Functional Research Of Exocyst Complex Subunit SEC6 In Polar Auxin Transport And Vacuole Biogenesis In Arabidopsis

Transport of material between organelles is mainly mediated by carrier vesicles.Tethering plays an important role in vesicle transport reaction,which determines the specificity of vesicle targeting.Exocyst is an an octameric vesicle-tethering complex which contains 8 subunits.Exocyst complex has multiple functions in plant cell such as cytokinesis,autophagy,cell polar localization and defence against pathogens et al.Previous research shows exocyst complex SEC6 participates in formation of contractile vacuoles in Chlamydomonas reinhardtii,cell wall integrity and is a determinant of hyphal branching in Candida albicans,cell plate formation in plant et al.In this study,we made use of two knockdown mutant of sec6(PRsec6)mutants to investigate the role of SEC6 in auxin transport and vacuole biogenesis.1.Exocyst complex subunit SEC6 participates in auxin transport and PIN proteinsrecyclingPolar auxin transport is an essential process for land plant development and plays key role in embryonic development,organ formation,apical dominance,gravitropism et al.Auxin polar transport maintains the auxin gradient from apex to base,generates higher auxin concentrations in specific cells/tissues maintain plants in different tissues auxin concentration difference,which regulates auxin gradients of many growth and development in plants.In this study,we made use of two pollen rescued sec6(PRsec6)mutants to investigate the role of exocyst complex SEC6 subunit in PIN proteins recycling and primary root development.We found that in PRsec6 mutants,primary root growth was retarded,lateral root initiation was compromised and auxin activity in primary roots was siginificant reduced.Primary roots were sensitive to exogenous auxin 1-napthalene acetic acid(NAA)but not 2,4-dichlorophenoxy(2.4-D).Recycling of PIN1 and PIN2 proteins from the Brefeldin A(BFA)compartment to the PM was delayed.Vesicles accumulated in the primary root tip cells,especially accumulated in the cytosol closed to the PM.These results further demonstrated that the exocyst complex plays an important role in PIN protein recycling and polar auxin transport in Arabidopsis primary root.2.Exocyst complex subunit SEC6 participates in vacuole biogenesisPlant vacuoles play roles in storing storage proteins,inorganic ions,nutrients,maintaining osmotic pressure,pH and turgor during the developing of plant cells.The vacuole morphology is essential to the basic function of vacuole.In our study,we used tonoplast marker VAMP711-YFP to observe the vacuole morphology in PRsec6-1.We found lytic vacuoles in the mutant were become small and fragment.The transmission electron microscopic images also showed obvious fragment vacuoles in PRsec6-1.We also used prevacuole compartment marker RHA1-mCherry and ARA7-GFP to observe the PVC morphology in PRsec6-1.We didn't find abnormal PVCs in PRsec6-1.We believe exocyst complex subunit SEC6 takes part in the formation of vacuole.In addition,we also found the abnormal vacuoles in PRsec8-1 and exo84b.We suppose these subunits also play a role in vacuole formation.There are two novel facets in our study.First,even through previous study showed EX070A1 participates in polar auxin transport and PIN protein recycling,but the function of SEC6 in Auxin polar transport has not been reported.Second,in our study,the function of exocyst complex subunit SEC6 in vacuole biogenesis has been analysis and explored.