Regulation Of Protein Kinases SOS2 And PKS5 By 14-3-3 Proteins Under Salt And Alkali Stress In Arabidopsis

Salt stress is a widespread abiotic stress that affects plant growth and reduces crop productivity worldwide.In Arabidopsis,the recognized and conserved Salt Overly Sensitive(SOS)pathway plays important roles in regulating sodium ion homeostasis under salt stress.In SOS pathway,SOS3 and SCaBP8 are calcium binding proteins.SOS2 is a Ser/Thr protein kinase.SOS1 encodes a plasma membrane Na+/H+ antiporter.Under salt stress,the calcium concentration in cytosol is increased.SOS3 and SCaBP8 perceive the Ca2+ signal,interact with and activate protein kinase SOS2.The activated SOS2 is recruited to the plasma membrane and it leads to the activation of SOS Na+/H+ antiporler.However,it is unclear that how SOS pathway was regulated in the absence of salt stress.We previously reported that phosphorylation of SOS2 Ser294 site is important for the 14-3-3-mediated(14-3-3? and ?)repression of SOS2 kinase activity in the absence of salt stress.However,the kinase that phosphorylates SOS2 Ser294 remains unknown and the regulation mechanisms of 14-3-3-SOS2 interaction under salt stress are still missing.In this study,we found that the Ser/Thr protein kinase PKS5 interacted with SOS2 and phosphorylated SOS2 at Ser294 site.In vivo kinase assay demonstrates that PKS5 negatively regulates the kinase activity of SOS2,and in the SOS-pathway reconstituted yeast system,we showed that PKS5 inhibits SOS2 activity via phosphorylating the Ser294 site in SOS2 leading to an enhanced-repression of SOS2 by 14-3-3 proteins.Noninvasive ion flux assay showed that the salt-induced vaa efflux of pks5-3 and pks5-4(two constitutive active PKS5 mutants)were decreased,and overexpression of SOS2S294A partly rescued the salt-sensitive phenotype of pks5-4.Taken together,PKS5 participates in and negatively regulates salt tolerance through phosphorylating the Ser294 site in SOS2 in Arabidopsis.This study found that 14-3-3? interacts with the kinase domain of PKS5 and salt stress enhances the interaction between 14-3-3 and PKS5 and represses PKS5 activity.Moreover,14-3-3 proteins bound to Ca2+,which enhances their interaction with PKS5 and reduces their interaction with SOS2.Beside activated by SOS3/SCaBP8-SOS2,the activation of SOS1 requires plasma membrane H+-ATPase activity to generate the proton gradient.It is likely that salt-induced calcium signal may be recognized by unknown calcium-binding proteins to inhibit the kinase activity of PKS5.Previous studies have found that 14-3-3 protein contains a calcium-binding domain and binds to Ca2+.Therefore,we hypothesized that elevated calcium signal was recognized by 14-3-3 proteins,and reduced the interaction between 14-3-3 and SOS2 to relieve its activity under salt stress.On the other hand,it enhanced the interaction between 14-3-3 and PKS5 to inhibit its kinase activity.In vitro calcium binding experiments showed that binding of 14-3-3 to Ca2+reduces the 14-3-3-SOS2 and increases 14-3-3-PKS5 interactions.The plasma membrane H+-ATP ase activity in 14-3-3?? double mutant was decreased compared with that in wild type,and 14-3-3?? showed a high pH sensitive phenotype for the release of PKS5 activity.Whereas,the Na4+/H+ antiport activity of 14-3-3?? was higher than that of wild type,and the salt tolerance phenotype of 14-3-3?? was due to the activation of SOS2 activity.Our results suggest that 14-3-3 proteins play critical roles in regulating salt and alkali stress.Here,we find that PKS5 phosphorylates SOS2 Ser294 site and enhances the 14-3-3-repressed SOS2 activity under normal conditions.In the presence of salt stress,salt-induced Ca2+ signal is decoded by 14-3-3 proteins,which reduces the 14-3-3-SOS2 interaction and releases the SOS2 activity,at the same time,calcium ions enhance the interaction between 14-3-3 and PKS5,which represses the PKS5 activity and leading to activation of SOS2 and PM H+-ATPase.Our results suggest that a salt-induced calcium signal is decoded by 14-3-3 and SOS3/SCaBP8 proteins,which selectively activate/inactivate the downstream protein kinases SOS2 and PKS5 to regulate Na+homeostasis by mediating plasma membrane Na+/H+ antiporter and H+-ATPase activity.