Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm

The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (C-m) to monitor membrane changes in protoplasts from Vicia faba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 mu M) at constant ambient osmotic pressure (pi(o)) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (f(FM,i)) increased with reference to the total fluorescence (f(FM,t)) by 7.4 x 10(-4)min(-1). This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of pi(o) by 200 mosmol kg(-1) caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7,9%. Over the same time f(FM,i)/f(FM,t) increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm, Combined measurements of C-m and total fluorescence of a protoplast (f(FM.p)) showed that an increase in pi(o) evoked a decrease in C-m but no change in fFM.p This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 mu m) with FM1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area.