Plant and Soil

, Volume 153, Issue 1, pp 61–69 | Cite as

Low molecular weight humic substances stimulate H+-ATPase activity of plasma membrane vesicles isolated from oat (Avena sativa L.) roots

  • Z. Varanini
  • R. Pinton
  • M. G. De Biasi
  • S. Astolfi
  • A. Maggioni
Research Article


The effect of <5 KDa (low molecular weight, LMW) and >5 KDa (high molecular weight, HMW) humic fractions on transport activities of isolated plasma membrane vesicles was studied. The K+-stimulated component of the ATP-hydrolyzing activity was considerably increased by LMW humic substances at concentrations ranging from 0.075 mg org CL-1 to 1 mg org CL-1. The stimulation was still evident when the detergent Brij-35 was added in the assay mixture, indicating a direct effect of LMW humic substances on plasma membrane ATPase activity. The LMW humic fraction stimulated ATP-dependent intravesicular H+-accumulation with a pattern similar to that recorded for ATP hydrolysis. LMW humic substances induced also an increase in passive membrane permeability to protons, as revealed by following the dissipation of an artificially imposed pH gradient. Membrane permeability to anions, as measured by the anion-dependent active proton accumulation was affected by LMW humic substances. In the presence of NO3- these molecules clearly enhanced proton transport, while Cl--dependent activity was almost unaffected, thus suggesting a specific action of LMW humic fraction on transmembrane NO3- fluxes. On the other hand, HMW humic substances decreased the passive permeability to protons and reduced the anion-dependent intravesicular H+-accumulation. The results suggest that the stimulatory effect of soil humic substances on plant nutrition and growth might be, at least in part, explained on the basis of both direct action of LMW humic molecules on plasma membrane H+-ATPase and specific modification of cell membrane permeability.

Key words

Avena sativa L. humic substances plasma membrane H+-ATPase proton gradient 



acridine orange


bovine serum albumine


bis-tris-propane (1,3-bis(tris(hydroxy-methyl)-methylamino)-propane)




ethylene glycol bis(β-aminoethyl ether)-N.N′-tetracetic acid


high molecular weight


iminodiacetic acid


low molecular weight


(2(N-morpholino) ethanesulfonic acid)


phenylmethylsulfonyl fluoride




sodium dodecyl sulfate


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Albuzio A and Ferrari G 1989 Modulation of the molecular size of humic substances by organic acids of the root exudates. Plant and Soil 113, 237–241.CrossRefGoogle Scholar
  2. Briskin D P 1987 Plasma membrane H+-transporting ATPase: Role in potassium ion transport? Physiol. Plant. 68, 159–163.Google Scholar
  3. Briskin D P and Hanson J 1992 How does the plant plasma membrane H+-ATPase pump protons? J. Exp. Bot. 43, 269–289.Google Scholar
  4. Carpita N, Sabularse D, Montezinos D and Delmer D P 1979 Determination of the pore size of cell walls of living plant cells. Science 205, 1144–1147.Google Scholar
  5. Chaminade R 1966 Physiological effects of soil organic matter constituents on the metabolism of plants. Report of the FAO/IAEA Meeting. Pergamon Press, Oxford.Google Scholar
  6. Dell'Agnola G and Nardi S 1987 Hormone-like effect and enhanced nitrate uptake induced by depolycondensed humic fractions obtained from Allolobophora rosea and A. caliginosa faeces. Biol. Fertil. Soils 4, 115–118.CrossRefGoogle Scholar
  7. DeMichelis M I, Pugliarello M C and Rasi-Caldogno F 1983 Two distinct proton translocating ATPases are present in membrane vesicles from radish seedlings. FEBS Lett. 162, 85–90.Google Scholar
  8. DeMichelis M I and Spanswick R M 1986 H+-pumping driven by the vanadate sensitive ATPase in membrane vesicles from corn roots. Plant Physiol. 81, 542–547.Google Scholar
  9. DeNobili M, Cercignani G, Leita L and Sequi P 1986 Evaluation of organic matter stabilization in sewage sludge. Commun. in Soil Sci. Plant Anal. 17, 1109–1119.Google Scholar
  10. Forbusch B 1983 Assay of the Na+K+-ATPase in plasma membrane preparations: Increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin. Anal. Biochem. 128, 159–163.Google Scholar
  11. Giannini J L and Briskin D P 1987 Proton transport in plasma membrane and tonoplast vesicles from red beet (Beta vulgaris L.) storage tissue. A comparative study of ion effects of δpH and δψ. Plant Physiol. 84, 613–618.Google Scholar
  12. Giannini J L, Gildensoph L H and Briskin D P 1987 Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue. Arch. Biochem. Biophys. 254, 621–630.CrossRefPubMedGoogle Scholar
  13. Giannini J L, Ruiz-Cristin J and Briskin D P 1987 Calcium transport in isolated vesicles from red beet (Beta vulgaris L.) storage tissue. Plant Physiol. 85, 1137–1142.Google Scholar
  14. Lew R R and Spanswick R M 1985 Characterization of anion effects on the nitrate-sensitive ATP-dependent proton pumping activity of soybean (Glycine max L.) seedling root microsomes. Plant Physiol. 77, 352–357.Google Scholar
  15. Maggioni A, Varanini Z, Nardi S and Pinton R 1987 Action of soil humic matter on plant roots: Stimulation of ion uptake and effects on (Mg2++K+) ATPase activity. Sci. Tot. Envir. 62, 355–363.CrossRefGoogle Scholar
  16. Nardi S, Concheri G, Dell'Agnola G and Scrimin P 1991 Nitrate uptake and ATPase activity in oat seedlings in the presence of two humic fractions. Soil Biol. Biochem. 23, 833–836.CrossRefGoogle Scholar
  17. Pinton R, Varanini Z, Vizzotto G and Maggioni A 1992 Soil humic substances affect transport properties of tonoplast vesicles isolated from oat roots. Plant and Soil 142, 203–210.Google Scholar
  18. Samson G and Visser S A 1989 Surface-active effect of humic acids on potato cell membrane properties. Soil Biol. Biochem. 21, 343–347.CrossRefGoogle Scholar
  19. Serrano R 1989 Structure and function of plasma membrane ATPase. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 61–94.CrossRefGoogle Scholar
  20. Vaughan D and Malcolm R E 1985 Influence of humic substances on growth and physiological processes. In Soil Organic Matter and Biological Activity. Eds. DVaughan and R EMalcolm. pp 37–75. Kluwer Academic Publishers, Dordrecht.Google Scholar
  21. Vaughan D and Ord B G 1981 Uptake and incorporation of 14C-labelled soil organic matter by roots of Pisum sativum L. J. Exp. Bot. 32, 679–687.Google Scholar
  22. Vaughan D and Ord B G 1985 Soil organic matter. A perspective on its nature, extraction, turnover and role in soil fertility. In Soil Organic Matter and Biological Activity. Eds. D.Vaughan and R.E.Malcolm pp. 1–35. Kluwer Academic Publishers, Dordrecht.Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Z. Varanini
    • 1
  • R. Pinton
    • 1
  • M. G. De Biasi
    • 2
  • S. Astolfi
    • 2
  • A. Maggioni
    • 1
  1. 1.Department of Plant Production and Agriculture TechnologyUniversity of UdineUdineItaly
  2. 2.Department of Agrobiology and AgrochemistryUniversity of ViterboViterboItaly

Personalised recommendations