Map-based Cloning Of Prig And Dpr1 And Gene Functional Analysis In Arabidopsis

Roots are important plant organs that not only acquire water and nutrients vital to plant growth,but also provide mechanical support to the aerial portion of the plant.Thus,overall plant survival relys on proper root growth,development and function.The study of root growth and development is of important theoretical value and practical significance.The Arabidopsis roots have a simple cellular organization and can be easily grown in nonsoil media,that facilitiates for studying the molecular mechanism of root development.The establishment and maintenance of the Arabidopsis root apical meristem are essentical for the normal growth of the root.In this study,we obtained two short root mutants prgi(primary root growth inhibition)and dpr1(defective primary root 1)in previous screens using EMS-mediated mutagenesis of seeds.We identified the mutation site of each mutant respectively by positional cloning and isolated PRGI and FPGS1 two candidate genes,and performed complemention test to verify gene function.Then we analyzed their spatiotemporal expression patterns,the mutant phenotypes and the expression of root stem cell markers and auxin reporters in the mutant.Our results reveal PRGI and FPGS1's important roles and molecular mechanism of PRGI and FPGS1 in the embryo and root development in Arabidopsis thaliana.The main results obtained are as follows:(1)A mutant library was constructed using EMS mutagenesis for Rop GEF7_pro:GUS transgenic seeds.Two short root mutants were isolated,and these two short root mutants were named as prgi and dpr1.(2)In the prgi mutant,defects in the QC specification and maintenance result in reduced root meristem cell division and root growth.In addition to the abnormal development of the root,embryo patterning was also affected.These results indicate that PRGI plays an important role in plant growth and development.(3)Genetic analysis showed that the prgi mutation was recessive.The mutation located between the two molecular markers T5P19?SNP?80875 and F28O9?SNP?4692 on chromosome 3 based on map-based cloning.Sequencing and analysis of the candidate genes revealed a single base change in the PRGI gene.Complementation test was carried out and all transgenic plants obtained were phenotypically indistinguishable from wild type plants,confirming that the root growth phenotype observed in prgi were caused by the disruption of PRGI.As PRGI encodes a key enzyme on Met biosynthetic pathway,exogenously supplementation of Met could restore the prgi root growth.(4)PRGI was expressed broadly in embryonic and postembryonic stages by analyzing the promoter-GUS activity and PRGI-GFP was localized in the plastids.(5)Expression of the root stem cell specific regulators,including SCR,SHR and PLT1/2,was investigated in prgi embryos and seedlings.The results show that the expression of PLT1/2,SCR and SHR is dramatically suppressed during embryogenesis and postembryonic development,suggesting that PRGI plays an important role in the maintenance of the root stem cell niche in Arabidopsis by regulating the PLT1/PLT2 and SHR/SCR pathways.(6)The activity of auxin response reporter DR5rev:GFP was suppressed in prgi and the formation of local auxin maxima was disturbed in the embryos and roots of prgi.We also found that the accumulation of auxin efflux carrier PINs was reduced in embryos and roots of prgi,leading to altered auxin transport.(7)The DNA methylation level detected by immunofluorescence decreased significantly in prgi root tips,showing that PRGI plays an important role in DNA methylation maintenance.(8)In another short root mutant dpr1,the root growth was inhibited,the embryo development was abnormal.Genetic analysis showed that the dpr1 mutation was recessive,gene mapping showed that the mutation was in the second exon of AT5G05980,which encodes folylpolyglutamatesynthetase 1 and acts in the folic acid metabolism process.The short root and defective embryo phenotypes of dpr1 can be rescued via the expression of FPGS1_pro:FPGS1-GFP.It indicates that FPGS1 plays an important role in embryonic and postembryonic development.(9)FPGS1 was expressed broadly in embryonic and postembryonic stages by analyzing the FPGS1_pro:GUS and FPGS1_pro:FPGS1-GFP transgenic lines and FPGS1 is located in plastids and cytoplasm.(10)The activity of auxin response reporter DR5rev:GFP was suppressed in dpr1 and the formation of local auxin maxima was disturbed in the roots of dpr1.We also found that the accumulation of auxin efflux carrier PINs was reduced in roots of dpr1,leading to altered auxin transport.The expression of the root stem cell specific regulators,PLT1/2,is dramatically suppressed in the roots of dpr1,suggesting that FPGS1 participates in auxin-regulated root apical meristem maintenance.Taken together,PRGI is mainly expressed in embryo,the QC and the surrounding stem cells of the root it regulates the embryonic and postembryonic growth and development,and is associated with auxin pathway.PRGI encodes a key enzyme on Met biosynthetic pathway,the DNA methylation level decreased significantly in prgi root,indicating that PRGI may affect the auxin dependent embryo and root growth by regulating DNA methylation.FPGS1 is strongly expressed in embryo and root meristem,and also involved in regulating the embryo and root growth.FPGS1 encodes folylpolyglutamatesynthetase 1that acts in the folate metabolism.The mutation in FPGS1 causes the failure in the formation of local auxin maxima and the reduced accumulation of auxin efflux carrier PINs,the expression of root stem cell transcription factor PLT1/2 was also reduced in prgi root tips,suggesting that FPGS1 participates in auxin-regulated root apical meristem maintenance.