However, the precise role and functional significance of the annexin5–actin interaction was difficult to establish in this study because the pollen grains and tubes in the Ann5-overexpressing lines displayed a phenotype similar to that of wild-type when Lat B was used to destroy the F-actin organization in the pollen cell. Pollen germination and tube growth require continued fusion with the plasma membrane by trafficking vesicles originating from the Golgi apparatus. To investigate the function of Ann5 in pollen cellular processes, we treated pollen cells in Ann5-overexpressing lines with BFA,Candesartan an inhibitor of secretion and vacuolar protein transport. BFA not only promotes a rapid release of COPI coat proteins from the Golgi apparatus into the cytosol and blocks ER to Golgi transport but also affects trafficking in the endocytic pathway resulting from disruption of the Golgi apparatus. Thus, BFA interferes with endomembrane trafficking and strongly inhibits pollen germination and pollen tube growth in a dosedependent manner. For example, BFA obstructs the secretion of cell wall material and leads to growth arrest in tobacco pollen tubes. Moreover, the inhibited secretion and enhanced endocytosis that are induced by BFA are responsible for the retarded growth of pollen tubes of Picea meyeri. On the other hand,Ipratropium Bromide BFA possesses some secondary effects that inhibit membrane traffic in the secretory and endocytic pathways. As has been previously reported, BFA can dissipate the typical, oscillating tip-focused calcium gradient that is normally associated with pollen cell growth. In this study, we demonstrated that both pollen germination and pollen tube growth of Ann5-overexpressing plants significantly increased their resistance to BFA treatment compared with the control line. Vesicles shuttle among the endoplasmic reticulum, Golgi apparatus, plasma membrane and endosomes in plant cells. In general, there are two steps for a pollen cell to transport its cargo. First, trafficking vesicles arrive at the target membrane along microfilaments that provide tracks for vesicle movement. It has been well established that the growing pollen tube possesses a ‘‘tip-focused‘‘ gradient of free calcium, in which the cytosolic concentration of free Ca2+ extends from 2–10 mM to 20–200 nM.