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 endocytot ic ('osmocytotic') vacuoles are formed by invagination of the plasma m embrane. In thin section these are indistinguishable from other small vacuoles ('mini-vacuoles') which are formed by fragmentation of the la rge central vacuole. However, the two types of vacuole can be individu ally 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. Osmocy totic vacuoles do not fuse with the plasma membrane nor with the mini- vacuoles during a subsequent swelling phase. After several hours, osmo cytosed Lucifer Yellow gradually leaks out of the endocytotic vacuoles when protoplasts are returned to hypotonic conditions. This leakage i s 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 sit uation in planta, intact guard cells in epidermal strips were first al lowed to accumulate neutral red in their vacuoles and then subjected t o osmotic shrinkage in the presence of external Lucifer Yellow. Osmocy totic vacuoles were not formed, although the production of mini-vacuol es was frequently observed.