SUBCELLULAR-LOCALIZATION OF ACTIN DEPOLYMERIZING FACTOR IN MATURE AND GERMINATING POLLEN

Mature pollen contains enzymes which display unique functions during pollen germination and tube growth. We have previously isolated pollen-specific cDNA clones from lily and Brassica napus which encode for a putative actin depolymerizing factor (ADF). Polyclonal antibodies were raised against a synthetic peptide which corresponds to the carboxy terminal region of the ADF proteins. Immune blot analysis showed that the protein is preferentially expressed in the male gametophyte and is not detectable in other reproductive or vegetative organs, The protein was first detectable in 6.5 cm-long Easter lily buds and continued to reach a maximal level in mature flowers. Transmission electron microscopy data revealed that the ADF was associated with electron dense inclusions which were embedded in the storage bodies of mature pollen. During a 30 min in vitro pollen germination, some of the ADF remained attached to the inclusions. However, after 1 h incubation, most of the ADF was dissipated into the cytosol. Subcellular fractionation experiments confirmed the attachment of ADF with a small subcellular particle. The protein was stably maintained in cytosol during 16 h incubation, suggesting that the ADF may play an important role during pollen tube growth, Our finding, along with the previous observation that the electron dense inclusion is a storage place for minerals and proteins, suggests that the inclusion may be a common location for storing nutrients and enzymes which are utilized during pollen tube growth and fertilization.