Endocytosis in elongating root tip cells of Lobelia erinus
Creators: name: Samuels, Anne Lacey
Publisher: name: University of British Columbia
Year: 2010

description: Endocytosis was measured along the axis of differentiation of epidermal and cortical root cells of Lobelia erinus from meristematic to fully expanded vacuolate cells. Both lanthanum and lead were tested as markers of endocytosis; lanthanum proved to be more effective. Lanthanum treatment produced electron dense deposits in the apoplast of the root, as well as coated pits, coated vesicles, smooth vesicles and multivesicular bodies within the cells. X-ray microanalysis was used to confirm the lanthanide nature of the deposits. In both secretory (meristematic and elongating cells actively depositing new cell wall material) and non secretory (mature vacuolate) cells the amount of endocytosis occurring was measured by counting the' number of lanthanum labelled vesicles/um² /cell. The amount of endocytosis correlated very well with the cell wall secretory activity. The highest amount of endocytosis was found in the elongating cells, with meristematic having an intermediate value. Mature, vacuolate cells had the least endocytosis. The relationship between endocytosis and secretory activity suggests that endocytosis may be acting to remove excess membrane material added during exocytosis of secretory vesicles. Cytochemical tests for polysaccharides were performed on both conventional transmission electron microscopy preparations and ultrarapidly frozen, freeze substituted preparations. The ultrastructural preservation was superior using the cryotechnique. The organelles involved in secretion of cell wall components were compared with the organelles associated with endocytosis in chapter 1. The Golgi showed distinct dictyosome polarity, small peripheral vesicles and an elaborate trans Golgi network. Vesicles in the cytoplasm displayed diverse staining properties. One population of larger, densely staining vesicles located on the trans Golgi and in the cortical cytoplasm was interpreted to be secretory vesicles. Microtubules were disrupted with colchicine to test the importance of these cytoskeletal elements in endocytosis. Immunofluorescence was used to determine the concentration of colchicine which disrupted cortical microtubules in these cells. After colchicine treatment, there were fewer endocytotic vesicles and less lanthanum label in the multivesicular body, suggesting microtubules are involved in endocytosis during elongation. Cell wall pore size was tested using electron dense apoplast markers. Partial enzyme digestions were used to find which components of the cell wall determine porosity. Pectin was found to be the most important component, while cellulose and protein did not seem to effect porosity in these primary roots. The porosity of the wall was interpreted in terms of the intact wall ultrastructure. Ultrarapid freezing, freeze substitution were used to reexamine endocytosis in elongating cells. Using lanthanum as a marker for endocytosis, the results of the earlier, conventional TEM study were supported and extended. In addition to coated and smooth vesicles, multivesicular bodies were labelled. The instantaneous preservation of the endomembrane system allowed the localization of label in the partially coated reticulum and in vesicles associated with the Golgi as well. The partially coated reticulum and multivesicular body are proposed to represent plant endosomes. When interpreted in conjunction with the secretion study, it can be suggested that the heterogenous populations of vesicles occur in the cytoplasm of the elongating Lobelia erinus root cells.