Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells

An investigation was conducted into the feasibility of using enzymically isolated protoplasts from suspension-cultured cells of Nicotiana glutinosa L. to study ion transport. Transport of K(+) ((86)Rb), (36)Cl(-), H(2) (32)PO(4) (-) and (45)Ca(2+) from 1 millimolar salt solutions was determined after separation of intact protoplasts from nonabsorbed tracers by centrifugation through a Ficoll step gradient. Influx of K(+), Cl(-), and H(2)PO(4) (-) measured over a 30-minute period was reduced (up to 99%) by respiratory inhibitors such as 5 micrograms per milliliter oligomycin, 0.1 millimolar dinitrophenol, 0.1 millimolar cyanide, or N(2) gas. In contrast, Ca(2+) influx was not tightly coupled to respiratory energy production. The influx of K(+) was highest between pH 6.5 and 7.5 whereas the influx of H(2)PO(4) (-) and Cl(-) was greatest between pH 4.5 and 5.5. Influx of K(+) and Cl(-) was maximal at 35 and 45 C, respectively, and was almost completely inhibited below 10 C. Fusicoccin (0.01 millimolar) stimulated K(+) influx by more than 200% but had no effect on the influx of either Cl(-) or H(2)PO(4) (-). Apparent H(+) efflux, as measured by decrease in solution pH, was enhanced by K(+), stimulated further by 0.01 millimolar fusicoccin, and inhibited by 0.1 millimolar dinitrophenol or 5 micrograms per milliliter oligomycin. The measured ionic fluxes into protoplasts were similar to those obtained with intact cultured cells. The results indicate that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast, and that it is feasible to use isolated protoplasts for studies on ion transport.