The Dynamics Of Endocytosis/Exocytosis With Reference To Their Effects On Cell Wall Construction In Pollen Tubes Of Picea Meyeri

Pollen tube, a carrier of sperm nuclei during the process of sexual plant reproduction in flowering plants, is a highly polarized, rapidly tip-growing plant cell, and thus is an ideal system for studying the molecular mechanism involved in the regulation of cell growth. As compared with angiosperm, pollen grains of gymnosperms germinate and growth slowly. However, the mechanism of pollen germination and tube growth in coniferous species remains speculative. Thus, with BFA treatment, the endocytosis/exosytosis process and cell wall construction in Picea meyeri pollen tube development was investigated by means of cytological, biochemical and physiological methods, with a view to revealing the regulation mechanism during this polarized tip growth. Firstly, the differences of cytoplasmic organization in pollen tubes between Picea meyeri and angiosperm were investigated. FM4-64 labeling showed a typical bright apical staining in normally growing Picea meyeri pollen tubes, while this FM4-64 staining pattern as detected in Picea meyeri was different from 'V-shaped apical staining' reported from angiosperms and the proportion of apical FM4-64 staining region to the whole pollen tube was much lower than that in angiosperms. Transmission electron microscopic (TEM) observation showed that the density of secretory vesicles at the tip zone in Picea meyeri pollen tube was much lower compared to angiosperm. In addition, unlike in angiosperm, the preferential occurrence of mitochondria was closed to tube wall, the dictyosomes did not show any specific zonation or accumulation in the tube, and the ER distribution was absent of orientation as well in Picea meyeri pollen tube. Secondly, We assessed the effects of Brefeldin A (BFA) on pollen tube development of Picea meyeri using FM4-64 as a membrane-inserted endocytic/recycling marker together with ultrastructural observation and Fourier transform infrared (FTIR) analysis. BFA inhibited pollen germination and pollen tube growth causing morphological changes in a dose-dependent manner. FM4-64 labeling showed a typical bright apical staining in normally growing Picea meyeri pollen tubes, while this apical staining pattern was rapidly lost in the presence of BFA. In contrast, the overall uptake of FM4-64 dye was about twofold over the control after BFA treatment, revealing that BFA stimulated endocytosis. Transmission electron microscopic (TEM) observation showed that the number of secretory vesicles at the tip zone dramatically decreased together with the disappearance of Paramural bodies, while the number of vacuoles and other larger organelles increased. Acid phosphatase assay (acPase) confirmed that the addition of BFA significantly inhibited secretory pathways. Therefore, BFA disturbed the secretory pathway through inhibiting exocytosis and stimulating during Picea meyeri pollen tube growth. Last, we measured the changes in the chemical composition of pollen tube walls after the disorder of secretory patmway induced by BFA treatment. FTIR microspectroscopy documented significant changes in the chemical composition of pollen tube walls that was grown in the presence of BFA. The content of proteins and polysaccharides decreased after BFA treatment, and in contrast to a lower reduction in protein content, there was a marked decrease in polysaccharide content among the three regions. Importantly, the reduction of polysaccharides in the newly formed tip region was far more obvious than in the already formed middle and basal regions. Further analysis indicated that BFA decreased the content of cell-wall glycoprotein to 60%, and SDS-PAGE analysis showed many cell-wall proteins did not express or had a lower content. Microscopic observations revealed that there was less cellulose in the walls when treated with BFA, while callose had accumulated in the tip regions of pollen tubes. Immunolabelling with LM6 antibody showed that in normal pollen tubes, AGPs deposited with a ring-like formation in cell wall, whereas pollen tubes treated with BFA exhibited a different fluorescently labeled pattern i.e the ring-like distribution lost. Acidic pectin, as detected by immunolabeling with JIM5, was observed at the whole pollen tubes grown in normal medium, however, in BFA-treated pollen tubes, the fluorescence labelled with JIM5 weakened, JIM5-reactive pectins accumulated within "BFA compartments". The above results suggest that that pollen tube growth rate mainly depends on the particular cellular organization at tip region. The smaller clear region and lowerdensity of secretory vesicles in Picea meyeri pollen tubes resulted in its slower tube growth. Although the cytoplasmic organization was different between gymnosperm and angiosperm, the vigourious endocytosis and exocytosis are required for normal pollen tubes in gymnosperm, as occurred in angiosperm. The disruption of secretory pathway further led to modifications in the composition of the pollen tube walls by affecting the deposition of proteins and polysaccharides and further resulted in the cease of pollen tube growth.