Establishment Of A Method For The Determination Of The Activity Of Hormone Transports And The Analysis Of The Activity Of Cytokinin Transporters

Cytokinins(CKs)are a major group of phytohormones regulating plant growth,development and stress responses.In recent years,the research about synthesis metabolism and signal transduction of cytokinin has been very clear,but the molecular basis of cytokinin transport is poorly understood.As it is known to all that is difficult to identify the biochemical activity of hormone transporters.AtABCG14,as the first half-molecule transporter of CK over long distances,plays an important role in transport trans-zeatin(tZ)-type cytokinins to the ground tissues.However,the biochemical activity of AtABCG14 remains to be further explored.Therefore,in order to study the biochemical activity of cytokinin transporter,especially AtABCG14,more conveniently and effectively,we established a feasible and effective method for detecting the activity of plant hormone transporter,which uses to detect the transport activity of AtABCG14.The concrete results are as following:(1)To establish a simple and effective method for the study of cytokinin transporter activity.Through studying the reported transport activities of ABA transporter AtABCG25,JA transporter AtABCG16 and cytokinin transporter AtPUP14,we proved the feasibility of this method,established by a tobacco transient expression system and a liquid chromatography-mass/mass spectrometry(LC-MS/MS)hormone detection system(tobacco-mass spectrometry).(2)The cytokinin transporter ZmABCG43 in maize was identified by the established tobacco-mass spectrometry.we found that ZmABCG43 had the biochemical activity of extracellular transporting of cytokinins,by transport activity assays using tobacco leaves and protoplasts,prepared from tobacco leaves expressing GFP-ZmABCG43.(3)ZmABCG43 has the biological function of transporting CK in Arabidopsis thaliana.we proved that ZmABCG43 could transport CK in plants,which the ZmABCG43 coding region was transformed into the atabcg14 mutant,and the root length and rosette leaf size of the transgenic lines returned to the wild type.(4)The biochemical activity and substrate specificity of AtABCG14 were demonstrated using tobacco-mass spectrometry.we found that AtABCG14 had the biochemical activity of extracellular transporting of trans-zeatin(t Z),trans-zeatin riboside(tZR),N~6-(?~2-isopentenyl)adenine(iP)and Isopenteny ladenosine riboside(iPR),by transport activity assays using tobacco leaves,prepared from tobacco leaves expressing EGFP-AtABCG14.(5)It was further demonstrated AtABCG14 biochemical activity of extracellular transporting CK in the tobacco suspension cell(By-2)with stable expression of AtABCG14.The results proved that AtABCG14 had the biochemical activity of extracellular transporting of tZ,tZR,cis-zeatin(cZ)and cis-zeatin riboside(cZR),by transport activity assays using BY-2,steadily expressing EGFP-AtABCG14.In summary,we established a simple and effective approach for the detection of the biochemical activity of hormone transporters,and proved that AtABCG14 has the biochemical activity of extracellular transporting CK.This method will be used for the screening of other plant hormone transporters,the validation of biochemical activity and screening of unknown hormone transporters.