Protein phosphatases 1 and 2A and the regulation of calcium uptake and pollen tube development in Picea wilsonii

To investigate the roles of protein phosphatases 1 and 2A in the development of pollen tubes of Picea wilsonii Mast., pollen grains were cultured in standard medium in the presence and absence of the protein phosphatase inhibitors okadaic acid and calyculin A. At nanomolar concentrations, these compounds blocked pollen tube growth, causing abnormal morphologies of the pollen tubes. Studies with Fluo-3 revealed that the inhibitors reduced the pollen-tube tip-to-base cytoplasmic calcium (Ca(2+)) gradient and arrested extracellular Ca(2+) uptake. The transmission electron microscope observations indicated that the fusion of paramural bodies with plasma membranes occurred frequently in the tip and sub-tip regions of control pollen tubes, but fusion rarely occurred in inhibitor-treated pollen tubes. Staining with aniline blue showed that callose accumulated in the tip regions of inhibitor-treated pollen tubes. Immunolabeling of pollen tubes revealed that acidic pectin epitopes recognized by the monoclonal antibody JIM5 were present in the tip region and on the flanks of the sub-tip in normal pollen tubes. In inhibitor-treated pollen tubes, these epitopes existed only in the extreme tip region and at higher concentrations than in control pollen tubes. The esterified pectin recognized by JIM7 was located preferentially at the extreme tip region in normal pollen tubes, but at basal sites in inhibitor-treated tubes. Fourier transform infrared (FTIR) analysis further confirmed the changes in acidic and esterified pectin distributions and their relative contents. These results suggest that protein phosphatase 1 or 2A, or both, are involved in the regulation of Ca(2+) uptake across the plasma membrane, in exocytotic activity and in the biosynthesis of cell wall components, all processes that occur in the tip region of pollen tubes and that control pollen tube development.