Identification And Charactrization Of An Root Cytokinin-accumulatied Mutant Zsr9 In Arabidopsis

Cytokinins,as important plant hormones,play an important role in plant growth,development,aging,disease resistance and abiotic stress.The previous researches on the synthesis,metabolism and signal transduction of cytokinins have been well clarified,but the molecular mechanism of transport has not been revealled.As the first long-distance cytokinin transporter in plants,AtABCG14 participates in trans-Zeatin(tZ)synthesized in roots transporting to the top.Therefore,in order to further study in the transport mechanism of cytokinins,we established EMS mutagenesis library by using forward genetic method.In this study,the results of mutant screening,map-based cloing and functional analysis are as follows:(1)According to the criteria of short root and fluorescence accumulation,zsr9 mutant were screened.The phenotype analysis showed that zsr9 had the smaller rosettes and inflorescence,thinner stem and lower chlorophyll content.Combined with qRT-PCR analysis,zsr9 had similar expression pattern with atabcg14,and that preliminarily confirmed the relationship between zsr9 and cytokinins.(2)Cloning of ZSR9 gene.zsr9 was hybridized with two ecotypes Col-4(Columbia-4)and Ler(Landsberg).The hybridized F2 generations were used for mapping,the backcrossed F2 generations were used for phenotypic observation.Finally we isolated the zsr9 by map-based cloning and sequencing,which is a protein phosphatase PP7.(3)Verification of the function of T-DNA insertion mutants.We observed that the two T-DNA insertion mutants had more obvious phenotypes than zsr9,which were mainly characterized by shorter roots,abnormal apical meristem and defective dark morphogenesis,smaller inflorescence,shorter plant height,lower chlorophyll content and weaker fertility,reflecting that the loss of PP7 may cause many growth and development problems.(4)PP7 genome complementary experiment.The full-length genome of PP7 was used to replenish the pp7 mutant.The phenotypes of root length,lotus leaf size,plant height and inflorescence were observed.It was found that it could compensate for the phenotype of pp7.(5)Expression patterns analysis of PP7 showed that it strongly expressed in root tip,adventitious root,true leaf,growth point,vein,inflorescence and young pods.Subcellular localization result showed that PP7 was localized in cytoplasm and nucleus.(6)GUS expression and GFP localization of pp7 mutant hybridized with cytokinin and auxin-related materials indicated that PP7 might be involved in the apical transport of cytokinins,the polar distribution of PIN protein and the cell division of apical meristem.(7)Analysis of hormone metabolism in the aboveground tissues of pp7 mutant.It was found that the amount of SA and JA in the pp7 increased significantly,the amount of ACC and IAA decreased slightly,and the amount of tZ and tZR decreased slightly.It proved that PP7 could affect the dynamic balance of hormones in plants,especially the transport of tZ and tZR.In summary,the loss function of PP7 can produce a series of phenotypes,such as smaller inflorescence,thinner stem and growth retardation,which lead to abnormalities in the whole stage of plants.PP7 is a specific protein phosphatase.It is preliminarily speculated that PP7 participates in the signal transduction of cytokinin,or affect polar transport of auxin,but the specific pathway need to be further studied.