Moreover, Ca2+ has been reported to affect organelle and vesicle motility along actin filaments by inactivating the myosin motor through binding to its calmodulin light chain. In addition to the results showing that the Ann5G26EG28E and Ann5G257EG259E proteins exhibited significantly less phospholipid binding compared with Ann5 in vitro, the pollen germination, pollen tube growth and velocity of cytoplasmic streaming in Ann5G26EG28E- and Ann5G257EG259E-overexpressing plants were less resistant to BFA treatment than those in Ann5-overexpressing plants. These results further confirmed that Ann5 promoted endomembrane trafficking in response to BFA treatment and that this cellular process was modulated by Ca2+ fluctuations occurring within pollen cells. In addition, the abortion of pollen grains in the Ann5UTR-RNAi lines could be only partially recovered by Ann5G26EG28E and Ann5G257EG259E, while Ann5 at a similar transcript level could fully rescue the sterile phenotype. Although Ann5,Aripiprazole Ann5G26EG28E and Ann5G257EG259E were diffusely distributed throughout the pollen tube, the quantitative fluctuation and polar distribution of Ca2+ determined the differences in the phospholipid membrane binding activity of Ann5 in distinct spatial positions and developmental phases of pollen cells. Tight regulation of the endomembrane trafficking of Ann5 in pollen involved Ca2+ as a second messenger. Ann5 might act as a Ca2+ sensor and thus play an important role in controlling the processes of pollen development, germination and tube growth. In summary, our results clearly demonstrated that pollen germination, pollen tube growth and cytoplasmic streaming are more resistant to BFA treatment in a Ca2+-dependent manner in Ann5-overexpressing plants. Considering the results that Ann5 binds to membranes,Naratriptan Ca2+ and actin filaments in vitro, we suggest that Ann5 exerts a major influence on the physiological processes involved in pollen development and growth by modulating membrane trafficking in a Ca2+-dependent manner. Porcine transmissible gastroenteritis coronavirus is an animal coronavirus that causes severe gastroenteritis in young TGEV-seronegative pigs.