The Molecular Mechanism Of Arabidopsis Root Development Regulated By Cytokinin Transporter AtABCG14

In Arabidopsis,root has important role in development as the main organ for fixation and absorption of nutrients.The growth and development of root is dependent on root apical meristem(RAM).In RAM,there are many undiferentiated stem cells,which maintain the RAM morphogenesis and function.As a plant hormone,cytokinin can regulate cell division,which is related to regulation of RAM morphogenesis.Meanwhile,cytokinin and auxin can inhibit each other in root,causing the change in rate of cytokinin and auxin.And the cell division and differentiation can be changed by this process.But the role of cytokinin in the root development is largely unknown.AtABCG14 is a cytokinin transporter,which is essential for the long-distance translocation and distribution of the root-synthesized tZ-type cytokinin species.Compared with wild type,atabcg14 mutant has shorter root,causing by the over-accumulation of tZ-type cytokinin in the root.Based on this phenotype,we investigated the mocular mechanism of root development regulated by cytokinin.According to molecular biology,genetics,cytology and transcriptome analysis,the major results were as following:(1)The over-accumulation of cytokinin in root of atabcgl4 mutant,indicated that atabcgl4 mutant impair the translocation of cytokinin.(2)The short-root phenotype of atabcgl4 mutant indicated that over-accumulation of cytokinin impaired the root development of atabcgl4.(3)The GUS activity promoted by the CYCB1;1 promoter,one of the marker for cell division,in atabcg14 mutant was significantly reduced.Moreover,The GUS activity was reduced by 6-BA treatment in the wild-type background.Meanwhile,cell number of RAM was also reduced compared with wild-type.These data suggested that the meristem activity could be inhibited by cytokinin,and this inhibition of cytokinin mainly lead to the reduced cell number.(4)The GUS activity promoted by the DR5 promoter,one of the marker for auxin,in atabcg14 mutant was also signficantly inhibited.The expression level of PINs,the auxin transporter genes,was detected to be changed.The fluorescence of PIN1 and PIN2 were reduced,on the contrary,PIN7 fluorescence was increased.These data indicated that the translocation of auxin in the RAM could be affected by cytokinin.(5)According to transcriptiome analysis,DA02,important for IAA oxidase,was increase significantly in atabcg14 seedlings.The result of qPCR analysis showed the same increase of DA02.These result suggested that the IAA oxidation was activated by cytokinin,which increased the expression of DA02.Meanwhile,it has been proven that AtABCG14 acts as a key component in cytokinin long-distance transportation.However,the regulation mechanism of AtABCG14 gene at transcriptional level is not poorly understood.Therefore,we performed the functional analysis of AtABCG14 promoter,and further try to find the transcription factors which regulate AtABCG14 expression at transcriptional levels.The major results are as following:(1)The GUS genes driven by different truncated promoters were detected by GUS staining and showed differential patterns.When we further expressed AtABCG14 drived by truncated promoters in atabcg14 mutant,the recovery of mutant phenotypes were depend on the length of the promoters.These results suggested the regulatory cis-elements in promoter region may determine the expression of AtABCG14 in different tissues.(2)Seven transcription factors were identified to be able to bind to the promoter of AtABCG14 by yeast-one-hybrid and dual luciferase assay.