| Cell polarity,broadly defined as asymmetric distribution of molecules within a cell,is one of the basic features to fulfill the cell development and environment regulation function.As the model plant,the single-cell pollen or multicellular epidermal systems in Arabidopsis have been the focus of the investigation of polarity generation.Previous studies suggested that in the Arabidopsis epidermal system,auxin could initiate the downstream plant-specific ROP6(Rho-related GTPase from plants 6)-RIC1(ROP-interactive CRIB motif-containing protein 1)signals transduction,which thereby affected the arrangement of cytoskeleton to control the cell polarity growth and morphological generation.In this thesis,we used the forward genetics to screen a rop6-2 mutation-based EMS-induced mutation pool and got a new component of ROP6-RIC1 signaling pathway controlling pavement cell polar development,named 507-2,which had largely reduced lobe number in the pavement cell.Finally,the homozygous recessive mutant 507-2 was out-crossed with Ler ecotype to create a mapping population,then next-generation sequencing and BSA(Bulked Sergeant Analysis)were used for analysis and four candidate causal mutations were found.After further analysis and identification,the mutation was identified lying in change of the G in the 877th nucleotide to A of the Angustifolia(AN)in CDS sequence and thereby led to change of the coded amino acid from E to K.The 507-2-SM with removed rop6-2 background by outcrossing wasnamed as ANE293K.The subcellular localization of AN and ANE293K was observed in the cytosol and trans-golgi network using transient expressed GFP-tagged fusion protein in the Nicotiana benthamiana.Also,AN and ANE293K were found forming large particle and moving in the cytosol,and the motility of AN was far faster than that of AN E293K protein.In comparisorn with wild type,the microtubule bundle in the ANE293K was thicker and distributed with higher density in the cortical region.It was hard to observe the perpendicular microtubule in the ANE293K.The cellular phenotype of 507-2 was more similar to ANE293K rathser than rop6-2 single mutant Therefore,AN was genetically supposed to be a downstream component of ROP6 signaling pathway.Because there were no differences in the transcriptional level between AN and AN E293K,it was deduced that the mutated phenotype of ANE293K was attributed to point mutation-causing changes in biochemical properties of AN. |
