The Preliminary Research Of Arabidopsis ECH And YIP Proteins In Regulation Of The Morphogenesis Of Cotyledon Epidermal Cells

Arabidopsis thaliana cotyledon epidermal pavement cells(cotyledon pavement cell,PC)have special shapes,with a jigsaw-puzzle appearance and interdigitation status.It was reported that interdigitation of plant PCs formed by anisotropic cell growth,which depended on ROP small GTPases(Rho-related GTPase of plants)regulation,mainly including ROP2 and ROP6 proteins.ECH(ECHIDNA)and YIP(YPT/RAB GTPase Interacting Protein)were found in the previous researches,which interacted with each other.The two proteins localized in the trans-Golgi network(TGN),forming a TGN complex in Arabidopsis thaliana,which regulated transportation of cell wall ingredients in cell wall growth process.They can influence cell elongation and change cell shapes,for this reason,we postulated that ECH/YIP may be associated with ROP in some ways.However,the mechanisms that ECH and YIP regulating leaf pavement cell morphogenesis via ROP were still poorly understood.In the study,we chose Arabidopsis cotyledon as a material to investigate the relationship between ECH/YIP and ROP2/ROP6,as well as cytoskeleton.First,we obtained ech/GFP-ROP2,yip4 a yip4b/GFPROP2,ech/GFP-ROP6,yip4 a yip4b/GFP-ROP6 by hybridization in the study,whose hybridization plants were dwarf and highly branched,besides cotyledons were slimsy and pointed,phenocopied the phenotypes of ech and yip4 a yip4b mutants.When ECH or YIP was absent,in consequence,we found that ROP2 or ROP6 expression was affected not only in the cytoplasm but also in the plasma membrane.To further test this conclusion,we verified the mRNA expression of ROP2 and ROP6 were reduced,which explain that ECH and YIP influcence expression of ROP2 and ROP6 directly or indirectly at transcriptional level.Meanwhile,we found that ROP6 total protein was reduced in ech and ROP2/ROP6 were reduced in yip4 a yip4b,except that ROP2 had increased expression level in ech.Afterwards,to investigate the ROP activity,we conducted Pull-down experiments and discovered that ROP2 activities were greatly decreased in ech and yip4 a yip4b mutant.Interestingly,ROP6 activities were greatly decreased in yip4 a yip4b while increased in ech mutant.Thus,the above results demonstrated that ECH and YIP were required for the activation of ROP2 and ROP6 in PCs.Because of ROP regulating the organization of the cytoskeleton,therefore,from the perspective of cytoskeleton,we further found that cortical microtubule become more orderly and actin microfilament become more uniform and slender in ech or yip4 a yip4b.The randomness of cortical cytoskeleton reduced in the ech and yip4 a yip4b mutants.Meanwhile,the research found a punctate pattern of ECH and YIP proteins that colocolized with TGN compartmants in transient expression experiment.In the respect of tissue expression pattern of ECH,YIP4 a and YIP4 b,we took advantage of GUS staining assays to track those genes expression at different stages of cotyledon development,which provided foundation for ECH and YIP functional research in PC morphogenesis.Taken together,inspired by ECH and YIP regulating cell elongation,with the knowledge that ROP influence the cell morphogenesis and interdigitation,our findings demonstrated that ECH and YIP play an important role in ROP expression and activation process.Furthermore,they altered the normal formation of PC lobes and indentations.Besides,we found that ECH and YIP can influence the alinement of microtubule and microfilament,and participate in cell morphogenesis.The research established a new horizone for further exploring the function of ECH and YIP in PC morphogenesis.