, Volume 196, Issue 3–4, pp 181–189 | Cite as

Dynamics of activity and structure of the tonoplast vacuolar-type H+-ATPase in plants with differing CAM expression and in a C3 plant under salt stress

  • J. -B. Mariaux
  • E. Fischer-Schliebs
  • U. Lüttge
  • R. Ratajczak


Differences in the activity and structure of the vacuolar H+-ATPase (V-ATPase, EC were investigated in the C3/CAM intermediate plantKalanchoë blossfeldiana Poellnitz cv. Tom Thumb, with lower or higher expression of CAM, andHordeum vulgare cv. Carina, grown with or without 150 mM NaCl. InK. blossfeldiana ATP-hydrolysis and H+-transport activity were higher with higher expression of CAM than in plants with very weak CAM. This was mainly due to a larger amount of V-ATPase. Statistical analysis of the diameter of intramembrane particles (IMPs) on freeze-fractures of tonoplast vesicles showed that IMPs were larger in tonoplast vesicle preparations ofK. blossfeldiana with strong CAM expression (9.1 nm) than in preparations ofK. blossfeldiana with low CAM expression (7.3 nm). As there is evidence that the majority of IMPs on freeze-fractures of tonoplast vesicles corresponds to the V0 domain of V-ATPase, the higher activity of V-ATPase inK. blossfeldiana with stronger CAM could be a result of additional structural changes in its membrane-integral domain. The higher activity of V-ATPase inK. blossfeldiana with stronger CAM is discussed in relation to the requirement for a higher proton pumping capacity for nocturnal malate accumulation in the vacuole. The ATP-dependent H+-pumping activity inH. vulgare was higher under salt stress than in control plants, while the rates of ATP-hydrolysis and the size of IMPs were not affected by the salt treatment. The data presented here indicate that different mechanisms might increase the transport capacity of V-ATPase to meet the higher requirements of secondary active transport related to CAM expression and adaptation to salt stress.


Kalanchoë daigremontiana Kalanchoë blossfeldiana Hordeum vulgare Freeze-fracturing Tonoplast Vacuolar ATPase 



adenosine triphosphate


crassulacean acid metabolism


intramembrane particles


vacuolar proton-translocating adenosine triphosphatase


domain membrane-integral domain of V-ATPase


domain membrane-peripheral domain of V-ATPase


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Arai H, Terres G, Pink S, Forgac M (1988) Topography and subunit stoichiometry of the coated vesicle proton pump. 263: 8796–8802Google Scholar
  2. Berndt E (1986) Untersuchungen zur Struktur, Funktion und Regulation der Tonoplasten-H+-ATPase. PhD thesis, Technische Hochschule Darmstadt, Darmstadt, Federal Republic of GermanyGoogle Scholar
  3. Betz M, Dietz K-J (1991) Immunological characterization of two dominant tonoplast polypeptides. Plant Physiol 97: 1294–1301Google Scholar
  4. Bremberger C, Haschke H-P, Lüttge U (1988) Separation and purification of the tonoplast ATPase and pyrophosphatase from plants with constitutive and inducible crassulacean metabolism. Planta 175: 465–470Google Scholar
  5. Cushman JC, DeRocher EJ, Bohnert HJ (1990) Gene expression during adaptation to salt stress. In: Katterman F (ed) Environmental injury to plants. Academic Press, San Diego, pp 173–203Google Scholar
  6. Forgac M (1989) Structure and function of vacuolar class of ATP-driven proton pumps. Physiol Rev 69: 765–796Google Scholar
  7. Futai M, Nuomi T, Maeda M (1989) ATP synthase (H+-ATPase): results by combined biochemical and molecular biological approaches. Annu Rev Biochem 58: 111–136Google Scholar
  8. Gräber P, Böttcher B, Boekema EJ (1990) The structure of the ATP-synthase from chloroplasts. In: Milazzo G, Blank M (eds) Biolectrochemistry III. Plenum, New York, pp 247–276Google Scholar
  9. Jochem P, Lüttge U (1987) Proton transporting enzymes at the tonoplast of leaf cells of the CAM plantKalanchoë daigremontiana. I. The ATPase. J Plant Physiol 129: 251–268Google Scholar
  10. Kane PM, Stevens TH (1992) Subunit composition, biosynthesis, and assembly of the yeast vacuolar proton-translocating ATPase. J Bioenerg Biomembr 24: 383–393Google Scholar
  11. —, Kuehn MC, Howland-Stevenson I, Stevens TH (1992) Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H+-ATPase. J Biol Chem 267: 447–454Google Scholar
  12. Klink R, Lüttge U (1991) Electron-microscopic demonstration of a “head and stalk” structure of the leaf vacuolar ATPase inMesembryanthemum crystallinum L. Bot Acta 104: 122–131Google Scholar
  13. — — (1992) Quantification of visible structural changes of the V0V1-ATPase in the leaf-tonoplast ofMesembryanthemum crystallinum by freeze-fracture replicas prepared during the C3 photosynthesis to CAM transition. Bot Acta 105: 414–420Google Scholar
  14. Koyro H-W, Stezler R, Huchzermeyer B (1993) ATPase activities and membrane fine structure of rhizodermal cells fromSorghum andSpartina roots grown under mild salt stress. Bot Acta 106: 110–119Google Scholar
  15. Lai S, Watson JC, Hansen JN, Sze H (1991) Molecular cloning and sequencing of cDNAs encoding the proteolipid subunit of the vacuolar H+-ATPase from a higher plant. J Biol Chem 266: 16078–16084Google Scholar
  16. Lee Taiz S, Taiz L (1991) Ultrastructural comparison of the vacuolar and mitochondrial H+-ATPases ofDaucus carota. Bot Acta 104: 117–121Google Scholar
  17. Leigh RA, Gordon-Weeks R, Steele SH, Koren'kov VD (1994) The H+-pumping inorganic pyrophosphatase of the vacuolar membrane of higher plants. In: Blatt MR, Leigh RA, Sanders D (eds) Membrane transport in plants and fungi: molecular mechanisms and control. Company of Biologists, Cambridge, pp 61–75Google Scholar
  18. Löw R, Rockel B, Kirsch M, Ratajczak R, Hörtensteiner S, Martinoia E, Lüttge U, Rausch T (1996) Early salt stress effects on the differential expression of vacuolar H+-ATPase genes in roots and leaves ofMesembryanthemum crystallinum. Plant Physiol 110: 259–265Google Scholar
  19. Lüttge U (1993) The role of crassulacean acid metabolism (CAM) in the adaptation of plants to salinity. New Phytol 125: 59–71Google Scholar
  20. — (1994) Plant cell membranes and salinity: structural, biochemical and biophysical changes. Rev Brasil Fisiol Veg 5: 217–224Google Scholar
  21. —, Smith JAC (1984) Mechanisms of passive malic acid efflux from vacuoles of the CAM plantKalanchoë daigremontiana. J Membr Biol 81: 149–158Google Scholar
  22. —, Ratajczak R, Rausch T, Rockel B (1995) Stress responses of tonoplast proteins: an example for molecular ecophysiology and the search for ecoenzymes. Acta Bot Neerl 44: 343–362Google Scholar
  23. Manolson MF, Proteau D, Jones EW (1992) Evidence for a conserved 95–120kDa subunit associated with and essential for activity of V-ATPases. J Exp Biol 172: 105–112Google Scholar
  24. Mariaux J-B, Becker A, Kemna I, Ratajczak R, Fischer-Schliebs E, Kramer D, Lüttge U, Marigo G (1994) Visualization by freeze fracture electron microscopy of intramembraneous particles corresponding to the tonoplast H+-pyrophosphatase and H+-ATPase ofKalanchoë daigremontiana Hamet et Perrier de la Bâthie. Bot Acta 107: 321–327Google Scholar
  25. Marquardt-Jarczyk O, Lüttge U (1990) PPiase-activated ATP-dependent H+-transport at the tonoplast of mesophyll cells of the CAM plantKalanchoë daigremontiana. Bot Acta 101: 203–213Google Scholar
  26. Myers M, Forgac M (1993) Assembly of the peripheral domain of the vacuolar H+-adenosine triphosphatase. J Cell Physiol 156: 35–42Google Scholar
  27. Nakamura Y, Kasamo K, Shimosato N, Sakata M, Ohta E (1992) Stimulation of the extrusion of protons and H+-ATPase activities with the decline in pyrophosphatase activity of the tonoplast in intact mung bean roots under high-NaCl stress and its relation to external levels of Ca2+ ions. Plant Cell Physiol 33: 139–149Google Scholar
  28. Narasimhan ML, Binzel ML, Perez-Prat E, Chen Z, Nelson DE, Singh NK, Bressan RA, Hasegawa PM (1991) NaCl regulation of tonoplast ATPase 70-kilodalton subunit mRNA in tobacco cells. Plant Physiol 97: 562–568Google Scholar
  29. Nelson N (1994) Organellar proton ATPases. Springer, Berlin Heidelberg New York TokyoGoogle Scholar
  30. Peterson GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83: 364–356Google Scholar
  31. Queiroz O (1965) Sur le métabolisme acide des Crassualacées. Action à long terme de la température de nuit sur la synthèse d'acide malique parKalanchoë blossfeidiana “Tom Thumb” placée en jours courts. Physiol Veg 3: 203–213Google Scholar
  32. Ratajczak R, Richter J, Lüttge U (1994) Adaptation of the tonoplast V-type H+-ATPase ofMesembryanthemum crystallinum to salt stress, C3-CAM transition and plant age. Plant Cell Environ 17: 1101–1112Google Scholar
  33. —, Hille A, Mariaux J-B, Lüttge U (1995) Quantitative stress responses of the V0V1-ATPase of higher plants detected by immuno-electron microscopy. Bot Acta 108: 505–513Google Scholar
  34. Reuveni M, Bennett AB, Bressan RA, Hasegawa PM (1990) Enhanced H+-transport capacity and ATP-hydrolysis activity of the tonoplast H+-ATPase after NaCl adaptation. Plant Physiol 94: 524–530Google Scholar
  35. Rockel B, Ratajczak R, Becker A, Lüttge U (1994) Changed densities and diameters of intra-membrane tonoplast particles ofMesembryanthemum crystallinum in correlation with NaCl-induced CAM. J Plant Physiol 143: 318–324Google Scholar
  36. Sze H, Ward JM, Lai S (1992) Vacuolar H+-translocating ATPases from plants: structure, function and isoforms. J Bioenerg Biomembr 24: 371–381Google Scholar
  37. Taiz L (1992) The plant vacuole. J Exp Biol 172: 113–122Google Scholar

Copyright information

© Springer-Verlag 1997

Authors and Affiliations

  • J. -B. Mariaux
    • 1
  • E. Fischer-Schliebs
    • 2
  • U. Lüttge
    • 2
  • R. Ratajczak
    • 2
  1. 1.Max-Plank-Institut für Züchtungs-forschungKölnFederal Republic of Germany
  2. 2.Institut für BotanikTechnische Hochschule DarmstadtDarmstadt

Personalised recommendations