Vacuoles: Isolation, Purification, and Uses

  • A. Maretzki
  • M. Thom
Part of the Biotechnology in Agriculture and Forestry book series (AGRICULTURE, volume 9)


Since plant vacuoles were first successfully isolated by Cocking in 1960, interest in them as a subject of research has steadily increased. Over the past few years the availability of vacuoles from an increasing number of plant species has narrowed gaps in our knowledge about intracellular compartmental distribution, electrochemical properties of the tonoplast, and membrane transport of primary and secondary metabolites associated with these properties. Vacuoles are being used extensively to investigate both enzymes functioning exclusively or primarily within the vacuole and enzymes associated with the tonoplast. Direct access to this organelle has at times merely confirmed prior inferences or conclusions reached from studies with cells or tissues; but, more frequently, new perspectives have been gained. Either way, access to isolated vacuoles has become an important asset for the plant physiologist.


Crassulacean Acid Metabolism Plant Chenopodium Rubrum DEAE Dextran Osmotic Lysis Tonoplast Vesicle 
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  1. Abel S, Glund K (1986) Localization of RNA-degrading enzyme activity within vacuoles of cultured tomato Lycopersicon esculentum cultivar lucullus cells. Physiol Plant 66: 79–86CrossRefGoogle Scholar
  2. Admon A, Jacoby B (1980) Assessment of cytoplasmic contaminations in isolated vacuole preparations. Plant Physiol 65: 85–87PubMedCrossRefGoogle Scholar
  3. Aerts JMFG, Schram AW (1985) Isolation of vacuoles from the upper epidermis of Petunia-hybrid petals. 1. A comparison of isolation procedures. Z Naturforsch 40c: 189–195Google Scholar
  4. Alibert G, Boudet AM (1982) Progrès, problèmes et perspectives dans l’obtention et l’utilization de vacuoles isolée. Physiol Veg 20: 289–302Google Scholar
  5. Alibert G, Carrasco A, Boudet AM (1982) Changes in biochemical composition of vacuoles isolated from Acer pseudoplatanus L. during cell culture. Biochim Biophys Acta 721: 22–29CrossRefGoogle Scholar
  6. Aoki K, Nishida K (1984) ATPase activity associated with vacuoles and tonoplast vesicles isolated from the CAM plant, Kalanchoë daigremontiana. Physiol Plant 60: 21–25CrossRefGoogle Scholar
  7. Barbier H, Guern J (1982) Transmembrane potential of isolated vacuoles and sucrose accumulation in Beta vulgaris roots. In: Marmé D, Marré E, Hertel R (eds) Plasmalemma and tonoplast: their function in the plant cell. Elsevier Biomedical Press, Amsterdam, pp 233–239Google Scholar
  8. Bentrup FW, Hoffmann B, Gogarten-Boekels M, Gogarten JP (1985) A patch clamp study of tonoplast electrical properties in vacuoles isolated from Chenopodium rubrum suspension cells. Z Naturforsch 40c: 886–890Google Scholar
  9. Bentrup FW, Gogarten-Boekels M, Hoffmann B, Gogarten JP, Baumann C (1986) ATP-dependent acidification and tonoplast hyperpolarization in isolated vacuoles from green suspension cells of Chenopodium rubrum L. Proc Natl Acad Sci USA 83: 2431–2433PubMedCrossRefGoogle Scholar
  10. Boller T, Alibert G (1983) Photosynthesis in protoplasts from Melilotus alba: Distribution of products between vacuole and cytosol. Z Pflanzenphysiol 110: 231–238Google Scholar
  11. Boller T, Kende H (1979) Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol 63:1123 —1132Google Scholar
  12. Boller T, Vögeli U (1984) Vacuolar localization of ethylene-induced chitinase in bean leaves. Plant Physiol 74: 442–444PubMedCrossRefGoogle Scholar
  13. Boller T, Wiemken A (1986) Dynamics of vacuolar compartmentation. Annu Rev Plant Physiol 37: 137–164CrossRefGoogle Scholar
  14. Boudet AM, Canut H, Alibert G (1981) Isolation and characterization of vacuoles from Melilotus alba mesophyll. Plant Physiol 68: 1354–1358PubMedCrossRefGoogle Scholar
  15. Briskin DP, Leonard RI’ (1980) Isolation of tonoplast vesicles from tobacco protoplasts. Plant Physiol 66: 684–687PubMedCrossRefGoogle Scholar
  16. Briskin DP, Thornley WR, Wyse R (1985) Membrane transport in isolated vesicles from sugar beet taproot. I. Isolation and characterization of energy dependent, H+-transporting vesicles. Plant Physiol 78: 865–870PubMedCrossRefGoogle Scholar
  17. Broglie KE, Gaynor JJ, Broglie RM (1986) Ethylene regulated gene expression: molecular cloning of the genes encoding an endochitisase from Phaseolus vulgaris. Proc Natl Acad Sci USA 83: 6820PubMedCrossRefGoogle Scholar
  18. Buser C, Matile P (1977) Malic acid in vacuoles isolated from Bryophyllum leaf cells. Z Pflanzenphysiol 82: 462–466Google Scholar
  19. Chatterton NJ, Harrison PA, Bennett JH (1986) Environmental effects on sucrose and fructan concentrations in leaves of Agropyron spp. In: Cronshaw J, Lucas WL, Giaquinta RT (eds) Phloem Transport. Plant Biology Vol I, Allan Liss, NY, pp 471–476Google Scholar
  20. Chedhomme F, Rona JP (1986) Isolation and electrical characterization of tonoplast vesicles from the Kiwi fruit (Actinidia chinensis). Physiol Plant 67: 29–36CrossRefGoogle Scholar
  21. Cocking EC (1960) A method for the isolation of plant protoplasts and vacuoles. Nature 187(4741):962— 963Google Scholar
  22. D’Auzac J (1975) Caracterisation d’une ATPase membranaire en presence d’une phosphatase acide dans les lutoides du latex d’Hevea brasiliensis. Phytochemistry 14: 671–675CrossRefGoogle Scholar
  23. Deus-Neumann B, Zenk MH (1984) A highly selective alkaloid uptake system in vacuoles of higher plants. Planta 162: 250–260CrossRefGoogle Scholar
  24. Doll S, Rodier F, Willenbrink J (1979) Accumulation of sucrose in vacuoles isolated from red beet tissue. Planta 144: 407–411CrossRefGoogle Scholar
  25. Dracup MNH, Greenway H (1985) A procedure for isolating vacuoles from leaves of the halophyte Suaeda maritima. Plant Cell Environ 8: 149–154CrossRefGoogle Scholar
  26. DuPont FM, Bennett AB, Spanswick RM (1982) Localization of a proton-translocating ATPase on sucrose gradients. Plant Physiol 70: 1115–1119CrossRefGoogle Scholar
  27. Findley GP, Hope AB (1976) Electrical properties of plant cells: methods and findings. In: Lüttge U, Pitman MG (eds) Transport in Plants II. Encyclopedia of Plant Physiology, New Series 2 ( A). Springer, Berlin Heidelberg New York, pp 53–92Google Scholar
  28. Frehner M, Keller F, Wiemken A (1984) Localization of fructan metabolism in the vacuoles isolated from protoplasts of Jerusalem Artichoke tubers (Helianthus tuberoses L.). J Plant Physiol 116: 197–208Google Scholar
  29. Gibrat R, Barbier-Brygoo H, Guern J, Grignon C (1985) Membrane potential difference of isolated plant vacuoles: Positive or negative I. Evidence for membrane binding of cationic probes. Biochim Biophys Acta 819: 206–214Google Scholar
  30. Graustedt RC, Huffaker RC (1982) Identification of the leaf vacuole as a major nitrate storage pool. Plant Physiol 70: 410–413CrossRefGoogle Scholar
  31. Grob K, Matile P (1979) Vacuolar location of glucosinolates in horseradish root cells. Plant Sci Lett 14:327 —335Google Scholar
  32. Guy M, Kende H (1984) Conversion of 1-amino cyclo propane-1-carboxylic acid to ethylene by isolated vacuoles of Pisum sativum. Planta 160: 281–287CrossRefGoogle Scholar
  33. Guy M, Reinhold L, Michaeli D (1979) Direct evidence for a sugar transport mechanism in isolated vacuoles. Plant Physiol 64:61— 64Google Scholar
  34. Hedrich R, Flügge UI, Fernandez JM (1986) Patchclamp studies of ion transport in isolated plant vacuoles. FEBS Lett 204: 228–232CrossRefGoogle Scholar
  35. Hopp W, Seitz HU (1987) The uptake of acylated anthocyanin into isolated vacuoles from a cell suspension culture of Daucus carota. Planta 170: 74–85CrossRefGoogle Scholar
  36. Hopp W, Hinderer W, Petersen M, Seitz HU (1985) Anthocyanin-containing vacuoles isolated from protoplasts of Daucus carota cell cultures. In: Pilet PE (ed) The Physiological Properties of Plant Protoplasts. Springer, Berlin Heidelberg New York, pp 122–132CrossRefGoogle Scholar
  37. Kaiser G, Heber U (1984) Sucrose transport into vacuoles isolated from barley mesophyll protoplasts. Planta 161: 562–568CrossRefGoogle Scholar
  38. Kaiser G, Martinoia E, Wiemken A (1982) Rapid appearance of photosynthetic products in the vacuoles isolated from barley mesophyll protoplasts by a new, fast method. Z Pflanzenphysiol 107: 103–113Google Scholar
  39. Keller F (1988) A large-scale isolation of vacuoles from protoplasts of mature carrot taproots. J Plant Physiol 132: 199–203Google Scholar
  40. Kenyon WH, Black CC (1986) Electrophoretic analysis of protoplast, vacuole, and tonoplast vesicle proteins in crassulacean acid metabolism plants. Plant Physiol 82: 916–924PubMedCrossRefGoogle Scholar
  41. Kringstad R, Kenyon WH, Black CC Jr (1980) The rapid isolation of vacuoles from leaves of crassulacean acid metabolism plants. Plant Physiol 66: 379–382PubMedCrossRefGoogle Scholar
  42. Kutchan TM, Rush M, Coscia CJ (1986) Subcellular localization of alkaloids and dopamine in different vacuolar compartment of Papaver bracteatum. Plant Physiol 81: 161–166PubMedCrossRefGoogle Scholar
  43. Leigh RA (1981) Methods, progress and potential for the use of isolated vacuoles in studies of solute transport in higher plant cells. Physiol Plant 57: 390–396CrossRefGoogle Scholar
  44. Leigh RA, Branton D (1976) Isolation of vacuoles from root storage tissue of Beta vulgaris L. Plant Physiol 58: 656–662PubMedCrossRefGoogle Scholar
  45. Le-Quoc K, Le-Quoc D, Pugin A (1987) An efficient method for vacuole isolation using digitonin for plasmalemma lysis. J Plant Physiol 126: 329–335Google Scholar
  46. Lörz J, Harms CT, Potrykus I (1976) Isolation of “vacuoplasts” from protoplasts of higher plants. Biochem Physiol Pflanz 169: 617–620Google Scholar
  47. Mandala S, Taiz L (1985) Proton transport in isolated vacuoles from corn coleoptile. Plant Physiol 78: 104–109PubMedCrossRefGoogle Scholar
  48. Mandala S, Taiz L (1986) Characterization of the subunit structure of the maize tonoplast ATPase. J Biol Chem 261: 12850–12855PubMedGoogle Scholar
  49. Maretzki A, Thom M (1987) UDP-glucose-dependent sucrose translocation in tonoplast vesicles from stalk tissue of sugarcane. Plant Physiol 83: 235–237PubMedCrossRefGoogle Scholar
  50. Marigo G, Bouyssou H, Belkoura M (1985) Vacuolar efflux of malate and its influence on nitrate accumulation in Catharanthus roseus cells. Plant Sci 39: 97–103CrossRefGoogle Scholar
  51. Martinoia E, Heck U, Wiemken A (1981) Vacuoles as a storage compartment for nitrate in barley leaves. Nature 289: 292–294CrossRefGoogle Scholar
  52. Martinoia E, Flügge UI, Kaiser G, Heber U, Heldt HW (1985) Energy-dependent uptake of malate into vacuoles isolated from barley mesophyll protoplasts. Biochem Biophys Acta 806: 311–319CrossRefGoogle Scholar
  53. Martinoia E, Schramm MJ, Kaiser G, Kaiser WM (1986) Transport of anions in isolated barley vacuoles. I. Permeability to anions and evidence for a Cl-uptake system. Plant Physiol 80: 895–901PubMedCrossRefGoogle Scholar
  54. Matern V, Reichenbach C, Heller W (1986) Efficient uptake of flavonoids into parsley Petroselinumhortense vacuoles requires acylated glycosides. Planta 167: 183–189CrossRefGoogle Scholar
  55. Matile P (1979) Biochemistry and function of vacuoles. Annu Rev Plant Physiol 29: 193–213CrossRefGoogle Scholar
  56. Mayne RG, Kende H (1986) Ethylene biosynthesis in isolated vacuoles of Vicia faba. Requirement for membrane integrity. Planta 167: 159–165Google Scholar
  57. Mettler IJ, Leonard RT (1979) Isolation and partial characterization of vacuoles from tobacco protoplasts. Plant Physiol 64: 1114–1120PubMedCrossRefGoogle Scholar
  58. Miller AJ, Brimelow JJ, John P (1984) Membrane-potential changes in vacuoles isolated from storage roots of red beet (Beta vulgaris L.). Planta 160: 59–65CrossRefGoogle Scholar
  59. Murphy TM, Widell S, Hellergren J (1986) On the use of phase partition to isolate vacuoles from cultured cells. Plant Cell Tissue Organ Cult 7: 205–216CrossRefGoogle Scholar
  60. Nishimura M (1982) pH in vacuoles isolated from castor bean endosperm. Plant Physiol 70:742–744PubMedCrossRefGoogle Scholar
  61. Nishimura M, Beevers H (1978) Hydrolases in vacuoles from castor bean endosperm. Plant Physiol 62: 44–48PubMedCrossRefGoogle Scholar
  62. Ohlrogge JB, Garcia-Martinez JL, Adams D, Rappaport L (1980) Uptake and subcellular compart- mentation of gibberelin Al applied to leaves of barley and cowpea. Plant Physiol 66: 422–427PubMedCrossRefGoogle Scholar
  63. Poole RJ, Briskin DP, Kratky Z, Johnstone RM (1984) Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet. Characterization of proton-transport and ATPase in tonoplast vesicles. Plant Physiol 74: 549–556Google Scholar
  64. Pugin A, Montrichard F, Le-Quoc K, Le-Quoc D (1986) Influence of the method for the preparation of vacuoles on the vacuolar ATPase activity of isolated Acer pseudoplatanus cells. Plant Sci 47: 165–177CrossRefGoogle Scholar
  65. Rausch I, Butcher DN, Taiz L (1987) Active glucose transport and proton pumping in tonoplast membrane of Zea mays L. coleoptiles are inhibited by anti H+-ATPase antibodies. Plant Physiol 85: 996–999PubMedCrossRefGoogle Scholar
  66. Renaudin JP, Brown SC, Barbier-Brygoo H, Guern J (1987) Quantitative characterization of protoplasts and vacuoles from suspension-cultured cells of Catharanthus roseus. Physiol Plant 68:695 — 703Google Scholar
  67. Salyaev RK, Kuzevanov VY, Ozolina NV (1983) Content of lipids, proteins, and carbohydrates in the membrane of isolated vacuoles of red beet. Sov Plant Physiol 29: 718–724Google Scholar
  68. Sandelius AS, Penel C, Auderset G, Brightman A, Millard M, Moiré DJ (1986) Isolation of highly purified fractions of plasma membrane and tonoplast from the same homogenate of soybean hypocotyls by free flow electrophoresis. Plant Physiol 81: 177–185PubMedCrossRefGoogle Scholar
  69. Saunders JA (1979) Investigation of vacuoles isolated from tobacco. I. Quantitation of nicotine. Plant Physiol 64:74— 78Google Scholar
  70. Schloss P, Walter C, Maeder M (1987) Basic peroxidases in isolated vacuoles of Nicotiana tabacum L. Planta 170: 225–229CrossRefGoogle Scholar
  71. Smith JAC, Uribe EG, Ball E, Lüttge U (1984) ATPase activity associated with isolated vacuoles of the crassulacean acid metabolism plant Kalanchoë daigremontiana. Planta 162: 299–304CrossRefGoogle Scholar
  72. Strack D, Sharma V (1985) Vacuolar localization of the enzymatic synthesis of hydroxycinnamic acid esters of malic acid in protoplasts from Raphanus sativus var sativus leaves. Physiol Plant 65:45 — 50Google Scholar
  73. Sze H (1980) Nigericin-stimulated ATPase activity in microsomal vesicles of tobacco callus. Proc Natl Acad Sci USA 78: 5578–5582CrossRefGoogle Scholar
  74. Thom M, Maretzki A (1985) Group translocation as a mechanism for sucrose transfer into vacuoles from sugarcane cells. Proc Natl Acad Sci USA 82: 4697–4701PubMedCrossRefGoogle Scholar
  75. Thom M, Maretzki A, Komor E (1982) Vacuoles from sugarcane suspension cultures. I. Isolation and partial characterization. Plant Physiol 69:1315 —1319Google Scholar
  76. Thom M, Leigh RA, Maretzki A (1986) Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet. Planta 167: 410–413CrossRefGoogle Scholar
  77. Wagner GJ (1979) Content and vacuole/extravacuolar distribution of neutral sugars, free amino acids, and anthocyanin in protoplasts. Plant Physiol 64: 88–93PubMedCrossRefGoogle Scholar
  78. Wagner GJ (1981) Vacuolar deposition of ascorbate-derived oxalic acid in barley. Plant Physiol 67: 591–593PubMedCrossRefGoogle Scholar
  79. Wagner GJ (1983) Higher plant vacuole and tonoplasts. In: Hall JL, Moore AL (eds) Isolation of Membranes and Organelles from Plant Cells. Academic Press, New York, pp 83–118Google Scholar
  80. Wagner GJ (1988) Isolation of higher plant mature vacuoles: General principles, criteria for purity and integrity. In: Methods in Enzymology, Academic Press, New York (in press)Google Scholar
  81. Wagner GJ, Lin W (1982) An active proton pump of intact vacuoles isolated from Ttdipa petals. Biochim Biophys Acta 689: 261–266CrossRefGoogle Scholar
  82. Wagner GJ, Siegelman HW (1975) Large scale isolation of intact vacuoles and isolation of chloroplasts from protoplasts of mature plant tissue. Science 192: 1298–1299Google Scholar
  83. Wang Y, Leigh RA, Kaestner KM, Sze H (1986) Electrogenic H + pumping pyrophosphatase in tonoplast vesicles of oat roots. Plant Physiol 81: 497–502PubMedCrossRefGoogle Scholar
  84. Weigel HJ, Weiss E (1984) Determination of the proton concentration difference across the tonoplast membrane of isolated vacuoles by means of 9-aminoacridine fluorescence. Plant Sci Lett 33: 163–175CrossRefGoogle Scholar
  85. Werner C, Matile P (1985) Accumulation of coumarylglucosides in vacuoles of barley mesophyll protoplasts. J Plant Physiol 118: 237–249CrossRefGoogle Scholar
  86. Willenbrink J, Doll S (1979) Characteristics of the sucrose uptake system of vacuoles isolated from red beet tissue. Kinetics and specificity of the sucrose uptake system. Planta 147: 159–162Google Scholar
  87. Yamaki S (1984) Isolation of vacuoles from immature apple fruit flesh and compartmentation of sug- ars, organic acids, phenolic compounds and amino acids. Plant and Cell Physiol 25: 151–166Google Scholar
  88. Yoshida S, Uemura M, Niki T, Sakai A, Gusta LV (1983) Partition of membrane particles in aqueous two-polymer phase system and its practical use for purification of plasma membrane from plants. Plant Physiol 72:105 —114Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1989

Authors and Affiliations

  • A. Maretzki
  • M. Thom
    • 1
  1. 1.Hawaiian Sugar Planters’ AssociationAieaUSA

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