Immunolocalization of PIP aquaporins in protoplasts from the suspension culture of sugar beet mesophyll under isoosmotic conditions and osmotic stress

Protoplasts from the suspension culture of sugar beet (Beta vulgaris L.) mesophyll were found to change their volume in response to short-term osmotic stress. When the sorbitol concentration in the external medium was increased 1.5-fold (from 0.4 to 0.6 M) or decreased from 0.4 to 0.25 M, the volume of protoplasts decreased and increased, respectively, by 55–60%. These changes started immediately after the shift in osmoticum concentration and completed within 1–3 min. In the presence of an endocytosis marker FM1-43, its fluorescence increased conspicuously after replacement of isotonic medium with the hypotonic solution but did not change after the substitution with hypertonic medium. At the same time, the hypertonic shrinkage of protoplasts was accompanied by accumulation of fluorescent material in the periplasmic space. The western blot analysis with the use of immune serum for conservative sequence of PIP-type aquaporins revealed their presence in the plasmalemma and intracellular membranes. This conclusion was confirmed by indirect immunofluorescence microscopy: the membrane-bound secondary antibodies labeled with a fluorescent probe Alexa-Fluor 488 were distributed comparatively uniformly on the boundary between the plasmalemma and the protoplast internal compartment. As evident from micrographs of protoplasts exposed to the hypotonic treatment, the fluorescence was smoothly distributed over the plasmalemma after protoplast swelling but its intensity was not so bright. The protoplast shrinkage during the hypertonic treatment resulted in heterogeneous alternate distribution of fluorescent and transparent plasmalemma regions, the fluorescence of stained regions being very intense. The results are interpreted as the evidence that the short-term osmotic stress activates exo-and endocytosis. The migrating regions of the plasmalemma were depleted of PIP-type aquaporins; hence, the induction of osmotic stress has no effect on the amount of this type aquaporins in the plasma membrane.