Genetics And Biochemistry Analysis Of Arabidopsis Transcription Factor EBP1 Negatively Regulate RALF1 In Response To RALF1-FER Signal

FERONIA(FER),a plant plasma membrane receptor-like kinase protein,plays critical roles in plant development,cell growth,integrating environment and cell signal pathway.Recent researches showed that,rapid alkalinization factor 1(RALF1)is an extracellular ligand of FER and critical in FER signal pathway.RALF1 could bind to FER extracellular domain,thus leading to series of downstream events,including inhibition of Arabidopsis H~+-ATPase 2(AHA2)activity to regulate cell wall acidification.Several FER-interacted downstream components in cytoplasm or plasmamembranehavebeenrevealed,includingmembraneco-receptor LORELEI-Like GPI-Anchored protein 1(LLG1)and RPM1-induced protein kinase(RIPK),cytoplasm located ROP/RAC family members,SAM1/2 and GAPC.However,the crucial mechanism that FER regulating downstream gene expression in nucleus remains unknown.Does any transcription factor exist downstream of FER signal to regulate gene expression in nucleus in response to RALF1-FER?Unveiling and investigating the definite molecular mechanism of the transcription factor member in RALF1-FER signal pathway is critical to demonstrate the refined working model of RALF1-FER responsing and transducing extracellular signal.Based on these,we proceed series research of genetics and biochemistry,the main conclusions are listed as below.(1)Genetics and phenotypic analysis showed that the animal-plant conserved EBP1 was revealed working in Arabidopsis root hair,hypocotyl elongation,plant and seed size,and ABA response.(2)EBP1 physically interacts with receptor-like kinase FER at plasma membrane,through yeast two hybrid,BiFC,GST pull-down and Co-IP assays analysis.(3)RALF1-FER signal pathway promotes EBP1 mRNA bound by polysomes to increase the EBP1 mRNA translation efficiency,through biochemistry methods of ribosome profiling assay,and use of mRNA translation inhibitor.(4)Through in vitro and in vivo phosphorylation assay,we found FER kinase couldphosphorylateEBP1protein.Usingmassspectrometry,tenEBP1phosphorylation sites regulated by FER were identified critical for EBP1 nucleus accumulation.Thus we suggest that RALF1-FER could phosphorylate EBP1 and further promote its nucleus accumulation.(5)Through genetics,physiological and biochemistry analysis,we found EBP1negatively regulate RALF1-FER mediated inhibition of root elongation.And EBP1 is also involved in RALF1-FER related ROS production,MAPK cascade and proton secretion.(6)Using RNA-seq,chromatin immune-precipitation(ChIP),electrophoretic mobility shift(EMSA)and transient transcription dual-luciferase assays,EBP1 was found binding downstream CML38 gene directly,leading to CML38 gene expression inhibition.Thus we established a working model that EBP1 negatively feeds back on RALF1-FER signaling pathway.