Functional Analysis Of COG Complex Subunit COG3 In Pollen Tube

Polarized and rapid pollen tube growth is essential for sexual reproduction of flowering plants. It is well known that many factors are involved in the regulation of tube growth, for example, calcium signal, small GTPases, the cytoskeleton, cell wall biosynthesis etc. Among the factors, the spatial distribution of cell wall polysaccharides are essential important for pollen tube growth. The conserved oligomeric Golgi (COG) complex is a multi-subunit hetero-oligomeric tethering complx that comprise eight subunits (Cogl-8),which mediate the Golg-associated retrograde transport in mammalian and yeast. But the research progress about the corresponding function advanced slowly in the plant cell.Previous studies showed that the cog7 null mutant didn't exhibit any apparent abnormalities until the late stage of embryogenesis in Arabidopsis. But the functions for the other subunits in the complex are largely unknown. Here we obtained a T-DNA insertion mutant by utilizing reverse genetics method, our finding suggested that Cog3, a subunit of COG complex, which was essential for the polar and rapid pollen tube growth.1. cog3 insertional mutations specifically block male gametophytic transmission by affecting pollen tube growthOur initial attempt to screen the mutant harboring T-DNA insertions within the gene AtCOG3 identifies no homozygous mutant line. The progeny of self-fertilized cog3/+showed segregation between mutants and wild type at a ratio close to 1:1. To clarify whether the defects were due to male or female transmission,reciprocal crosses between wild type and mutants were made. Mutations in COG3 completely suppressed the genetic transmission of male gametophytes and had no discernible influence on female gametophyte function. The time and site of gene expression often imply its function. So, it was necessary to determine the initiation time of COG3 expression in the male gametophytes before exploring the reason for the male sterility in cog3. The GUS staining wasn't observed until binucleate pollen stage and increasing gradually at trinucleate pollen stage. Strong GUS signals were also detected in pollen tube and embryo. Therefore we focused on the later stage of microsporegensis. For characterization of mutant phenotypes, cog3 were introgressed into the quartet1 (qrtl) mutant background. A quartet produced by the cog3 heterozygous mutant plant contained two mutant pollen grains (cog3) and two wild-type pollen grains (COG3). Pollen morphology, nuclear division, and pollen viability of the mutant plants were examined by SEM, DAPI staining, and Alexander staining, respectively.No obvious defects in the four pollen grains in the mature quartets from cog3/+;qrtl/qrt were found compared with those from qrtl/qrtl plant, indicating that the cog3 mutants don't affect pollen grain formation. Microscopic observations showed that cog3/+ pollen had nearly the same germination rate as wild-type pollen, but the pollen tube produced by the cog3 pollen grains grew abnormaly in vitro. The abnormal phenotype could be divided into three groups: ruptured pollen tube, short aberrant pollen tube and deformity pollen tube and the abnormally growing pollen tube occupied nealy 50% of the total population. Pollen germination was next investigated in vivo, wild-type pollen grains can produce three normal pollen tube. In contrast, the pollen grains from cog3/+; qrtl/qrtl produced no more than two normal pollen tube. To confirm that the phenotypes of cog3 were attributable to defects in COG3, complementation experiments were performed with cDNA of COG3. We used pollen-specific promoter LAT52 to drive COG3 and GFP expression and introduced this construct to cog3 heterozygous plant. LAT52::COG3-GFP could complement the phenotypes of cog3, indicating that the C-terminal GFP fusion does not affect the normal function of COG3. This suggests that the GFP fusion protein probably represent the correct localization of COG3 in vivo. Accordingly, transgenic Arabidopsis lines expressing LAT52:COG3-GFP was subjected to further analysis using confocal laser scanning microscopy (CLSM). The green fluorescence signals of COG3-GFP show the same localization pattern as the red fluorescence signals of Golgi marker RPA-DsRed2, indicting that COG3 were indeed present in the Golgi apparatus. In the transgenic offsprings, we still can't obtain the cog3 homozygous seedlings. Therefore, we tested immature siliques from heterozygous cog3 plants for the presence of abnormal seeds. We noticed there were greater heterogeneity in the stage of development within the same silique in contrast to the wild-type silique. Thus, COG3 seems to be essential for plant development.2. cog3 insertional mutations affected both Golgi morphology and intra-Golgi retrograde traffickingThe acute depletion of Cog3 subunit of mammalian COG complex resulted in the fragmented Golgi apparatus and mislocation of Golgi resident proteins. We wonder whether At COG3 play a similar role in maintaining Golgi structure and function at the molecular level. Therefore, we examined whether two fluorescently labled Golgi marker protein, GAUT14-GFP and GFP -EMP12, were properly located in the Golgi apparatus in the cog3 pollen grains. In wild-type pollen grains, we detected the fluorescence of GAUT14-GFP and GFP -EMP12 as desks and bars, which reflected typical Golgi morphology. By contrast, the fluorescence pattern was approximate to circle in cog3 pollen grains. Thus, the cog3 mutation might cause deformation of Golgi apparatus. To characterize more details about Golgi structural changes, the morphology of the mutant pollen grains was examined using TEM. Ultrastructural images of high-pressure frozen and freeze-substituted mutant cells showed changes in Golgi morphology. The changes can be divided into two categories: 74% of Golgi basically maintained the original Golgi structure,but the length of the cisternae got shorter and the gap between the neihbouring cisternae got wilder compared with the compact organization of the Golgi apparatus in wild-type pollen grains; 26% of Golgi appear to have reduced number of cisternae and we nearly couldn't distinguish their polarity. Immunogold electron microscopy was performed to confirm the localization of EMP12 in wild-type and mutant cell. Majority of gold particles were found in the cis- and medial-Golgi in wild-type pollen grains, whereas the corresponding particles spread throughout Goigi in the mutant cells. These observations suggested that COG complex in the plant cell also play a role in maintaining Golgi structure and mediating the retrograde transport within Golgi apparatus.3. Cell wall materials needed for pollen tube growth are targeted to wrong sites of the cog3 pollen tubesThe Golgi apparatus is not only a central sorting point within the secretory pathway.This compartment also plays a central biosynthetic role in processing of complex carbohydrate structure. Therefore, we investigated the cell wall characteristics of in vitro-grown Arabidopsis pollen tubes using a combination of immunocytochemical labeling and chemical staining techniques. In the mutant cells, immunocytochemistry confirmed that, the characteristic spatial profiles of de-esterified pectins and esterified pectins distribution in the pollen tube wall altered. Callose was observed not only in the cell wall of the flank but also in that of the tip of the pollen tube. By contrast, cellulose deposition was unaltered. Mislocation of callose and pectin altered the distribution of the mechanical properties in pollen tube cell wall, which resulted in various abnormal phenotypes in the mutant cells, such as ruptured pollen tube, short aberrant pollen tube and deformity pollen tube.4. Subunit architecture of the COG complexThe COG,an evolutionarily conserved protein complex, is required for the tethering of vesicle on the rib of Golgi apparatus. The complex comprised of eight subunits that divided into two sub-complexes in yeast and mammalian cell, which taken on bi-lobed structure which was determined using various approaches. Bidirectional yeast two hybrid assay were used to determine the spatial interaction patterns between the different COG subunits, we tested all of the 91-1=90 possible pairwise combinations of COG subunits. The interactions obtained so far identify five possible COG subassemblies,all of them containing three subunits(Cog2/3/4,Cog3/5/8,Cog5/6/7,Cogl/3/8 and Cog5/6/8),the subassembly,Cog3/5/8, seemed to play a central role in the organization of the plant COG complex and the assemble pattern of COG subunits(Cog2/3/4, Cog5/6/7 and Cogl/8) seemed to conserved in the whole eukaryote.