Alterations in cell membrane structure and expression of a membrane-associated protein after adaptation to osmotic stress

Cultured tobacco cells (Nicotiana tabacum L. cv. Wisconsin 38) adapted to NaCl exhibited significant morphological and physiological changes. Adapted cells remained smaller and more isodiametric at maturity than unadapted cells. The vacuole increased in size relative to the cytoplasm and an extensive network of transvacuolar membrane strands developed. These changes altered the surface contact area between the cytoplasm and the vacuole substantially. In addition, the network of Hechtian strands that anchor the cortical structure to the cell wall became more extensively branched possibly facilitating surface contact of the cytoplasm to the extracellular matrix. Many changes in membrane proteins could also be identified after NaCl adaptation. In particular, a 50-kDa protein that is associated with the plasma membrane and tonoplast was induced during adaptation. Immunocytochemical localization indicated that this 50-kDa protein is associated with Golgi vesicles. By immunoscreening using anti-50-kDa antibody, a 1.71-kb cDNA clone (p50C) was isolated from a λ-ZAP cDNA expression library. The sequence of p50C did not show any significant identity with other genes. Because of the very low abundance of the p50C message, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze p50C gene expression. Immunoblot and quantitative RT-PCR analyses indicated that the expression of this gene is regulated developmentally since the mRNA and protein increased with age in salt-adapted cells but decreased with age in unadapted cells. Also in tobacco plants, p50C mRNA was more abundant in younger leaves than in older leaves. The gene was responsive to NaCl in tobacco cells and to ABA in tobacco seedlings.