The SOS Pathway Generates And Fine-tunes An AtANN4-dependent Calcium Signature Under Salt Stress

Calcium(Ca2+)serves as a ubiquitous second messenger that regulates many cellular activities in plant cells.For an ever increasing number of environmental stresses,pathogens attack,drought stress,cold/heat stress,oxidative stress and salt stress,it has been found that temporally and spatially defined rapid changes of Ca2+ concentration in the cytoplasm([Ca2+]cyt)differs in elevation duration,intensity,amplitude,frequency and other aspects.The environmental stresses induced the special changes in[Ca2]cyt is referred to as Calcium signature or Calcium signal,which could be sensed,recognized and decoded by many calcium-binding proteins and thus activates the downstream transcriptional regulation or related protein/enzyme activity to maintain normal life activities.However,little is known about how specific calcium signals are generated and controlled.The Salt Overly Sensitive(SOS)is a core mechanism in plant salt tolerance,which is regarded as a Ca2+-dependent activation pathway.However,the mechanism of Ca2+-dependent activation in vivo is still elusive.Moreover,the generation and regulation mechanism of specific calcium signals that activate SOS pathway under salt stress need to be further explored.Here,we found that salt stress-induced[Ca2+]cyt elevation indeed involved in the activation of SOS pathway and SCaBP8,SOS2 negatively regulated the salt stress-induced[Ca2+]cyt.AtANN4 was isolated as a potential regulator through yeast two-hybrid screening and the interaction between SCaBP8 and AtANN4 was proved by in vitro and in vivo protein-protein interaction assays.Aequorin-based or YC3.6-based calcium testing assay showed that the AtANN4-mediated increase in[Ca2+]cyt promotes SOS2 kinase activity.Subsequently,I found that SOS2 directly phosphorylated AtANN4 at the Ser46 of N-terminal variable region and further in vitro kinase assay,yeast three-hybrid assay and Split-LUC assay indicated that SCaBP8 promoted the interaction between SOS2 and AtANN4,which enhanced the SOS2-mediated AtANN4 phosphorylation.Results of co-immunoprecipitation and yeast two-hybrid assay showed that phosphorylation modified AtANN4 enhanced the interaction with SCaBP8.Therefore,salt stress can promote the formation and stabilize SCaBP8-AtANN4-SOS2 complex.Acorrding to the ATP-induced calcium imaging assay,we found both the interaction with and phosphorylation of AtANN4 repressed its activity and alter calcium transients and signatures.In a summary,I determine that AtANN4,a putative calcium-permeable transporter,and its interacting proteins,SCaBP8 and SOS2,generate a specific calcium signal,which activates the Salt Overly Sensitive(SOS)pathway in response to long time salt stress.These results reveal how downstream targets are required to create a specific calcium signal via a negative feedback regulatory loop,thereby enhancing our understanding of the regulation of calcium signaling.