The Application Of Biosensors And Quadruple Mutants To Explore The Regulation Mechanism Of SnRK2/OST1 Activity On Stomata Closure Signaling

Protein kinase plays an important role in the signal transduction of eukaryotic cells,which widely involved in the growth,development and abiotic resistance of animals and plants through phosphorylation.In addition,the protein phosphorylation is strictly regulated in time and space,which differ in each cells and time.However,investigation of protein kinase activities remains a challenge,particularly real-time measurements in living cells.It is difficult to track dynamic kinase activity in specific cell types or tissues,and time course measurements in living cells and subcellular analyses are not feasible.To overcome this drawback,f?rster resonance energy transfer（FRET）biosensor developed and could report protein kinase activity in spatial and temporal resolution,which becomes critical tools for cellular processes research in plants.Abiotic stresses,including drought and salinity,trigger a complex osmotic-stress and abscisic acid（ABA）signal transduction network to regulate the stomatal movement.The core ABA signaling components are snf1-related protein kinase2s（Sn RK2s）including the Sn RK2.6（also named OST1,OPEN STOMATA 1）.And the regulation mechanisms of Sn RK2 protein kinase remains unclear.In order to investigate the regulation mechanism of Sn RK2,the second biosensor based on fluorescence resonance energy transfer（FRET）technology was developed to report the activity of Sn RK2 kinase in plants,which was named SNACS（Sn RK2 activity sensor）.Taken use of SNACS,the effects of methyl jasmonate（Me JA）and elevated CO₂on Sn RK2s activity were investigated in guard cells,which could provide a strong tool and evidence for guard cell signaling.In parallel,mitogen-activated protein kinase kinase kinase family was reported to be required for Sn RK2-kinase activation for abscisic acid signal transduction.M3Kδ1、M3Kδ6 and M3Kδ7could strongly activate Sn RK2.6/OST1 functioning in the regulation of Sn RK2.To better understand the M3Ks-denpendent Sn RK2-kinase activation in ABA signaling,we created M3Ks high order mutant.In this study,m3kδ1/δ5/δ6-1/δ7 quadruple mutants were isolated and the phenotype was also identified,which could provide an important genetic material for ABA signaling.The results are as follows:1.In this study,DNA encoding for amino acids 1-48 of the Arabidopsis AKS1（ABA-responsive kinase substrate 1）protein and full length 14-3-3 protein,GF14phi（267amino acids）were inserted in the biosensor（named SNACS）,which could use as the reporter of the activity of Sn RK2 kinase.The work model of SNACS:Sn RK2 phosphorylate the AKS1 region of SNACS.The 14-3-3 domain was hypothesized to bind to the phosphorylated triggering the YPet to Turquoise GL fluorescent closer induce the fluorescent protein energy transfer.2.Based on this working model,we tested this sensor to determine whether it can function as a readout of Sn RK2 protein kinase activity in vitro.We performed in vitro phosphorylation assays and FRET experiment.The results demonstrated that the SNACS could be phosphorylated by Sn RK2.6/OST1,which result in an increase in the ratio of Turquoise GL to YPet fluorescence emission in vitro due to the fluorescent protein energy transfer.Furthermore,we tested whether the SNACS could report the Sn RK2 kinase activity in planta.In N.benthamiana leaf cells and Arabidopsis guard cells,ABA rapidly activates Sn RK2 protein kinase in cells was observed.These data provide strong evidence that time-dependent Sn RK2 activity can be detected in single live cells by SNACS both in N.benthamiana and Arabidopsis.In addition,these data also suggest that SNAS could work as a reporter of Sn RK2 activity.3.The movement of the stomata is directly regulated by the guard cells.We also demonstrate the effects of protein kinase inhibitors on SNACS in planta duing kinase inhibitors treatment.The results showed that 10μM K252a application resulted in a time-resolved drop in the FRET emission ratio in Arabidopsis guard cells.The protein kinase inhibition decreases FRET efficiency in guard cells,providing direct experimental evidence that basal Sn RK2 activity prevails in guard cells.Moreover,Me JA and CO₂,as the stimuli of stomatal movement.In this study,using this sensor,we show that Me JA and CO₂do not cause SNACS FRET ratio increases in guard cells in real-time in vivo analyses suggesting no-activate influence of Sn RK2.4.MAP kinase kinase kinaseδ5 has a higher expression in guard cells.Using the T-DNA single mutant of m3kδ5 crossing with the previously identified triple mutant,the m3kδ1/δ5/δ6-1/δ7 quadruple mutants were isolated.In addition,the ABA phenotype was identified.The result showed that the m3kδ1/δ5/δ6-1/δ7 quadruple mutants showing stronger ABA insensitive phenotype comparing with the m3kδ1/δ6-1/δ7 triple mutant suggestion the function in ABA signaling.The response of crops to adversity stresses is the result of the coordinated expression of various intracellular biological processes.As a stress hormone,ABA can also be used as an indicator of crop drought resistance.The development and successful application of SNCAS in this study is conducive to mining and monitoring the activity of Sn RK2,the basic concentration of ABA and the state of stomata movement under different stresses（such as extreme temperature and hypertonicity）,providing a powerful tool for future crop resistance research technical approach.