The Crosstalk Mechanism Of Receptor-like Kinase FER And Phosphatase ABI2 Interaction To Mediate ABA And RALF1 Signaling In Arabidopsis

Plants are constantly bombarded with environmental signals and they must integrate these cues into plant growth control in order to adapt to the changing environment.Plant growth and development are tightly controlled by a battery of hormones and peptides working together in a delicate balance.The phytohormone ABA plays a central role in these responses.Plant ABA content increases under drought or salinity stress conditions,inducing guard cell closure,and accumulation of osmo-compatible solutes,and stress response gene expression,thus enhancing the capacity to cope with stress conditions.Given the importance of ABA to plant responses to environmental stress conditions,understanding the signal transduction processes linking the hormone to target responses has been a focus of abiotic stress research.ABI2-type PP2C phosphatase is the core regulatory factor in ABA response,which can inhibit the kinase activity of SnRK2s and lead to the inactivation of ABA pathway.Previous studies in our laboratory have found that FER activates ABI2 phosphatase activity through the FER-GEF1/4/10-ROP11-ABI2 pathway and mediates the inhibition of ABA pathway.However,how ABA mediates the regulation of FER signaling pathway is indistinct,and the molecular mechanism of crosstalk between ABA and RALF pathway that regulate plant growth and development and stress response is not clear.In 2014,Sussman's lab found that FER can bind to a polypeptide ligand called RALF1 to activate the kinase activity of FER,thus inhibiting AHA2-mediated acidification,resulting in the inhibition of root growth.ABA signaling,on the other hand,inhibits the phosphatase activity of PP2CAs by ABA binding to its receptors,leading to the release of SnRK2s kinase activity,the active SnRK2s that directly inhibits the root growth induced by the AHA2-mediated acidification of extracellular space.Based on this,we want to further study how RALF1 and ABA pathways corporate mediate the inhibition of root growth,study the node of the crosstalk between RALF1 and ABA pathways,and study the molecular mechanism of the interaction between RALF1 and ABA pathways in stress response.Based on this,a series of molecular,biochemical and genetic experiments were performed.The specific research results of this study are as follows:?1?Using E.coli prokaryotic expression system,GST-tagged fusion recombinant protein GST-FER_1-446aa-446aa was produced and immunized rabbit using purified recombinant protein GST-FER_1-446aa as an antigen for FER polyclonal antibody preparation.Using gel shift protein migration in combination with western blot method,FER was phosphorylated in vivo and in vitro through phosphorylation experiment by RALF and ABA,the experimental results show that the FER can respond to RALF1 and ABA.?2?FER can regulate abiotic stress response by cold,heat,salt,osmotic pressure and other abiotic stress treatments on genetic materials of fer mutants.fer mutants are hypersensitive to cold,heat and salt stress,but less sensitive to osmotic stress.?3?ABI2 can partially restore hypersensitive responses of fer mutants to ABA and NaCl stress?stomatal opening and root growth inhibition analysis?through studies on abi2-1,a gain-of-function mutant of ABI2 in which ABI2 phosphatase activity is insensitive to inhibition by ABA,indicating that abi2-1 mutants compensate for the functional deficiency of fer mutants in ABA response.?4?Through yeast two-hybrid?Y2H?,GST pull-down,Co-IP,bimolecular fluorescence complementation?BiFC?analysis demonstrated that ABI2 can specifically interact with FER receptor protein kinase,and ABI2 interact with FER to inhibit the kinase activity of FER.?5?By rebuilding the ABA/PYL1/ABI2/FER signaling pathway in vitro,we found ABA promoted the phosphorylation of FER by inhibiting phosphatase activity of ABI2through ABA-PYR/PYL/RCARPP2CAs signal pathway,resulting in the release of inhibition of FER kinase activity by ABI2.?6?Through root growth inhibition analysis of abi2-1,ralf1-RNAi/ralf1?ralf1?and RALF1 and ABA dose response analysis,found that ABA enhances RALF1 action in root growth inhibition,and RALF1 suppresses ABA responses in root growth.?7?A working model of ABA cross-talk with RALF1 signaling pathway through the interaction between FER and ABI2 was established:ABA inhibits AHA2 directly by activating SnRK2s.RALF1-FER pathway inhibits cell growth by inhibiting AHA2activity,while RALF1-FER activates ABI2 phosphatase by activating GEF1/4/10-ROP11 pathway,thereby inhibiting ABA reaction.Meanwhile,ABI2 interacts with FER and dephosphorylates FER to inhibit the FER-GEF1/4/10-ROP11 pathway.In conclusion,those results of this study showed that plants can jointly regulate the growth and stress response through the interaction between FER and ABI2 mediated the crosstalk of ABA and RALF1 signal.