Functional Analysis Of Arabidopsis Thaliana COG4 And Glo3p Type ArfGAP In Pollen Tube Growth

Vesicle transport is an important form of intracellular transport of eukaryotic cells.It can be divided into four steps:vesicle formation,transport,tethering and fusion.Tethering is the process by which the vesicles are initially contacted and identified by the target membrane,and is mainly mediated by tethering factors.The COG complex(Conserved Oligomeric Golgicomplex)is a multi-subunit tethering factor complex located on the Golgi apparatus.In Arabidopsis,the COG3 and COG8 subunits play a role in the maintenance of Golgi morphology and the polar growth of pollen tubes.In this paper,the interaction between Arabidopsis COG4 and Glo3P ArfGAP protein and the growth of pollen tube were studied.The main results are as follows:1.COG4 gene expression and COG4 protein localizationThe results of RT-PCR and GUS staining show that COG4 gene was highly expressed in pollen.COG4-GFP was localized on the Golgi apparatus in tobacco epidermal cells..2.cog4 mutation leads to pollen tube growth defectsGenetic analysis of the cog4/+mutant showed that the male gametophyte was sterile and the transmission efficiency of the female gametophyte was also significantly reduced.Transfer the full-length gene of COG4 into the cog4/+mutant can complement the male gametophytic sterility and the semi-sterile phenotype of the female gametophyte,indicating that the cog4/+mutant gametophytic dysplasia is caused by the COG4 gene mutation.Scanning electron microscopy(SEM),Alexander staining and DAPI staining were used to observe the morphology,pollen activity and nuclear division of pollen grains in cog4/+mutants.There was no significant difference compared with wild type,indicating that the development of cog4/+pollen was normal.The cog4/+ mutant pollen germinated normally,but 60%of the pollen tube showed a deformity and burst phenotype in the growth of pollen tube.Therefore,male gametophytic sterility of the cog4/+mutant is caused by defects in pollen tube growth.3.cog4 mutation leads to abnormal Golgi morphologyBy fluorescence observation,we found that the EMP12-GFP-labeled Golgi has a rod-like structure in wild-type pollen,86%of the pollen with an Golgi aspect ratio of 2.0-3.5,and 18%of the cog4/+mutant pollen.It is indicated that in the mutant pollen,the morphology of the Golgi apparatus has changed.Transmission electron microscopy of high pressure freeze-fixed-frozen replacement samples revealed that the Golgi cistemea width became shorter in the cog4/+ mutant pollen,and the distance between each bubble became larger,and the Golgi bubble lost cis-trans polarity.Mutations in the COG4 gene result in abnormal morphological structure of the Golgi apparatus.The localization of COPI vesicle-coated protein y-COP-GFP and Arfl family small G protein Arf1c-GFP in cog4/+mutant pollen was affected and dispersed in the cytoplasm.4.Interaction between Arabidopsis COG4 protein and Glo3P-type ArfGAPIn order to further explore the function of COG4 gene,we screened the Arabidopsis thaliana yeast two-hybrid DNA library with COG4 protein as a bait,and obtained an interaction protein AGD8(ArfGAP domain 8).In Arabidopsis,AGD8 is homologous to AGD9,AGD10 and closely related to Glo3p in yeast,and is called Glo3p-type AGD.Yeast two-hybrid,luciferase complementary imaging(LCI),GST Pull-down experiments demonstrated that COG4 protein interacts with Glo3P-type ArfGAP,and bi-molecular fluorescence complementation(BiFC)indicates mutual interaction between COG4 protein and Glo3P-type ArfGAP.The site is located in the Golgi apparatus.The pLAT52::AGD8-GFP was transformed into cog4/+ mutant,and the transgenic plants were selected.The results showed that cog4 mutation resulted in AGD8 to diffuse in the cytoplasm.This study demonstrates that Arabidopsis COG4 plays an significant role in pollen tube growth and Golgi morphology maintenance,and that COG4 protein interacts with Glo3P-type ArfGAP in the Golgi apparatus.