Abstract
Cultured tobacco (Nicotiana tabacum var Wisconsin 38) cells adapted to grow under osmotic stress synthesize and accumulate a 26 kilodalton protein (osmotin) which can constitute as much as 12% of total cellular protein. In cells adapted to NaCl, osmotin occurs in two forms: an aqueous soluble form (osmotin-I) and a detergent soluble form (osmotin II) in the approximate ratio of 2:3. Osmotin-I has been purified to electrophoretic homogeneity, and osmotin-II has been purified to 90% electrophoretic homogeneity. The N-terminal amino acid sequences of osmotins I and II are identical through position 22. Osmotin-II appears to be much more resistant to proteolysis than osmotin-I. However, it cross-reacts with polyclonal antibodies raised in rabbits against osmotin-I. Osmotin strongly resembles the sweet protein thaumatin in its molecular weight, amino acid composition, N-terminal sequence, and the presence of a signal peptide on the precursor protein. Thaumatin does not cross-react with antiosmotin. An osmotin solution could not be detected as sweet at a concentration at least 100 times that of thaumatin which could be detected as sweet. Immunocytochemical detection of osmotin revealed that osmotin is concentrated in dense inclusion bodies within the vacuole. Although antiosmotin did not label organelles, cell walls, or membranes, osmotin appeared sparsely distributed in the cytoplasm.