Cross Talk Between Abscisic Acid And Ethylene In Stomatal Regulation In Arabidopsis Thaliana

As sessile living organisms,plants often suffer from biotic and abiotic stress such as pathogen diseases,low temperature,drought and high salt.In order to adapt to these adverse growing conditions,plants formed the relative systematic responds and protection mechanism through long-term evolution.Stomata are plant-specific structure which plays an important role in plant environmental stress response,under drought stress,plant reduces the loss of water by closing stomatal pores.When subjected to insect or pathogen against,plants prevent pathogen entering through closed stomata and generate defense signal for immune response.Reports shown that plant synthesize many abscisic acid(ABA)to promote stomatal close,thereby reducing loss of water and improving drought resistance.To dates,ABA-dependent drought resistant pathway has been deeply and systematically revealed.Functions of ABA receptor and its corresponding signaling components have been cloned and analyzed.Ethylene also is an important plant hormone.To dates,most of research about ethylene focus on senescence and developmental regulation,seldom report on drought conditions.Nevertheless,there are two controversial opinions on stomatal regulation,which are promoting stomatal closure and suppressing stomatal closure.In view of this,through genetic,physiology,biochemistry and molecular biology,we try to investigate whether exist an ethylene-dependent drought resistant pathway and the possibility of cross talk between ABA and ethylene.This study takes a phenotype analysis based on ethylene synthetic mutants and ethylene signal transduction mutants.Under drought stress,compared with wild type Col-0,ethylene sensitive mutant ebf1-1,ebf2-1,eto1 showed slower loss of water,mutants recover to normal growth after 6 h of watering,while ethylene insensitive mutants ein2-5,etr1,ein3-1 rapidly loss of water and wilting seriously,mutants did not recover to normal growth.Microscopic observation of epidermal peel stomata revealed that compared with Col-0,the stomata of ethylene sensitive mutant ebf1-1 all most closed,while the stomata of ethylene insensitive mutant ein3-1 remains half opened.These results indicated that ethylene contributes to drought-resistant ability through ethylene signaling regulating stomatal close in Arabidopsis thalina.To dates,few reports focus on the cross talk mechanism between ABA and ethylene on stomatal regulation.Given the protein kinase SnRk2.6/ OST1 is an important member of the ABA signaling.In this study,we try to examine the possibility of SnRk2.6/ OST1 served as a cross point between ABA signaling and ethylene signaling.Whether OST1 interaction with a series of ethylene signaling components such as ETR1,EBF1,EBF2,EIN2,EIN3,MKK1,MKK4,MPK6 and MKK9.Results of yeast two-hybrid assay and bimoecular fluorescence complement experiments indicated OST1 interact with MKK1,MKK4 and MPK6,maybe involved in stomatal regulation during drought response.At last,we want to determine the relationship between OST1 with MKK1,MKK4,MPK6 by kinase assays and fusion protein expression vector was constructed with His-tag,GST-tag and MBP-tag,respectively.MKK4 fusion Protein-induced condition has been confirmed that 12?,0.3 mmol/L IPTG induction for 26 h,a certain amount of the target protein exist in the supernatant by western blot.Kinase assay have yet to be carried out.To gain genetic evidence,we constructed double mutants ost1ctr1,ost1ebf1,aba2ebf1,aba2ctr1,aba2ein3,stomatal phenotype analysis and drought experiment are being carried on.