phosphorylation sites (S102 and T118) of OsCaM1 were identified, and found to affect the ability to bind to Ca2+in vitro and the kinase activity of OsCCaMK in vivo. OsCCaMK specifically interacted with OsMKK1/6 in a Ca2+/CaM-dependent manner. In vitro kinase and in vivo dual-luciferase assays revealed that OsCCaMK phosphorylated OsMKK6 while OsMKK1 phosphorylated OsCCaMK. Overexpression and antisense-RNA repression expression of OsCaM1-1, and CRISPR/Cas9-mediated gene editing mutations of OsMKK1, OsMKK6, and OsMKK1/6 proved that OsCaM1-1, OsMKK1, and OsMKK6 enhanced the auxin content in roots and lateral root growth under salt stress. Consistently, OsCaM1-1, OsMKK1, and OsMKK6 regulated the transcript levels of the genes of this cascade, and salt stress-related and lateral root growth-related auxin signaling under salt stress in rice roots. These findings demonstrate that the OsCaM1-associated OsCCaMK-OsMKK1/6 cascade plays a critical role in recruiting auxin signaling in rice roots. These results also provide new insight into the regulatory mechanism of the CaM-mediated phosphorylation relay cascade to auxin signaling in lateral root growth under salt stress in plants.