Effects Of Ethylene On Adventitious Root Development And Differential Protein Analysis In Cucumber Explants

Adventitious roots development is a critical and complex process in the life cycle of plants.Ethylene is a gaseous phytohormone which promotes adventitious root formation,but the molecular mechanism of ethylene induced adventitious root formation in cucumber remains unclear.Therefore,the regulation mechanism of ethylene on adventitious root formation is importantin both theoretics and practic.Cucumber（Cucumis sativus L.）explants were chosen as the experimental materials to observe the anatomical changes in three different adventitious root development phases.The i TRAQ technique was used to identify the different specific proteins that were expressed during the adventitious root development process.Meanwhile,the differentially expressed protein during the ethylene-induced the adventitious root development of explants was studied by iTRAQ,and verified by transcriptional,Western-blot,physiological and biochemical level.Finally,the physiological and molecular mechanism of development of adventitious roots was theoretically discussed.This will provides a theoretical basis and a technical support for the regulation of the adventitious root development in cucumber.The main results were as follows:1.The adventitious root development in the base of cucumber explants was observed by paraffin section technique,which was divided into three stages:the induction of adventitious root primordia（IRP;0-12 h）,the formation of adventitious root primordium（FRP;12-24 h）and the elongation of adventitious root（EAR;24-72 h）.Ethylene releasing compound,ethrel,had significant positive effect on adventitious root development and ethrel at the concentration of 0.5μM had the best promotive effect.Ethylene inhibitors,aminoethoxy vinylgly-cine（AVG）and（Ag NO₃）,could inhibit adventitious root formation in cucumber and reversed the promotive effects of ethrel partly.Thus,it was proved that the rooting process of cucumber explants was regulated by ethylene.2.A comparative proteomic analysis of the IPR,FRP and EAR of cucumber（Cucumis sativus L.）explants was performed to identify related proteins that may be involved in regulating adventitious rooting.Out of the 3622 differentially expressed proteins（DEPs）detected,57 were considered as adventitious root development DEPs,and 20 of which DEPs are interesting proteins abundance.Most of the proteins identified were mainly related to photosynthesis,carbohydrate and energy metabolism,lipid metabolism,stress response and amino acid metabolism.Nineteen of the differentially expressed proteins,including LOX and TUA were further analyzed at the transcription level,most of which were in accordance with the results of iTRAQ,accounting for 89%of the proteins.The Threonine dehydratase（Csa₆G448740）and Ferredoxin-1（Csa₃G146700）were not consistent with the result of DEPs.Moreover,the protein abundance changes of LOX and TUA are supported by western blot experiments.The LOX activity and ethylene evolution increased significantly during the three rooting stages.Ethylene evolution in different rooting phases closely followed that of LOX activity.Besides,the development of adventitious roots activated cell cycle with the transition of cell from G1 to S phrase and induced cell cycle-related genes CycA,CycB,CDKA and CDKB expression in the three phases.These results suggested that lipoxygenase and Tubulin alpha chain may participate in adventitious root development during the different rooting phases.3.Out of the 5014 differentially expressed proteins（DEPs）detected,115 were considered as adventitious root development DEPs,and 24 of which DEPs were interesting proteins abundance.Compared with CK（distilled water）,ethrel treatment identified with proteins were mainly involved in photosynthesis,transcription,translation,carbohydrate and energy metabolism,amino acid metabolism,and protein folding.A few kinds of functional category play a key role in the process of ethylene induced adventitious root formation.Twenty-three of the differentially expressed proteins,including SAMS and AtpA were further analyzed at the transcription level,nineteen of them were consistent with the results of iTRAQ.In addition,the level of Aspartokinase（Csa₅G457770）,Folylpolyglutamate synthase（Csa₅G630860）,Methionine aminopeptidase（Csa₂G172500）and Serine/threonine-protein Phosphatas（Csa₆G426880）were different from those of protein expression.Moreover,the protein abundance changes of SAM and AtpA were detected in cucumber explants by western blot experiments.The activity of ACO and ACS enzyme in ethylene treatment and the relative expression of gene were similar to that of ethylene production.Ethylene application significantly increased ethylene synthase activity and ethylene synthesis gene expression.Therefore,this promoted the synthesis of endogenous ethylene which subsequently promoted the adventitious root development.Ethylene treatment improved chlorophyll content,chlorophyll fluorescence parameters（Fv/Fm,ΦPSII and qP）,activities of Calvin cycle enzymes（Rubisco,GAPDH,FBPase,FBA and TK）,expression levels of genes coding for Calvin cycle enzymes（rbc S,GADPH,FBA and TK）,increased the content of soluble sugar and soluble protein as well as reduced the starch content in the explants.These substances interacted with each other and regulated the formation of the adventitious roots in the cucumber explants.Therefore,it is speculated that SAMS as a precursor of ethylene directly participated in the development and regulation of adventitious roots.At the same time,AtpA may be positively correlated with the development of ethylene-induced adventitious roots.