A study of cell wall regeneration by Douglas-Fir [Pseudotsuga menziesii (Mirb.) Franco] protoplasts from suspension cultures.

by Robinson, Kim William

An examination was made of the constituents deposited in the regenerating cell wall of nondividing Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] protoplasts. Suspension cultures of Douglas-fir cells derived from both needle and hypocotyl tissues were treated with cell wall degrading fungal enzyme preparations to generate uniform populations of protoplasts. Both L-(U-14C)-proline and D-(U-14C)-glucose were separately fed to the protoplasts and their uptake into the regenerating cell wall was monitored for up to 6 days. Proline was observed to incorporate into the wall immediately, but the glucose uptake was initiated only after about 30 hours. Uptake of the two precursors suggested that both protein and polysaccharide components were deposited during the period studied. The beginning of glucose incorporation coincided with a measurable linear increase in cell wall weight. The major sugars detected in the hydrolyzed regenerating wall were glucose, galactose, mannose and arabinose; glucose was dominant. All of the sugars except mannose were deposited only after two days of protoplast culturing. X-ray diffraction analysis of wall samples revealed amorphous patterns, suggesting the absence of crystalline cellulose. Further analysis of acetolysis demonstrated that cellulose was not present. Microscopical techniques, however, revealed the probable deposition of callose and pectin. Wall-bound protein was extracted by two different methods, one of which removed two proteins which isoelectrically focused at pH 4.2 and 4.4 but sedimented as one during ultracentrifugation. Amino acid and sugar analysis revealed that the regenerated wall glycoprotein was abundant in glycine, alanine, glucose, and galactose. The general conclusion of this study is that the regenerated cell wall of Douglas-fir protoplasts originating from suspension cells was synthesized in response to the stress of cell wall removal. Callose, usually associated with wound tissue, -2was detected in the material found on the protoplast surface. The cell wall glycoprotein isolated from the regenerating wall was compared with and found to be similar to a wound-induced cell wall glycoprotein reported in Phaseolus vulgaris, [Brown, R. G. and Kimmins, W. C., Phytochem. 17:29-33(1978)]. Given the absence of cell division and cellulose, it appears that the regenerated wall material of these cultured protoplasts differs considerably from normal cell walls. -3-