Endocytosis in soybean protoplasts and synchronous suspension cultures of soybean cells

Abstract

The double phosphate starvation method for inducing cell division synchrony in suspension cultures was applied to a rapidly growing suspension culture of soybean (Glycine max (L.) Merr.) cells. The synchrony of the culture was confirmed by changes in the mitotic index (NE) which were monitored using epifluorescence of the DNA stain, 4',6-dianiidine-2-phenylindole (DAPI). Synchrony was also assessed by examining the increase in cell fresh weight which was found to be restricted to the interphase portion of the cell cycle. Two rounds of synchronous cell division were obtained. The synchronous system thus established can be used to examine cell cycle related events in higher plants, such as endocytosis. Uptake of the fluorescent anion, Lucifer Yellow CH (LYCH), was investigated using a synchronized suspension culture of soybean. LYCH uptake increased during a short period immediately following cell division, reaching its peak as NU apparently decreased. As the cells started to divide, uptake of this anion was blocked. Using ethidium bromide, a vital dye of nucleic acids, cells determined to be mitotic did not accumulate LYCH in the vacuole. LYCH uptake changes may have resulted from changes in either fluid-phase endocytosis or anion transport capability of cells. Further studies relating fluid-phase endocytosis to the cell cycle are proposed using more reliable marker, e.g. BSA-gold in protoplasts and nanogold in walled plant cells. The FITC-polylysine was used as a fluorescently-labeled probe to measure the pH changes in endocytic compartment in soybean suspension culture. The ratio image of FITC-polylysine-labeled soybean protoplasts indicated that the earliest pH change following uptake of plasma membrane was an apparent alkalization. The pH near the plasma membrane, presumably in endocytic vesicles, remained alkaline. The pH of endosomes in the more distant cortical cytoplasm and in the trans-vacuole strands dropped to a relatively low value. Finally, the pH of larger organelles in the vicinity of the nucleus was more alkaline. The internalization of FITC-polylysine may follow an endocytic pathway similar to that taken by canonized fenitin (CF). The mechanism of alkalinization occurring during the early stage of interaction in soybean protoplasts is discussed in terms of changes in the activity of a H+/K AT'Pase at the plasma membrane and endosomes.