Stable NaCl Tolerance of Tobacco Cells Is Associated with Enhanced Accumulation of Osmotin

Abstract

Osmotin is a major protein which accumulates in tobacco cells (Nicotiana tabacum L. var Wisconsin 38) adapted to low water potentials. Quantitation of osmotin levels by immunoblots indicated that cells adapted to 428 millimolar NaCI contained 4 to 30 times the level of osmotin found in unadapted cells, depending on the stage of growth. Unadapted cells accumulated low levels of osmotin with apparent isoelectric points, (pi) of 7.8 and >8.2. Upon transfer of NaCI-adapted cells to medium without NaCI and subsequent growth for many cell generations, the amount of osmotin declined gradually to a level intermediate between that found in adapted and unadapted cells. NaCI-adapted cells grown in the absence of NaCI accumulated both pi forms; however, the form accumulated by cells adapted to NaCI (pi > 8.2) was most abundant. Adapted cells grown in the absence of NaCI exhibited absolute growth rates and NaCI tolerance levels which were intermediate to those of NaCI-adapted and unadapted cells. The association between osmotin accumulation and stable NaCI tolerance indicates that cells with a stable genetic change affecting the accumulation of osmotin are selected during prolonged exposure to high levels of NaCI. This stable alteration in gene expression probably affects salt tolerance.NaCl stress evokes changes in the levels of several plant gene products (8,12,14,21,(24)(25)(26)(27)(28). Since none of these gene products have been shown to be unequivocally involved in physiological adaptation to NaCl stress, additional information on the regulation of their expression is needed (11). Adaptation of tobacco to high levels of NaCl induces the accumulation of several proteins, particularly one with an apparent mol wt of 26 kD termed osmotin (6). Osmotin is a cationic protein occurring in tobacco cells in water soluble (osmotin-I) and insoluble (osmotin-II) forms. Osmotin accumulates in vacuolar inclusion bodies of cells which have adapted to NaCl (23). It is localized also in the cytoplasm at much lower levels (23) The biological function of osmotin is not yet known. However, analysis of the amino acid composition and sequence of osmotin indicates substantial homology to the sweet protein thaumatin, the bifunctional maize a-amylase/proteinase inhibitor, and a pathogenesis related protein induced by tobacco mosaic virus infections (22,23). These latter three proteins are hypothesized to be involved in defense reactions of plants to pathogens (22).In this report, we show that NaCl-adapted cells grown in the absence of NaCl are able to grow and adapt to salt more rapidly than cells never exposed to salt. These cells continue to overaccumulate predominantly the higher pI form of osmotin. These results further support the hypothesis that enhanced tolerance of NaCl-adapted cells is at least in part related to accumulation of osmotin. These data also support the hypothesis that selection for salt-tolerant cells has occurred during prolonged growth in medium containing high NaCl concentrations. MATERIALS AND METHODS Culture ...

Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures

“…It is hypothesized that these putative osmoprotectants may provide relief from the osmotic and/or salt stress (9, 12, 31). In suspension culture cells of both glycophytes and halophytes, these compounds accumulate, as do, presumably, osomprotectant proteins, after salt stress (2,14,18). This strongly suggests that the ability to osmoregulate via the accumulation of these products is accommodated by individual cells (5,25,27).…”

Osmotin Gene Expression Is Posttranscriptionally Regulated

“…Hybridization, filter washing, and autoradiography were performed as described elsewhere (16). NEPHGE, SDS-PAGE, and Immunoblots of Gels NEPHGE or SDS-PAGE was performed essentially as described by LaRosa et al (12), with the following provisos: The samples were run on duplicate gels, one gel was stained with Coomassie blue to test for even gel loading and protein integrity, the other was used for immunoblotting. The osmotin antibodies were partially purified chicken immunoglobulin Y.…”

Characterization of a Salt-Responsive 24-Kilodalton Glycoprotein in Mesembryanthemum crystallinum

“…Indeed, polypeptides serologically related to SRgp24 were detected on immunoblots under several stress conditions, including aging, wounding, and treatment with chemical inducers (Yen, 1993). After the involvement of osmotin in osmotic adaptation in tobacco was suggested (LaRosa et al, 1989), several reports indicated that osmotin and osmotin-like proteins were induced during protoplast isolation (Grosset et al, 1990a(Grosset et al, , 1990b, in vitro flower formation (Neale et al, 1990;Richard et al, 1992), and pathogen infection (Pierpoint et al, 1987;Woloshuk et al, 1991). One study revealed that a PR-5 protein, AP24, exhibited antifungal activity and was highly homologous to osmotin (Woloshuk et al, 1991).…”