Research On The Interaction Of Ropgefs And Phosphatidic Acid And The Function In Aba Regulated Stomatal Movement In Arabidopsis

No matter in plants or animals,small G proteins function as "molecular switches".They are activated by GTP,and inactivated by hydrolyzing GTP to GDP,which is the cycle model of ROPs.This resulting cycles of activation and inactivation of small GTP-binding proteins represents a ubiquitous regulatory mechanism in eukaryotic cells.Guanine nucleotide exchange factors(GEFs),which promote the exchange of GDP for GTP is the important mediator of the context-specific,spatio-temporal activation of ROP GTPase.Besides,every GEFs in Arabidopsis display difference tissue and subcellular localization and are regulated by binding to upstream factors,which makes the signaling pathway complex.Recent research reveal that PA,produced by PLD,is a hub in cell signaling transduction,and can interact with many proteins because of its specific biochemistry feature.One way of PA involving ABA signal pathway is directly interacting with proteins,which recruits proteins to cell membrane and change proteins function.PA interacts with a defined site in the Sos PH domain with high affinity and specificity,which is essential for Sos membrane recruitment and activating Ras.Above all,we wonder whether there are GEFs of plants interacting with PA,and how to be regulated by PA in ABA signaling.By molecular biochemical and physiological approaches,we found that it was probable that RopGEF8 bound to PA,and participated in ABA-induced stomatal closure.The results are as follows.Firstly,we extracted the RNA of many tissues of Arabidopsis,and cloned ten GEF genes of the RopGEF family after reverse transcription.These cDNA sequences were tested right and inserted into expression vector(pET-28a)with HIS tag.After expressed in Escherichia coli strain BL21,purified proteins were used in fat-western assay.We found that it was that RopGEF8 probably bound to PA,and preferred to bind to distearoyl-dilinoleoyl PA(18:0-18:2 PA),compared other PA isoforms.To further identify the interaction with PA,we used the ITC assay,showing the interaction sites between the two proteins.In order to identify the fragment of GEF8 involved in PA binding,we constructed several truncated fragments of RopGEF8 according to its function motif used for immunoblotting in fat-western in vitro.We found the N terminal was important,referring to the theory of PA's binding.Otherwise,after sequence alignment of RopGEF8 and RopGEF9 and using the site mutantions in fat-western assay,the result suggested that the residues K(13,18)were vital in this binding.To identify the influence on the activity of RopGEF8 after combining with PA,we got two ROPs(ROP7 and ROP10)as substrates in vitro and in vivo GEF8 activity,by Pull-down and split-YFP assay.The data reflect that PA has different effect on the activity of GEF8 with different ROPs.PA could up-regulate the activity of GEF8,if ROP7 as substrate,while could down-regulate it,if ROP10 as substrate.To identify the role of GEF8 and PA in ABA-induced stomatal closure,we next constructed gef8/pld?1,gef8-COM and gef8 mutants.We measured stomatal apertures of guard cells using the epidermal strips from rosette leaves of mutants,after exogenously applying ABA and total soybean PA.The results show the interaction between PA-GEF8 is important in the ABA activation of the stomatal closure.We found one GEF protein(GEF8)interacting with PA and its possible combination,that it was likely that GEF8 was in the downstream of PLDal-derived PA in the ABA-mediated stomatal closure.