Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1976 Nov;58(5):656–662. doi: 10.1104/pp.58.5.656

Isolation of Vacuoles from Root Storage Tissue of Beta vulgaris L. 1

Roger A Leigh a,2,3, Daniel Branton a
PMCID: PMC542277  PMID: 16659738

Abstract

Morphologically intact and osmotically active vacuoles were isolated from root storage tissue of the red beet Beta vulgaris L., and the factors influencing both yield and stability of the vacuoles were determined. Successful isolation depended upon slicing the tissue in an apparatus specifically designed to cut open plant cells without the use of high shear forces and to liberate cellular organelles into an undisturbed reservoir of osmoticum. The resulting brei was centrifuged at 2,000g for 10 min to yield a pellet which contained many vacuoles but which also contained tissue fragments, nuclei, mitochondria, and plastids. The vacuoles were further purified by accelerated flotation through a Metrizamide step gradient. Biochemical assays, light microscopy, and electron microscopy confirmed that there was only trace contamination of the final vacuole preparation by other organelles. Isolated vacuoles were intact and retained their in vivo coloration.

Full text

PDF
656

Images in this article

660Image
on p.660

Selected References

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

  1. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Branton D., Bullivant S., Gilula N. B., Karnovsky M. J., Moor H., Mühlethaler K., Northcote D. H., Packer L., Satir B., Satir P. Freeze-etching nomenclature. Science. 1975 Oct 3;190(4209):54–56. doi: 10.1126/science.1166299. [DOI] [PubMed] [Google Scholar]
  3. Douce R., Mannella C. A., Bonner W. D., Jr The external NADH dehydrogenases of intact plant mitochondria. Biochim Biophys Acta. 1973 Jan 18;292(1):105–116. doi: 10.1016/0005-2728(73)90255-7. [DOI] [PubMed] [Google Scholar]
  4. Fineran B. A. Organization of the tonoplast in frozen-etched root tips. J Ultrastruct Res. 1970 Dec;33(5):574–586. doi: 10.1016/s0022-5320(70)90182-6. [DOI] [PubMed] [Google Scholar]
  5. Fisher K., Branton D. Application of the freeze-fracture technique to natural membranes. Methods Enzymol. 1974;32:35–44. doi: 10.1016/0076-6879(74)32006-x. [DOI] [PubMed] [Google Scholar]
  6. Hodges T. K., Leonard R. T. Purification of a plasma membrane-bound adenosine triphosphatase from plant roots. Methods Enzymol. 1974;32:392–406. doi: 10.1016/0076-6879(74)32039-3. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Matile P., Wiemken A. The vacuole as the lysosome of the yeast cell. Arch Mikrobiol. 1967 Feb 20;56(2):148–155. doi: 10.1007/BF00408765. [DOI] [PubMed] [Google Scholar]
  9. Matile P., Wiemken A. Vacuoles and spherosomes. Methods Enzymol. 1974;31:572–578. [PubMed] [Google Scholar]
  10. Miflin B. J. The location of nitrite reductase and other enzymes related to amino Acid biosynthesis in the plastids of root and leaves. Plant Physiol. 1974 Oct;54(4):550–555. doi: 10.1104/pp.54.4.550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Moore T. S., Lord J. M., Kagawa T., Beevers H. Enzymes of phospholipid metabolism in the endoplasmic reticulum of castor bean endosperm. Plant Physiol. 1973 Jul;52(1):50–53. doi: 10.1104/pp.52.1.50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Moreau F., Lance C. Isolement et propriétés des membranes externes et internes de mitochondries végétales. Biochimie. 1972;54(10):1335–1348. doi: 10.1016/s0300-9084(72)80074-9. [DOI] [PubMed] [Google Scholar]
  13. Philipp E. I., Franke W. W., Keenan T. W., Stadler J., Jarasch E. D. Characterization of nuclear membranes and endoplasmic reticulum isolated from plant tissue. J Cell Biol. 1976 Jan;68(1):11–29. doi: 10.1083/jcb.68.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Strobel G. A., Hess W. M. Evidence for the presence of the toxin-binding protein on the plasma membrane of sugarcane cells. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1413–1417. doi: 10.1073/pnas.71.4.1413. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES