After the first mitotic division, a large vacuole is split into smaller vacuoles by invaginations of the tonoplast at several points from one side to the opposite side of the vacuole. The small vacuoles then disperse throughout the cytoplasm, and some fuse with the plasma membrane, which becomes intensely convoluted. The observation that many membrane-bound structures appeared between the convoluted plasma membrane and the intine implicated that exocytosis seems to occur in pollen. Our previous data demonstrated that Ann5 is predominantly expressed in pollen after the bicellular stage and pollen tube formation. In addition, downregulation of the function of Ann5 in transgenic RNAi lines led to severely sterile pollen grains at the bicellular stage of pollen development. However,Pancuronium dibromide the mechanisms underlying the function of Ann5 in pollen remain largely unclear. In this study, biochemical analyses indicated that Ann5 binds to phospholipid membrane and this association is stimulated by Ca2+. Moreover, Ann5 interacts with actin filaments in vitro. The pollen germination, pollen tube growth and cytoplasmic streaming of plants overexpressing Ann5 were more resistant to BFA treatment in a Ca2+-regulated manner in vivo. The defects observed in pollen grains of the Ann5UTR-RNAi lines could be only partially recovered by Ann5 mutants at Ca2+-binding sites, which further indicated that the Ann5 Ca2+-dependent phospholipid-binding activity is essential for pollen development. In plant cells, annexins represent approximately 0.1% of the total protein. Despite many years of investigation since the identification of the first plant annexin from tomato,Tolterodine tartrate the main physiological significance of plant annexins remains largely unknown, and most of our knowledge about plant annexins is focused on their structure and in vitro protein function. Ann5 is expressed abundantly in mature pollen grains and elongating pollen tubes. Furthermore, downregulation of Ann5 leads to a sterile pollen grain phenotype. Here, we provide both biochemical and physiological evidence to interpret the molecular mechanisms underlying the function of Ann5 in plant pollen development and pollen tube growth.