INDUCTION OF MEMBRANE LEAKAGE IN PLANT CELLS
Development of a method of inducing leakage of cellular membranes would be of great advantage for studies of the differential osmotic adjustments of the main cellular compartments. In the case of micro-organisms the formation of small holes is induced in the cell membrane by suitable techniques, the simplest being 5 min incubation in 1% toluene in water or 1% toluene + 4% ethanol in water. This treatment is particularly mild: low molecular weight cellular solutes are induced to leak out without the general structural organization of the cell being disturbed.
We have exploited the fact that all cellular solutes absorb at 198 nm to monitor the separation of the high and low molecular weight solutes during gel filtration on Sephadex G25. Cellular solute solutions were lyophylized before the gel filtration in order to eliminate the toluene and the ethanol.
The profile of the gel filtration of the supernatant of crude Atriplex nummularia root homogenate. It shows 2 maxima, one around the void volume, the other about 5 fold greater around the total volume. A shoulder between these 2 peaks is also present.
The solutes retained in Atriplex roots after 5 min incubation in toluene-ethanol were examined by gel filtration of the supernatant of root homogenates. The Sephadex G25 profile showed 2 peaks, but the peak for the low molecular weight solutes was diminished.
The gel filtration profile of solutes leaked from Atriplex root by 5 min incubation in toluene-ethanol. Only the low molecular weight solute peak can be observed, indicating that toluene-ethanol leaked only low molecular weight solutes. Thin layer chromatography indicated that sugars, amino acids and short-chain organic acids were among the leaked solutes.
In the case of micro-organisms, reactions catalyzed by enzymes located in the cytoplasm have been studied, after toluene treatment, by the diffusion of substrates into the leaky cells and concomitant spectrophotometric measurement of the rate of formation of the products diffusing out. We have examined whether substrates may similarly diffuse through the "pores" produced in the cellular membranes of plant roots by toluene treatment.