Showing papers by "Ray A. Bressan published in 1989"

Molecular Cloning of Osmotin and Regulation of Its Expression by ABA and Adaptation to Low Water Potential

Abstract: In response to adaptation to NaCl, 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 osmotic shock to unadapted cells does not increase the amount of message or protein present most likely because this treatment does not induce immediately the accumulation of abscisic acid. The increased expression of osmotin in adapted cells is not correlated with an increase in osmotin gene copy number. Osmotin is homologous to a 24 kilodalton NaCl-induced protein in tomato, as well as thaumatin, maize α-amylase/trypsin inhibitor and a tobacco mosaic virus-induced pathogenesis-related protein.

Alteration of the physical and chemical structure of the primary cell wall of growth-limited plant cells adapted to osmotic stress.

Abstract: Cells of tobacco (Nicotiana tabacum L.) adapted to grow in severe osmotic stress of 428 millimolar NaCl (−23 bar) or 30% polyethylene glycol 8000 (−28 bar) exhibit a drastically altered growth physiology that results in slower cell expansion and fully expanded cells with volumes only one-fifth to one-eighth those of unadapted cells. This reduced cell volume occurs despite maintenance of turgor pressures sometimes severalfold higher than those of unadapted cells. This report and others (NM Iraki et al [1989] Plant Physiol 90: 000-000 and 000-000) document physical and biochemical alterations of the cell walls which might explain how adapted cells decrease the ability of the wall to expand despite diversion of carbon used for osmotic adjustment away from synthesis of cell wall polysaccharides. Tensile strength measured by a gas decompression technique showed empirically that walls of NaCl-adapted cells are much weaker than those of unadapted cells. Correlated with this weakening was a substantial decrease in the proportion of crystalline cellulose in the primary cell wall. Even though the amount of insoluble protein associated with the wall was increased relative to other wall components, the amount of hydroxyproline in the insoluble protein of the wall was only about 10% that of unadapted cells. These results indicate that a cellulosic-extensin framework is a primary determinant of absolute wall tensile strength, but complete formation of this framework apparently is sacrificed to divert carbon to substances needed for osmotic adjustment. We propose that the absolute mass of this framework is not a principal determinant of the ability of the cell wall to extend.

Cell Walls of Tobacco Cells and Changes in Composition Associated with Reduced Growth upon Adaptation to Water and Saline Stress.

Abstract: The relative mass of the cell walls of tobacco (Nicotiana tabacum L.) cells adapted to grow in medium containing 30% polyethylene glycol 8000 or 428 millimolar NaCl was reduced to about 50% of that of the walls of unadapted cells. Cellulose synthesis was inhibited substantially in adapted cells. The proportions of total pectin in walls of unadapted and adapted cells were about the same, but substantial amount of uronic acid-rich material from walls of cells adapted to either NaCl or polyethylene glycol was more easily extracted with cold sodium ethylenediamine tetraacetic acid solutions (NM Iraki et al. [1989] Plant Physiol. 91: 39-47). We examined the linkage composition of the pectic and hemicellulosic polysaccharides to ascertain chemical factors that may explain this difference in physical behavior. Adaptation to stress resulted in the formation of a loosely bound shell of polygalacturonic acid and rhamnogalacturonan. Pectins extracted from walls of adapted cells by either cold sodium ethylenediamine tetraacetic acid or hot ammonium oxalate were particularly enriched in rhamnose. Compared to pectins of unadapted cells, rhamnosyl units of the rhamnogalacturonans of adapted cells were more highly substituted with polymers containing arabinose and galactose, but the side groups were of greatly reduced molecular size. Possible functional roles of these modifications in cell wall metabolism related to adaptation to osmotic stress are discussed.

Extracellular Polysaccharides and Proteins of Tobacco Cell Cultures and Changes in Composition Associated with Growth-Limiting Adaptation to Water and Saline Stress

Abstract: The chemical composition of extracellular polymers released by cells of tobacco (Nicotiana tabacum L. cv W38) adapted to a medium containing 30% polyethylene glycol 8000 (-28 bar) or 428 millimolar NaCl (-23 bar) was compared to the composition of those released by unadapted cells. Unadapted cells released uronic acid-rich material of high molecular weight, arabinogalactan-proteins, low molecular weight fragments of hemicellulosic polysaccharides, and a small amount of protein. Cells adapted to grow in medium containing NaCl released arabinogalactan and large amounts of protein but not the uronic acid-rich material, and cells adapted to grow in polyethylene glycol released only small amounts of an arabinogalactan of much lower molecular weight and some protein. Secretion of all material was nearly blocked by polyethylene glycol, but when cells were transferred to a medium containing iso-osmolar mannitol, they again released extracellular polymers at rates similar to those of unadapted cells. Like cells adapted to NaCl, however, these cells released arabinogalactan and large amounts of protein but only small amounts of the uronic acid-rich material. Media of NaCl-adapted cells were enriched in 40, 29, and 11 kilodalton polypeptides. CaCl(2) extracted the 40 and 11 kilodalton polypeptides from walls of unadapted cells, but the 29 kilodalton polypeptide was found only in the medium of the NaCl-adapted cells. Accumulation of low molecular weight polysaccharide fragments in the medium was also substantially reduced in both NaCl- and polyethylene glycol-adapted cells, and specifically, the material was composed of lower proportions of xyloglucan fragments. Our results indicate that adaptation to saline or water stress results in inhibition of both the hydrolysis of hemicellulosic xyloglucan and release of uronic acid-rich material into the culture medium.

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 NaCl 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, (pl) of 7.8 and >8.2. Upon transfer of NaCl-adapted cells to medium without NaCl 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. NaCl-adapted cells grown in the absence of NaCl accumulated both pl forms; however, the form accumulated by cells adapted to NaCl (pl > 8.2) was most abundant. Adapted cells grown in the absence of NaCl exhibited absolute growth rates and NaCl tolerance levels which were intermediate to those of NaCl-adapted and unadapted cells. The association between osmotin accumulation and stable NaCl tolerance indicates that cells with a stable genetic change affecting the accumulation of osmotin are selected during prolonged exposure to high levels of NaCl. This stable alteration in gene expression probably affects salt tolerance.

Mechanisms of Adaptation to Salinity in Cultured Glycophyte Cells

Abstract: Cultured cells of Nicotiana tabacum var Wisconsin 38 can be adapted to grow in levels of NaCl similar to those tolerated by many halophytes. Adaptation of these cells to high levels of salinity is associated with reduced cell expansion even though turgor is maintained, a result similar to that commonly reported for whole plants exposed to salinity and/or drought. Glycophytic cells adapted to salinity apparently utilize many of the same biochemical and physiological processes to deal with salinity as do halophytes. Na+ and Cl- are the principal solutes contributing to the extensive osmotic adjustment these cells undergo in response to salinity, although organic solutes, particularly proline, accumulate as well. Adapted cells accumulate less Na+ than unadapted cells when grown in comparable levels of NaCl, suggesting that the ability to regulate ion accumulation may be an important component of the salinity tolerance of these cells. The majority of Na+ and Cl- accumulated by the cells is compartmentalized in the vacuole, such that cytosolic levels of these ions remain near 100 mM at external NaCl concentrations of 428 mM. Current research is directed at examining changes in membrane transport properties associated with salinity in order to assess their contribution to the ability of the cells to tolerate salt.

Osmotin: A Protein Associated with Osmotic Stress Adaptation in Plant Cells

Abstract: Osmotin is a cationic protein which accumulates (up to 12% of total cell protein) in cells adapted to grow in the medium with low water potentials. The synthesis of osmotin is developmentally regulated and is induced by abscisic acid (ABA) in cultured cells. In whole plants, both the synthesis and accumulation of osmotin is tissue specific. The highest rate of synthesis occurs in outer stem tissue and the highest level of accumulation occurs in roots. ABA induced synthesis of osmotin is transient in cells and NaCl stabilizes its synthesis and accumulation. NaCl adapted tobacco cells exhibit a stable increase in both their ability to tolerate salt and to produce osmotin in the absence of NaCl. Osmotin is localized in vacuolar inclusions but also appears to be loosely associated with the tonoplast and plasma membrane. Osmotin is also found in the culture medium of adapted cells during all stages of cell growth. The molecular weight of mature osmotin deduced from the cDNA nucleotide sequence is 23,984 daltons. Osmotin is synthesized as a preprotein 2.5 kD larger than the mature protein. Three proteins, thaumatin, TPR and MAI, exhibit a very high level (52% to 61%) of sequence homology with osmotin. Osmotin mRNA synthesis is induced by ABA. The level of osmotin mRNA increases after NaCl adaptation.

Reduced Growth Rate and Changes in Cell Wall Proteins of Plant Cells Adapted to NaCl

Abstract: Plant cells which are exposed to sufficient osmotic stress will lose turgor and stop growing. However, within limits plant cells will osmotically adjust to such stress and restore turgor but growth remains inhibited. We have hypothesized that osmotically adapted cells have restricted growth rates because of alterations in cell wall metabolism. We demonstrate here that the amounts of hydroxyproline rich glycoproteins are greatly reduced in adapted cells and, hence, cannot be involved in restricted growth. Adapted cells do have an altered pattern of ionically-bound cell wall proteins and proteins released outside of the cell. Any role of these protein changes in restricting cell growth remains to be proven. Plants regenerated from adapted cells exhibit a genetically stable reduction in leaf cell enlargement rate. Alterations in cell wall properties of these leaf cells remain to be determined.