Function Analysis Of The Arabidopsis COG Complex Subunit COG6 In Pollen Tube Growth

Pollen tube growth is a polarized growth process whereby the pollen tube delivers sperm cells to the ovules for fertilization.In the process of pollen tube elongation,it is necessary to transport the new plasma membrane and cell wall components to the pollen tube apex to ensure the expansion of the apical cell wall.And Golgi apparatus plays a key role during this process.In plants,the molecular mechanisms of intra-Golgi vesicle transport and maintenance Golgi structure and fuction remain largely unclear.The conserved oligomeric Golgi(COG)has been implicated in tethering of retrograde intra-Golgi vesicles in yeast and mammalian cells.COG complex plays an important role in the development of embryo and the polar growth of pollen tube in Arabidopsis thaliana,and in penetration resistance of barley to the barley powdery mildew fungus.In this study,using genetic and cryptologic approaches,we demonstrate that Arabidopsis COG complex COG6 subunit which plays an very important role in the rapid growth of pollen tube.1.COG6 gene mutation affected the growth of pollen tube,resulting in male-specific transmission defect.Our initial attempt to screen the mutant harboring T-DNA insertions within the gene AtCOG6 identifies no homozygous mutant line.The progeny of self-fertilized cog6/+showed segregation between mutants and wild type at a ratio close to 1:1.Further genetic analysis revealed that the inheritance of male gametophyte was absolutely blocked,but the female gametophyte transmission was not affected.Alexander's staining showed that almost all cog6/+qrtl/-quartets had four viable pollen grains,scanning electron microscopy(SEM)and 4',6-Diamidino-2-phenylindole(DAPI)staining revealed that these pollen grains are normal in appearance and each contains two sperm cells and one vegetative cell.Therefore,pollen grain development was not affected by the cog6 mutations.In vitro germination,it was found that the germination rate was similar to wild type pollen,but the morphology of the mutants pollen tube was aberrant.The abnormal pollen tubes exhibit deformity and bursting phenotypes.In vivo germination also showed that it was unable to elongate in the style.The defects of pollen tube blocked male-specific transmission results in male sterility.In addition,the male sterile phenotype can be complemented by expression of COG6-GFP from the LAT52 pollen-specific promoter and COG6 genome DNA expression,indicating phenotypic male sterile mutant is caused by mutations in the COG6.2.COG6 gene was strongly expressed in pollen and pollen tubes,and COG6 was localize to the Golgi apparatus.The expression pattern of genes often indicates that it may function as a tissue site,the GUS staining experiment showed that the COG6 gene is constitutively expressed in Arabidopsis.COG6 were not expressed during pollen development until the binucleated stage and in the mature pollen and pollen tube was expressed strongly.The result of subcellular localization showed that the green fluorescence signals of COG6-GFP exhibited the same localization pattern as the red fluorescence signals of y-COP-mCherry,indicating that COG6 was located in the Golgi apparatus before and after germination.3.cog6 insertion mutant affects the morphology of Golgi apparatusWe analyzed the morphology of the Golgi apparatus labeled by EMP12-GFP and ERD2-GFP in cog6-/+plants using confocal microscopy.Results showed that Golgi in the mutant cells is small dotes or connected rings,while in the wild type is a typical long rod.These results suggest that COG6 mutation affects the morphology of Golgi apparatus,and the localization of protein in Golgi has changed.In addition,TEM results showed that the structure of Golgi apparaus is changed in mutant pollen grains,which the length of Golgi cisternae got shorter and Golgi cistemae polarity was not obvious.4.Cell wall materials required for pollen tube growth are deposited incorrectly in the cog6 pollen tubesThe Golgi apparatus plays a crucial role in synthesis of cell wall polysaccharides and vesicle transport progress.Accordingly,we investigated the cell wall characteristics of in vitro germination pollen tubes using chemical staining approaches.In the mutant plants,deposition of callose and pectin in the mutant pollen tube wall is abnormal,and the distribution of cellulose was not changed.Therefore,we determined that the callose and pectin deposition of the pollen tube wall resulted in the abnormal morphology of pollen tubes,which causes an absolute,male-specific transmission defect and male sterility.