How sterols affect protoplasts plasma membrane water permeability and their volume under osmotic shock

Protoplasts isolated from Arabidopsis leaves were used to study the initial stages of the plant cell response to osmotic stress. The role of sterols in these processes was investigated by their extraction from the protoplast plasma membrane in the presence of the oligosaccharide - methyl-β-cyclodextrin (MβCD). Depletion of membrane sterols caused by MβCD treatment did not alter protoplast volume under isosmotic conditions; however, volumes changed significantly when protoplasts were exposed to osmotic stress. Estimation of the plasma membrane water permeability coefficient (P_os), calculated from the initial rate of protoplast osmotic shrinkage, showed that control suspension is characterized by a high dispersion of the P_os values. However, P_os became more homogeneous after plasma membrane sterol depletion. Protoplasts were stained with FM 1-43 to assess how sterol extraction affects vesicular transport under osmotic shock. In order to determine the protoplast non-osmotic volume (V_b) steady-state volumes at different external osmolarities were fitted with linear dependences of the Boyle-van't Hoff (BVH) plot. It was found that sterol extraction is accompanied by a change in the slope of the BVH plot and a decrease in the apparent V_b. Several possible mechanisms behind the change in the protoplast volume and plasma membrane P_os regulation by sterols under osmotic stress are discussed.