, Volume 225, Issue 6, pp 1583–1595 | Cite as

Indolacetic and humic acids induce lateral root development through a concerted plasmalemma and tonoplast H+ pumps activation

  • Daniel Basílio Zandonadi
  • Luciano Pasqualoto Canellas
  • Arnoldo Rocha Façanha
Original Article


Increasing evidences have indicated that humic substances can induce plant growth and productivity by functioning as an environmental source of auxinic activity. Here we comparatively evaluate the effects of indole-3-acetic acid (IAA) and humic acids (HA) isolated from two different soils (Inseptsol and Ultisol) and two different organic residues (vermicompost and sewage sludge) on root development and on activities of plasmalemma and tonoplast H+ pumps from maize roots. The data show that HA isolated from these different sources as well as low IAA concentrations (10−10 and 10−15 M) improve root growth through a markedly proliferation of lateral roots along with a differential activation not only of the plasmalemma but also of vacuolar H+-ATPases and H+-pyrophosphatase. Further, the vacuolar H+-ATPase had a peak of stimulation in a range from 10−8 to 10−10 M IAA, whereas the H+-pyrophosphatase was sensitive to a much broader range of IAA concentrations from 10−3 to 10−15 M. It is proposed a complementary view of the acid growth mechanism in which a concerted activation of the plasmalemma and tonoplast H+ pumps plays a key role in the root cell expansion process driven by environment-derived molecules endowed with auxinic activity, such as that of humic substances.


Cell expansion H+-PPase Humic substances Lateral root initiation Plant growth regulators V-ATPase 



Humic acids


Membrane-bound pyrophosphatase


Indole-3-acetic acid


Plasma membrane H+-adenosine triphosphatase


Vacuolar H+-adenosine triphosphatase


Vacuolar H+-pyrophosphatase



The authors would like to thank Prof. Anna L. Okorokova-Façanha (UENF, Rio de Janeiro, Brazil) for critical revision of the manuscript. We are also indebted to two anonymous referees for valuable comments and suggestions. This work was supported by the International Foundation for Science (IFS), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).


  1. Atiyeh RM, Lee CA, Edwards CA, Arancon NQ, Metzger JD (2002) The influence of humic acids derived from earthworm-processed organic wastes on plant growth. Bioresour Technol 84:7–14PubMedCrossRefGoogle Scholar
  2. Bennett AB, O’Neill SD, Spanswick RM (1984) H+-ATPase activity from storage tissue of Beta vulgaris. Plant Physiol 74:538–544PubMedGoogle Scholar
  3. Bertosa B, Kojic´-Prodic B, Wade RC, Ramek M, Piperaki S, Tsantili-Kakoulidou A, Tomic S (2003) A new approach to predict the biological activity of molecules based on similarity of their interaction fields and the log p and log d values: application to auxins. J Chem Inf Comp Sci 43:1532–1541CrossRefGoogle Scholar
  4. Bhalerao RP, Eklof J, Ljung K, Marchant A, Bennett M, Sandberg G (2002) Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings. Plant J 29:325–332PubMedCrossRefGoogle Scholar
  5. Blakely LM, Blakely RM, Colowit PM, Elliott DS (1988) Experimental studies on lateral root formation in radish seedling roots II. Analysis of the dose-response to exogenous auxin. Plant Physiol 87:414–419PubMedGoogle Scholar
  6. Bottomley WB (1917) Some effects of organic-promotion substances (auximones) on the growth of Lema minor in mineral cultural solutions. Proc Royal Soc London Series B Biol Sci 89:481–505Google Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantification of micrograms quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  8. Cacco G, Dell’Agnola G (1984) Plant growth regulator activity of soluble humic complexes. Can J Soil Sci 64:225–228CrossRefGoogle Scholar
  9. Canellas LP, Façanha AR (2004) Chemical nature of soil humified fractions and their bioactivity. Pesq Agropec Bras 39:233–234Google Scholar
  10. Canellas LP, Olivares FL, Okorokova-Façanha AL, Façanha AR (2002) Humic acids isolated from earthworm compost enhance root elongation, lateral root emergence, and plasma membrane H+-ATPase activity in maize roots. Plant Physiol 130:1951–1957PubMedCrossRefGoogle Scholar
  11. Canellas LP, Zandonadi DB, Médice LO, Peres LEP, Olivares FL, Façanha AR (2005) Bioatividade de substâncias húmicas – ação sobre o desenvolvimento e metabolismo das plantas. In: Canellas LP, Santos GA (eds) Humosfera: tratado preliminar sobre a química das substâncias húmicas. Campos dos Goytacazes, Rio de Janeiro, pp 238–239Google Scholar
  12. Casimiro I, Marchant A, Bhalerao RP, Beeckman T, Dhooge S, Swarup R, Graham N, Inzé D, Sandber G, Casero PJ, Benett M (2001) Auxin transport promotes Arabidopsis lateral root initiation. Plant Cell 13:843–852PubMedCrossRefGoogle Scholar
  13. Charleton WA (1991) Lateral root initiation. In: Waisel Y, Eshel A, Kafkafi U (eds) Plant roots: the hidden half. Marcel Dekker, New York, pp 103–128Google Scholar
  14. Chen Y, Aviad T (1990) Effects of humic substances on plant growth. In: MacCarthy P, Clapp CE, Malcolm RL, Bloom PR (eds) Humic substances in soil and crop science selected readings. American Society of Agronomy Inc., Soil Science of America Inc., Madison, pp 161–186Google Scholar
  15. Chen Y, Senesi N, Schnitzer M (1977) Information provide on humic substances by E4/E6 ratios. Soil Sci Am J 41:352–358CrossRefGoogle Scholar
  16. Cosgrove DJ (2000) Expansive growth of plant cell walls. Plant Physiol Biochem 38:109–124PubMedCrossRefGoogle Scholar
  17. Coulthup NB, Daly LH, Wiberley SE (1964) Introduction to infrared and NMR spectroscopy. Academic, New YorkGoogle Scholar
  18. Dolan L, Davies J (2004) Cell expansion in roots. Curr Opin Plant Biol 7:33–39PubMedCrossRefGoogle Scholar
  19. Evans ML, Ishikawa H, Estelle MA (1994) Responses of Arabidopsis roots to auxin studied with high temporal resolution: comparison of wild type and auxin response mutants. Planta 194:215–222CrossRefGoogle Scholar
  20. Façanha AR, de Meis L (1998) Reversibility of H+-ATPase and H+-Pyrophosphatase in tonoplast vesicles from maize coleoptiles and seeds. Plant Physiol 116:1487–1495CrossRefGoogle Scholar
  21. Façanha AR, Façanha ALO, Olivares FL, Guridi F, Santos GA, Velloso ACX, Rumjanek VM, Brasil F, Schripsema J, Braz-filho R, Oliveira MA, Canellas LP (2002) Bioatividade de ácidos húmicos: efeito sobre o desenvolvimento radicular e sobre a bomba de prótons da membrana plasmática. Pesq Agropec Bras 37:1301–1310Google Scholar
  22. Fiske CF, Subbarow Y (1925) The colorometric determination of phosphorus. J Biol Chem 66:375–383Google Scholar
  23. Gogarten JP, Fichmann J, Braun Y, Morgan L, Styles P, Taiz SL, DeLapp K, Taiz L (1992) The use of antisense mRNA to inhibit the tonoplast H+-ATPase in carrot. Plant Cell 4:851–864PubMedCrossRefGoogle Scholar
  24. Govindasmy R, Chandrasekaran S (1992) Effect of humic substances on the growth, yield, and nutrient content of sugar cane. Sci Total Environ 117/118:575–581CrossRefGoogle Scholar
  25. Guminski S (1968) Present days view on physiological effects induced in plant organisms by humic compounds. Soviet Soil Sci 9:1250–1255Google Scholar
  26. Hager A, Debus G, Edel HG, Stransky H, Serrano R (1991) Auxin induces exocytosis and rapid synthesis of a high-turnover pool of plasma-membrane H+-ATPase. Planta 185:527–537CrossRefGoogle Scholar
  27. Huss M, Ingenhorst G, König S, Gabel M, Dröse S, Zeeck A, Altendorf K, Wieczore H (2002) Concanamycin A, the specific inhibitor of V-ATPases, binds to the Vo subunit c. J Biol Chem 277:40544–40548PubMedCrossRefGoogle Scholar
  28. Johnston CT, Aochi YO (1996) Fourier transform infrared and raman spectroscopy. In: DL Spark (ed) Methods of soil analysis. Chemical methods. American Society of Agronomy, Madison, pp 269–321Google Scholar
  29. Kononova MM (1966) Soil organic matter, its nature, its role in soil formation and in soil fertility. Pergamon, New YorkGoogle Scholar
  30. Kononova MM (1982) Materia orgánica del suelo: su naturaleza, propiedades y métodos de investigación. Oikos-tau, BarcelonaGoogle Scholar
  31. Leyser O, Fitter A (1998) Roots are branching out in patches. Trends Plant Sci 3:203–204CrossRefGoogle Scholar
  32. Li JS, Yang HB, Peer WA, Richter G, Blakeslee J, Bandyopadhyay A, Titapiwantakun B, Undurraga S, Khodakovskaya M, Richards EL, Krizek B, Murphy AS, Gilroy S, Gaxiola R (2005) Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development. Science 310(5745):121–125PubMedCrossRefGoogle Scholar
  33. Maeshima M, Nakanishi Y, Matsuura-Endo C, Tanaka Y (1996) Proton pumps of the vacuolar membrane in growing plant cells. J Plant Res 109:119–125CrossRefGoogle Scholar
  34. Malamy JE, Ryan KS (2001) Environmental regulation of lateral root initiation in Arabidopsis. Plant Physiol 127:899–909PubMedCrossRefGoogle Scholar
  35. Martin-Neto L, Cruvinel P, Mattoso LHC, Colnago LA (1996) Espectroscopias de infravermelho, ultravioleta-visível e pixe: alguns resultados disponíveis. In: Crestana S, Cruvinel PE, Mascarenhas S, Biscegli CI (eds) Instrumentação agropecuária: contribuições no limiar do novo século. Empresa Brasileira de Pesquisa Agropecuária, São Carlos, pp 51–90Google Scholar
  36. Masciandaro G, Ceccanti B, Garcia C (1999) Soil agro-ecological management: fertirrigation and vermicompost treatments. Bioresour Technol 59:199–220CrossRefGoogle Scholar
  37. Mato MC, Olmedo MG, Méndez J (1972) Inhibition of indoleacetic acid-oxidase by soil humic acids fractioned on sephadex. Soil Biol Biochem 4:469–473CrossRefGoogle Scholar
  38. Mulkey TJ, Kuzmanoff KM, Evans ML (1982) Promotion of growth and hydrogen ion efflux by auxin in roots of maize pretreated with ethylene biosynthesis inhibitors. Plant Physiol 70:186–188PubMedGoogle Scholar
  39. Muscolo A, Cutrupi S, Nardi S (1998) IAA detection in humic substances. Soil Biol Biochem 30:1199–1201CrossRefGoogle Scholar
  40. Nardi S, Pizzeghello D, Muscolo A, Vianello A (2002) Physiological effects of humic substances in higher plants. Soil Biol Biochem 34:1527–1537CrossRefGoogle Scholar
  41. Ozolina NV, Pradedova EV, Salyaev RK (1996) Phytohormone effects on hydrolytic activity of phosphohydrolases in red beet (Beta vulgaris L.) tonoplasts. Plant Growth Regul 19:189–191CrossRefGoogle Scholar
  42. Padmanaban S, Lin X, Perera I, Kawamura Y, Sze H (2004) Differential expression of vacuolar H+-ATPase subunit c genes in tissues active in membrane trafficking and their roles in plant growth as revealed by RNAi. Plant Physiol 134:1514–1526PubMedCrossRefGoogle Scholar
  43. Piccolo A (2001) The supramolecular structure of humic substances. Soil Sci 166:810–832CrossRefGoogle Scholar
  44. Pinton R, Varanini Z, Vizzoto G, Maggioni A (1992) Soil humic substances affect transport-properties of tonoplast vesicles isolated from oat roots. Plant Soil 142:203–210CrossRefGoogle Scholar
  45. Pizzeghello D, Nicolini G, Nardi S (2001) Hormone-like activity of humic substances in Fagus sylvaticae forests. New Phytol 151:647–657CrossRefGoogle Scholar
  46. Quaggiotti S, Ruperti B, Pizzeghello D, Francioso O, Tugnoli V, Nardi S (2004) Effect of low molecular size humic substances on nitrate uptake and expression of genes involved in nitrate transport in maize (Zea mays L.). J Exp Bot 55:803–813PubMedCrossRefGoogle Scholar
  47. Rayle DL, Cleland R (1992) The acid growth theory of auxin-induced cell elongation is alive and well. Plant Physiol 99:1271–1274PubMedCrossRefGoogle Scholar
  48. Salyaev RK, Ozolina NV, Pradedova EV (1999) Effects of exogenous phytohormones and kinetin on the hydrolytic activity of proton pumps in the tonoplast of red beet at different stages of plant development. Russ J Plant Physiol 46:1–4Google Scholar
  49. Schnitzer M, Gupta UC (1965) Determination of acidity in soil organic matter. Soil Sci Soc Am Proc 27:274–277CrossRefGoogle Scholar
  50. Schnitzer M, Poapst PA (1967) Effects of a soil humic compound on root initiation. Nature 213:598–599CrossRefGoogle Scholar
  51. Schnitzer M, Skinner SIM (1982) Organic matter characterization. In: American Society of Agronomy/Soil Science Society of America Agronomic Monograph (eds) Method of soil analysis, part 2. Chemical and mineralogical properties. ASA/SSSA Publishers, Madison, pp 581–597Google Scholar
  52. Schnitzer M (1991) Soil organic matter: the next 75 years. Soil Sci 151:41–58CrossRefGoogle Scholar
  53. Schumacher K, Vafeados D, McCarthy M, Sze H, Wilkins T, Chory J (1999) The Arabidopsis det3 mutant reveals a central role for the vacuolar H+-ATPase in plant growth and development. Genes Dev 13:3259–3270PubMedCrossRefGoogle Scholar
  54. Simpsom AJ (2002) Determining the molecular weight, aggregation, structures and interactions of natural organic matter using diffusion ordered spectroscopy. Magnet Reson Chem 40:572–582Google Scholar
  55. Smart LB, Vojdani F, Maeshima M, Wilkins TA (1998) Genes involved in osmoregulation during turgor-driven cell expansion of developing cotton fibres are differentially regulated. Plant Physiol 116:1539–1549PubMedCrossRefGoogle Scholar
  56. Stepanova AN, Hoyt JM, Hamilton AA, Alonso JM (2005) A link between ethylene and auxin uncovered by the characterization of two root-specific ethylene-insensitive mutants in Arabidopsis. Plant Cell 17:2230–2242PubMedCrossRefGoogle Scholar
  57. Stevenson FJ (1994) Biochemistry of the formation of humic substances. In: Stevenson FJ (ed) Humus chemistry, genesis, composition, reactions. John Wiley, New York, pp 188–211Google Scholar
  58. Swarup R, Parry G, Graham N, Allen T, Bennett M (2002) Auxin cross-talk: integration of signalling pathways to control plant development. Plant Mol Biol 49:411–426PubMedCrossRefGoogle Scholar
  59. Sze H (1985) H+-translocating ATPases: advances using membrane vesicles. Annu Rev Plant Physiol 36:113–122CrossRefGoogle Scholar
  60. Vaughan D, Malcom RE (1985) Influence of humic substances on growth and physiological process. In: Vaughan D, Malcolm RE (eds) Soil organic matter and biological activity. Kluwer, Dordrecht, pp 37–75Google Scholar
  61. Whitehead DC (1963) Some aspects of the influence of organic matter on soil fertility. Soils Fert 26:217–223Google Scholar
  62. Zhao H, Hertel R, Ishikawa H, Evans ML (2002) Species differences in ligand specificity of auxin-controlled elongation and auxin transport: comparing Zea and Vigna. Planta 216:293–301PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Daniel Basílio Zandonadi
    • 1
  • Luciano Pasqualoto Canellas
    • 2
  • Arnoldo Rocha Façanha
    • 1
  1. 1.Laboratório de Biologia Celular e Tecidual (LBCT), Centro de Biociências e Biotecnologia (CBB)Universidade Estadual do Norte Fluminense (UENF)Rio de JaneiroBrazil
  2. 2.Laboratório de Solos, Centro de Ciências e Tecnologias AgropecuáriasUniversidade Estadual do Norte FluminenseRio de JaneiroBrazil

Personalised recommendations