Efforts were made to estimate the water potential difference that is required, between rapidly growing oat coleoptile cylinders and dilute medium, to support the rate of water uptake involved in elongation, (a) by the traditional method of determining the concentration of mannitol in which the tissue neither gains nor loses water, and (b) by measuring the rates of osmotic exchanges induced by treating the tissue with different hypotonic mannitol concentrations. Both methods indicated large water potential differences (3 to 10 atm), in some cases approaching the osmotic pressure of the cells. However, indication was obtained that the rates of osmotic exchanges induced by mannitol solutions, and presumably also the equilibrium response sought in (a), are governed by the rate of diffusional exchange of mannitol with the free space rather than by the permeability of the tissue to water. Osmotic swelling of the tissue measured by immersing it in water after its turgor pressure had been reduced by evaporation, was at least two to four times more rapid than when mannitol was involved. The permeability to water estimated by the evaporation-immersion method indicated that rapidly elongating cylinders have water potentials between --0.8 and --2.5 atm, or between 10 and 25 per cent of their osmotic pressure.
ABSTRACrThe pasma s of protoplasts rekased from carrot s culture cel were labeled with I14Clacetyl-concanavalin A. After homogenizatio a single labeled e faction was isolated in a contiuous isopycic Renografin gradient. The labeled membranes peaked at an apparet density of 1.14 grams per cubic centimeter between the Golgi fraction at a denity of 1.11 grams per cubic centmeter as determined by latent IDPase activity and the mitochondria at a density of 1.16 grams per cubic centimeter as determined by the cytochrome c oxidase activity. Thi method provied a very discrete peak of putative asma On discontinuous Reogrfin gradients a relatively pure fimction of beled plasm m bras could be readly isolated at the 1.122 to 1.146 grams per cubic centmeter interface. Tbe labeled fraction was enriced in both a ATPase (pH 6.5) and a glum synthetase with a pH optimum of 6.5 whose actvity was promoted by iu and ceflobiose. Enzyme activities were not altered by the m e labeLAlthough the details of the role of the plasma membrane in plant growth and development remain a mystery, the plasma membrane has been implicated in such functional roles as cellulose biosynthesis (21), the mediation of auxin responses (13,25), and polar auxin transport (11). Based on toxin binding to an isolated membrane fraction, Albersheim (2) hypothesized that the plasma membrane may be involved in a host-pathogen response. To elucidate the roles of the plasma membrane in plant growth and development, a method of isolating relatively large quantities of pure plasma membrane is needed. Such an isolation is dependent upon having a specific marker for the plasma membrane.The most commonly used markers for the plasma membrane have been the phosphotungstic acid-chromic acid stain used in electron microscopy (27) and K+-stimulated ATPase measured at pH 6.5 (17); however, there is much controversy as to the reliability of these markers. In addition to being quite tedious and slow, the phosphotungstic acid-chromic acid stain is sometimes nonspecific (12,30 This paper builds upon a preliminary report of labeling with by detailing the improved techniques for specifically labeling and isolating plasma membranes from protoplasts by relatively gentle means, by describing the unique properties of Renografin as a gradient medium for membrane isolation, and by characterizing the ATPase and glucan synthetase found with the plasma membrane. MATERIALS AND METHODSProtoplast Isoation. A cell suspension culture of carrot, Daucus carota L., was obtained from Howard Bonnett and grown in a defined medium (7) containing 1.0 mg/l naphthalene acetic acid and 0.02 mg/l kinetin, with subculturing every 4 days. To release protoplasts, approximately 1.0 ml packed volume of carrot suspension culture cells was incubated in 20 ml of2% (w/v) Driselase in 0.4 molal sorbitol for 2 h at 28 C on a rotary shaker (150 rpm). The released protoplasts were filtered through glass wool to remove large fragments of cytoplasmic debris and centrifuged out of the enzyme at 40g for 5 to 10 min. The osmoti...
Avena coleoptile sections were treated with a fraction of a fungal filtrate containing a potent cellulase. Elongation rate was not affected although turgor pressure remained constant and wall extensibility was increased. These data show that the simple weakening of cell walls is not sufficient to promote growth and suggest that endogenous polysaccharidases are not the means by which the growth rate of the coleoptile is regulated.
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