Protein Kinase C Is Involved In Regulating Polarized Growth Of Pollen Tube In Pciea Wilsonii

Reversible protein phosphorylation in cytoplasm is centralpast-translation reaction of regulating activity of enzymes and isubiquitous involved in various physiological processes. Recently,research on animal cell and yeast show that protein kinase C (PKC)regulates formation of cell morphology such as polarized growth viacalcium channels, actin skeleton and cell wail etc. However, there are fewreports concerning the mechanism of PKC regulating polarized growth ofpollen tube. In the present study, the effects of BIM on pollengermination and tube growth, ultrastructure, actin skeleton and cell wallwere studied using statistic analysis, ultrastructure observation,immunofluorescent labeling, as well as FTIR analysis. The main resultsobtained from the experiment were summarized as follows:1. Based on observation and statistic analysis of pollen germinationrate and tube growth, we optimized culture medium of pollendevelopment in vitro: 12% sucrose, 0.02% boron and 0.03% calcium.2. Using western immunoblotting, we detected a 55kDa peptide bymonoclonal antibody and confirmed that PKC was present in Piceawilsonii Mast. In contrast, treatment with BIM prevents the strongaccumulation of PKC in pollen tubes. The activity assays of PKC furtherconfirmed the result of western immunoblotting.3. Fluorescent labeling with Fluo-3/AM revealed that control tubesof P. wilsonii displayed a typical tip-focused cytosolic free Ca²⁺ gradientwhich was disturbed by BIM treatment, indicating that PKC is involvedin regulating Ca²⁺ influx and sustain Ca²⁺ gradient in tip zone.4. Fluorescent labeling with FITC-phalloidin showed a dense arrayof fine filaments, and a few thick bundles of filaments extended along thelength of the pollen tube beyond the collar into the apex. In the presence of BIM, the organization of filaments was obviously disturbed, resultingin a meshwork of short actin filaments.5. Transmission electron microscopy (TEM) revealed that thereexists a zone filled with numerous secretory vesicles at the extreme apicalzone of pollen tube growing in control medium. BIM induced: sharpdecline in the number of secretory vesicles and the increase of vacuole. Inaddition, the Golgi apparatus disintegrated and mitochondria membranesswelled, indicating that the secretory system was interfered.6. Using monoclonal antibody JIM5 and JIM7, we found thatesterified pectin was limited to the growing apex in pollen tubes whereasacidic pectin was deposited along the tube, except in the apical region.Fluorescence labeling demonstrated the presence of callose and cellulosein the apex and along the entire flanks of control pollen tubes. However,BIM treatment induced the decrease of esterified pectin, acidic pectin,cellulose and deposition of callose in tip zone. The decline of cell wallcomponents responded to BIM treatment was confirmed by FTIRanalysis, thus demonstrating that inhibitor induced weakening, of cellwalls and then the construction was disturbed.Taken together, these results suggest that PKC may participate in theregulation of cytoplasmic calcium (Ca²⁺) gradient and the organization ofactin, in secretory system as wellas the biosynthesis of cell wallcomponents, all processes that occur in the tip region of pollen tubes,providing a mechanistic framework for the functions of PKC in the tipgrowth of pollen tubes.