EAR1 Negatively Regulates ABA Signaling By Enhancing 2C Protein Phosphatase Activity In Arabidopsis

Reversible protein phosphorylation,executed by kinases and phosphatases,is crucial in many eukaryotic cellular processes,including developmental growth and the response to environmental stimuli.In the early 1960s,the plant hormone ABA has been discovered,but the mechanism of its signaling pathway has made great progress in the recent decade.By the discovery of ABA receptors,the ABA core signaling pathway is gradually established.In the ABA core signaling pathway,the clade A type 2C protein phosphatases(PP2Cs)are key negative factors,which play an important regulatory role in the activation of ABA signaling.The ABA receptor PYRl/PYLs/RCARs can bind to and inhibit the phosphatase activity of PP2Cs when the ABA level elevates in plants,then initiating the ABA signal response in vivo.The ABA receptors,as negative modulators of clade A PP2Cs,play key role in inhibiting the phosphatase activity of PP2Cs.However,whether these PP2Cs can be positively regulated by other proteins,which working synergistically with PYR1/PYLs/RCARs receptors,is still unclear.In this study,we identified a new gene EAR1(ENHANCER OF ABA CO-RECEPTOR 1)by forward genetic screening.The earl-1 mutant exhibits pleiotropic ABA-hypersensitive phenotypes in germination,seedling root growth and stomatal movement compared to wild type.The leaf temperature of earl-1 is higher than that of wild type and the mutant in soil shows more drought resistant phenotype,indicating that EAR1 plays a negative regulatory role in the ABA signaling.By CRISPR/Cas9 technology we create an earl-c mutant,and this mutant also has ABA-hypersensitive phenotypes.Histochemical staining results show that EAR1 is expressed in most tissues of Arabidopsis,such as roots,hypocotyls,leaves,flowers and guard cells,indicating that EAR1 has a wide range of roles.Analysis of protein interactions by bimolecular fluorescence complementation,co-immunoprecipitation and pull-down reveal that EAR1 can interact with 6 members of clade A PP2C family.In vitro and in vivo phosphatase activity analysis show that EAR1 can specifically enhance the clade A PP2C protein phosphatase activity.Protein truncation analysis indicates EAR1 and clade A PP2C interaction region locates in the PP2C protein N-terminal,further study finds that EAR1 can enhance the clade A PP2C phosphatase activity by releasing the N-terminal self-inhibition of PP2C protein.In vitro phosphorylation assay results show that EAR1 can indirectly inhibite the downstream OST1 kinase activity by enhancing the phosphatase activity of clade A PP2C.In addition,in-gel kinase assay showes that the activity of OST1 in earl-1 mutant is higher than that in wild type,whereas the activities of OST1 in EAR1 overexpression lines are significantly lower than that in wild type.Subcellular localization results show that EAR1 protein mainly locates on endoplasmic reticulum,but ABA promotes EAR 1 protein translocation from endoplasmic reticulum to nucleus.The ear1-1/abil-2/abi2-2/hab1-1 mutant shows more ABA-hypersensitive phenotypes,and the ear1-1/snrk2s mutants can partly rescue the ABA-insensitive phenotypes of snrk2s.In summary,this study identifies a new component involved in the regulation of ABA signaling pathway,EAR1 can enhance the phosphatase activity of clade A type 2C protein phosphatases.When the ABA level increases in plants,the ABA receptors PYR1/PYLs/RCARs can inhibit the activity of PP2C and activate ABA signaling,while the EAR1 can enhance the activity of PP2C and repress ABA signaling.EAR1 and ABA receptors PYR1/PYLs/RCARs can synergistically modulate the phosphatase activity of clade A PP2Cs to ensure that the ABA signaling maintains at an appropriate level in vivo,which balances the plant developmental growth and the resistance to stress response.