OSMOCYTOSIS AND VACUOLAR FRAGMENTATION IN GUARD-CELL PROTOPLASTS - THEIR RELEVANCE TO OSMOTICALLY-INDUCED VOLUME CHANGES IN GUARD-CELLS

Guard cell protoplasts were prepared from young leaves of pea plants. Under hypertonic conditions they shrink and large numbers of endocytotic ('osmocytotic') vacuoles are formed by invagination of the plasma membrane. In thin section these are indistinguishable from other small vacuoles ('mini-vacuoles') which are formed by fragmentation of the large central vacuole. However, the two types of vacuole can be individually recognized by labelling the central vacuole with neutral red and by performing the osmotic shrinkage with fluorochromes such as Lucifer Yellow-CH or Cascade Blue present in the extracellular medium. Osmocytotic vacuoles do not fuse with the plasma membrane nor with the mini-vacuoles during a subsequent swelling phase. After several hours, osmocytosed Lucifer Yellow gradually leaks out of the endocytotic vacuoles when protoplasts are returned to hypotonic conditions. This leakage is not prevented by probenecid at concentrations (20-50 mmol m-3) which do not give rise to pathological changes in protoplast ultrastructure. In order to determine the relevance of these observations to the situation in planta, intact guard cells in epidermal strips were first allowed to accumulate neutral red in their vacuoles and then subjected to osmotic shrinkage in the presence of external Lucifer Yellow. Osmocytotic vacuoles were not formed, although the production of mini-vacuoles was frequently observed.