The Study On Arabidopsis Adaptor Protein-like Mutant Defective Pollen Tube Growth1

The interaction between pollen and stigma in angiosperm is the key step for pollination recognition, which determines the germination of pollen and the growth of pollen tube on the stigma. The study on the interaction mechanism between pollen/pollen tube and stigma is the basis for understanding the sexual reproduction and pollination recognition in angiosperm.Adaptor proteins (APs) serve to connect the clathrins with cargo molecules functioning in the process of endocytosis. In plant species, AP proteins are involved in various development regulations and physiological activities, such as in pollen tube growth. In this study, we identified an Arabidopsis T-DNA insertion mutant, defective pollen tube growth1 (dgl). Sequence analysis of protein showed that DG1 was an AP-like protein. It is conserved in plant, but has no homolog protein in animal species. In the self-pollinated progenies of the heterozygotic mutant plants, the segregation ratio of heterozygous to wild-type plants was about 1:1. However, the number of seeds in the silique of heterozygous mutant has no obvious difference with that of the wild type. The mutant allele could not be transmitted when the heterozygous mutant was the male parent, indicating that the dgl mutation led to the defective male gametophyte. The mutant pollen grains could not germinate and showed retarded growth both in vivo and in vitro.To verify the phenotype of mutant resulted from the dg1 mutation, we made the Lat52::amiDGl construct which was transformed into Arabidopsis. We found that the abnormal pollen phenotypes in amiDGl transgenic lines are similar with those of the dgl mutant.The qRT-PCR and GUS staining analysis indicated that DG1 was expressed at the apex of shoot and root of Arabidopsis, as well as the vascular tissues in leaf and root. However, we did not detect DG1 expression in developing and mature pollen. During the pollen germination, the expression of DG1 in the hydrating pollen and the growing pollen tube was increased, and was maintained at a higher level at the apex of growing pollen tube. In the transgenic pollen tube expressing pDG1::DG1-GFP, DG1-GFP signal was mainly located at the plasma membrane of the subapical region in growing pollen tube, and showed diffuse and faint distribution in the cytoplasm.After treated with the membrane traffic inhibitor Tyrphostin A23 (Tyr A23), the DG1-GFP signal disappeared in the pollen tubes expressing pDG1::DG1-GFP, but not treated with Wm and BFA. These results indicate that TyrA23 treatment selectively disrupts DG1 localization to the plasma membrane through breaking up the interaction with cargo proteins.