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ABSTRACEOsmotin is a cationic protein with a pl2 value >8.2 and accounts for 10 to 12% of total cellular protein (as determined by spectrophotometric measurement of Coomassie stained gels) in NaCl and polyethylene glycol adapted tobacco cells (23). In unstressed unadapted cells, an immunologically related 26 kD protein with a lower pl is also synthesized but is not accumulated under normal growth conditions (23). Osmotin synthesis is regulated by abscisic acid, but its accumulation depends on adjustment of the cells to NaCl or to water stress (24). Others using cells obtained from our laboratory, have confirmed our observation of the accumulation of this protein in salt adapted cells (10, 16). We have isolated osmotin from NaCl adapted tobacco cells in sufficient quantities for further characterization. In this report we describe a simple and rapid procedure for purification oftwo forms ofosmotin, some oftheir properties and localization within the cell, and the occurrence of significant amino acid sequence homology between osmotin, the sweet protein, thaumatin, and two other stress-related proteins.MATERIALS AND METHODS Cell Culture. Cell suspension cultures of tobacco (Nicotiana tabacum L. var Wisconsin-38) adapted to 25 g/L NaCl (S-25 cells) were maintained in our laboratory as described (12). These cells were harvested and stored as dry powders after washing in cold acetone (23

In response to adaptation to NaCI, cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) synthesize a major 26 kilodalton protein which has been named osmotin due to its induction by low water potentials. To help characterize the expression of osmotin in adapted cells, a cDNA clone for osmotin has been isolated. Abscisic acid induces messenger RNA encoding osmotin. Levels of this mRNA in adapted cells are approximately 15-fold higher than in unadapted cells. Message for osmotin is present at constant levels through the growth cycle of adapted cells, while in unadapted cells, the level decreases during exponential phase of growth and increases again when the cells approach stationary phase. While abscisic acid induces the message for osmotin, a low water potential environment appears to be required for accumulation of the protein. An osmotin mRNA, a low water potential environment is required for preferential translation of osmotin mRNA or to reduce osmotin turnover (21).We report here the isolation of a cDNA clone encoding osmotin. We confirm the hypothesis that ABA induces transcription or stabilization of osmotin mRNA. We also confirm that some posttranscriptional event allows the enhanced accumulation of osmotin after adaptation to osmotic stress. We show that the stable altered expression of osmotin is not related to gene amplification or rearrangement and compare the complete amino acid sequence of osmotin with four other homologous proteins.

Cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands (molecular weights of 58, 37, 35.5, 34, 26, 21, 19.5, and 18 kilodaltons) increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands (54, 52, 17.5, and 16.5 kilodaltons) are reduced. Enhanced levels of 43-and 26-kilodalton polypeptides are present in both NaCl and PEG-induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced levels of 29-, 17.5-, 16.5-, and 1 1-kilodalton polypeptides and reduced levels of 58-, 54-, 52-, 37-, 35.5-, 34-, 21-, 19.5-, and 18-kilodalton polypeptide bands.Synthesis of 26-kilodalton polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate 3sS into a 26-kilodalton polypeptide during the later stage of culture growth beginning at midlog phase. The 26-kilodalton polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunologically cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26-kilodalton polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. From our results, we suggest an involvement of the 26-kilodalton polypeptide in the adaptation of cultured tobacco cells to NaCl and water stress.

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