Protein Phosphatase 1 And/or 2A Is Involved In The Regulation Of Pollen Germination And Tube Growth In Picea Wilsonii

Pollen tube, a carrier of sperm cell during the process of sexual reproduction in seed plants, exhibits a typical polarized tip growth, and so its growth attracts much attention from scientists. A great number of reports have demonstrated that protein phosphatases (PPases) are involved in transduction pathway for ABA, pathogen infection, stresses and developmental signals. While the roles of PPases in pollen tube growth remain unclear due to the limited evidence. Therefore, by means of cytobiological methods and FTIR analysis, we conducted the investigation with a view to revealing the mechanism underlying which PP1/PP2A regulate the pollen tube development.The results showed that pollens contain abundant Ca~2+, and vacuole was the most important Ca~2+ resource in the Picea wilsonii pollens. When pollens were cultured in standard medium, the cytoplasmic Ca~2+ level decreased. But the decrease was efficiently blocked by treatment with 30 nM OA or 30 nM CalA. At nanomolar concentrations, OA and CalA clearly inhibited pollen germination and tube growth and led to abnormal morphology of pollen tubes.Normally growing pollen tubes of P. wilsonii displayed a typical tip-focused cytosolic free Ca~2+ gradient, whereas almost half (~45%) of the 30 nM OA- or 30 nM CalA-treated pollen tubes emitted faint fluorescence with a relatively leveled cytosolic Ca2+ gradient from the tip to the base of the tube. In some (-35%) of the 30 nM OA- or 30 nM CalA-treated tubes, the Ca2+ gradient had completely dissipated. One of important findings in the present study was that exogenous Ca2+ rapidly enhanced fluorescence intensity in the apex of control pollen tubes, but much more slowly in inhibitor-treated pollen tubes, indicating that the PP1 and/or PP2A was involved extracellular calcium uptake.TEM 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 the fusion rarely occurred in inhibitor-treated pollen tubes. FM4-64 labeling revealed that the endocytosis in tip region of pollen tube changed with the OA or CalA treatments, and a hypothesis is proposed to explain endocytotic mode in the growth of Picea wilsonii pollen tube.Callose was observed to be greatly accumulated in the tip regions of inhibitor-treated pollen tubes. The 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 concentration than in control pollen tube. 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. FTIR analysis further confirmed the changes in both pectin distributions and their relative contents.These data strongly indicate that the PPland/or PP2A is essential to Ca~2+ dynamics, exocytotic activity, and the biosynthesis and deposition of cell wall components, and that Ca~2+ dynamics presumably play a central role in the PP1 and/or 2A-modulated series of events in pollen tube development.