Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1979 Jan;63(1):183–190. doi: 10.1104/pp.63.1.183

Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells

I. General Characteristics 1

Irvin J Mettler a,2, Robert T Leonard a
PMCID: PMC542791  PMID: 16660675

Abstract

An investigation was conducted into the feasibility of using enzymically isolated protoplasts from suspension-cultured cells of Nicotiana glutinosa L. to study ion transport. Transport of K+ (86Rb), 36Cl−, H232PO4− and 45Ca2+ from 1 millimolar salt solutions was determined after separation of intact protoplasts from nonabsorbed tracers by centrifugation through a Ficoll step gradient. Influx of K+, Cl−, and H2PO4− measured over a 30-minute period was reduced (up to 99%) by respiratory inhibitors such as 5 micrograms per milliliter oligomycin, 0.1 millimolar dinitrophenol, 0.1 millimolar cyanide, or N2 gas. In contrast, Ca2+ influx was not tightly coupled to respiratory energy production. The influx of K+ was highest between pH 6.5 and 7.5 whereas the influx of H2PO4− and Cl− was greatest between pH 4.5 and 5.5. Influx of K+ and Cl− was maximal at 35 and 45 C, respectively, and was almost completely inhibited below 10 C. Fusicoccin (0.01 millimolar) stimulated K+ influx by more than 200% but had no effect on the influx of either Cl− or H2PO4−. Apparent H+ efflux, as measured by decrease in solution pH, was enhanced by K+, stimulated further by 0.01 millimolar fusicoccin, and inhibited by 0.1 millimolar dinitrophenol or 5 micrograms per milliliter oligomycin. The measured ionic fluxes into protoplasts were similar to those obtained with intact cultured cells. The results indicate that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast, and that it is feasible to use isolated protoplasts for studies on ion transport.

Full text

PDF
184

Images in this article

184Image
on p.184
185Image
on p.185
186Image
on p.186

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Carraway K. L. Covalent labeling of membranes. Biochim Biophys Acta. 1975 Dec 29;415(4):379–410. doi: 10.1016/0304-4157(75)90005-2. [DOI] [PubMed] [Google Scholar]
  2. Cleland R. E. Rapid stimulation of K -H exchange by a plant growth hormone. Biochem Biophys Res Commun. 1976 Mar 22;69(2):333–338. doi: 10.1016/0006-291x(76)90526-x. [DOI] [PubMed] [Google Scholar]
  3. Cocking E. C. Uptake of foreign genetic material by plant protoplasts. Int Rev Cytol. 1977;48:323–343. doi: 10.1016/s0074-7696(08)61748-9. [DOI] [PubMed] [Google Scholar]
  4. Galbraith D. W., Northcote D. H. The isolation of plasma membrane from protoplasts of soybean suspension cultures. J Cell Sci. 1977 Apr;24:295–310. doi: 10.1242/jcs.24.1.295. [DOI] [PubMed] [Google Scholar]
  5. Guy M., Reinhold L. Membrane transport of sugars and amino acids in isolated protoplasts. Plant Physiol. 1978 Apr;61(4):593–596. doi: 10.1104/pp.61.4.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  7. Leonard R. T., Nagahashi G., Thomson W. W. Effect of lanthanum on ion absorption in corn roots. Plant Physiol. 1975 Mar;55(3):542–546. doi: 10.1104/pp.55.3.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Mettler I. J., Leonard R. T. Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: II. Selectivity and Kinetics. Plant Physiol. 1979 Jan;63(1):191–194. doi: 10.1104/pp.63.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Poste G., Papahadjopoulos D., Vail W. J. Lipid vesicles as carriers for introducing biologically active materials into cells. Methods Cell Biol. 1976;14:33–71. doi: 10.1016/s0091-679x(08)60468-9. [DOI] [PubMed] [Google Scholar]
  10. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Spurr A. R. A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res. 1969 Jan;26(1):31–43. doi: 10.1016/s0022-5320(69)90033-1. [DOI] [PubMed] [Google Scholar]
  12. Uchimiya H., Murashige T. Evaluation of parameters in the isolation of viable protoplasts from cultured tobacco cells. Plant Physiol. 1974 Dec;54(6):936–944. doi: 10.1104/pp.54.6.936. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES