Functional Analysis Of Arabidopsis EXO70C1 In Pollen Tube Growth

Vesicle trafficking is a fundamental process participated in a wide range of biological processes in all eukaryotic cells,which mainly consists of budding,translocation,tethering and membrane fusion.The tethering mediates the initial contact of the transport vesicle with the target membrane,spatio-temporally regulating SNARE-mediated membrane fusion,and therefore is the key to regulate the polarized membrane trafficking.Nine multisubunit tethering complexes(MTCs)had been identified to tether vesicles at distinct cellular compartments or plasma membrane,and the exocyst complex facilitates the tethering of post-Golgi vesicles to the plasma membrane.Exocyst complex consists of eight subunits,among which the EXO70 subunit interacts with membrane lipids and Rho GTPase,mediating the assembly of the complex at the target membrane.Multiple EXO70 genes are present in genomes of terrestrial plants and supposed to involve in cell-specific exocytosis.Our preliminary results show that EXO70C1,a member of the EXO70 family in Arabidopsis thanliana,exhibited a high expression in mature pollen,but with its function unknown.In this thesis,the reverse genetics was employed to investigate the biological function of EXO70C1 by observing the phenotypes of T-DNA insertion mutant during pollen tube growth.And the localization and dynamics of EXO70C1 were examined in growing pollen tubes.The candidate proteins interacted with EXO70C1 was screened by yeast two-hybrid technique.The main results and conclusions are as follows:(1)Genetic analysis of exo70c1-2By analyzing the phenotypes of the exo70c1-2 mutant,we found that the in vitro germination rate of the exo70c1-2 mutant was significantly higher than that of the wild type,but the growth rate of the pollen tube was significantly lower than the wild type,indicating that EXO70C1 is a negative regulator of pollen germination,but a positive regulator in pollen tube growth.We further tested the self-crossing separation ratio and pollen transmitting efficiency in vivo,our results showed that the self-crossing separation ratios of the heterozygous mutants appeared to be normal and segregated in a 1:2:1 ratio,and the pollen transmitting efficiency was not significantly different from that of the wild type.It is speculated that the compromised pollen tube growth carrying EXO70C1 mutation can be neutralized by the enhanced pollen germination of mutant.To confirm the mutant phenotypes are caused by defective EXO70C1,the fragment of EXO70C1p::EXO70C1-GFP were introduced into exo70c1-2 plants,we found both phenotypes of pollen germination and growth rate were restored,indicated that the phenotype of the mutant is due to the EXO70C1 mutation,and the GFP tagged protein can replace the function of endogenous EXO70C1.(2)Cytological study of EXO70C1-GFPThe cytological observation of EXO70C1-GFP transgenic plant pollen were performed by laser confocal microscopy,and results showed that EXO70C1-GFP was found in the cytosol of mature pollen grains and pollen tubes,accumulated GFP signals were observed at the tip of the growing pollen tube.In order to confirm the membrane localized EXO70C1,we observed the plasma membrane of the tip by total internal reflection fluorescence technique.It was found that EXO70C1-GFP was spotted on the plasma membrane at the tip of the pollen tube,and the signal was retained at the plasma membrane for about 0.1 second.To further investigate the role of EXO70C1 in pollen tube,we used FM4-64 dye to label the endocytic pathway and found that EXO70C1-GFP did not co-localize with FM4-64 signal,indicating that EXO70C1 may not function in the endocytic pathway.(3)Screening the interacting protein of EXO70C1The identification of the EXO70C1 interacting proteins will help us to understand the regulation mechanism of EXO70C1.Since EXO70C1 is a cytoplasmic protein with transient localization on plasma membrane,we chose the Split-ubiquitin yeast two-hybrid methods to screen EXO70C1 interacting proteins.We created c DNA library with mature pollen,and screened the library with EXO70C1 as bait.A positive clone was identified to show identical sequence with fragment of SYP131.SYP131 is a putative t-SNARE protein specifically expressed in pollen.Thus,it is speculated that EXO70C1 and SYP131 synergistically regulate vesicle tethering and fusion processes.(4)Linkage analysis exo70c1-1Previous reports showed that exo70c1-1,the first identified allele of EXO70C1,exhibited defects in pollen development and primary root growth.Further cytological phenotypic analysis found that there were huge pollen grains and abortive pollen grains in exo70c1-1,and the transmitting efficiency of the mutant pollen decreased.However,our results revealed that the defective phenotypes of exo70c1-1 were not restored by wild type EXO70C1.Linkage analysis experiments demonstrated that the phenotype of primary root growth and pollen development defects of exo70c1-1 was not tightly linked to the EXO70C1 deletion,indicating that the observed phenotypes were possibly caused by other allele.