Skip to main content
SpringerLink
Log in

Auxin levels and auxin binding protein availability inrolB transformedBeta vulgaris cells

  • Original Papers
  • Published:
Biologia Plantarum

Abstract

The final biological effect of auxin depends both on free auxin levels and on auxin perception capacity.RolB transformedBeta vulgaris L. hairy roots provide a system for studying both factors. Highly purified plasma membrane fractions were prepared with aqueous two-phase partitioning. Individual hairy root clones were assessed for the binding activities of plasma membrane-bound auxin binding proteins and for their free intracellular indole-3-acetic acid levels. The presence of a high affinity auxin binding protein with a dissociation constant of 9.07 x 10−7 M was detected in the plasma membrane fractions isolated from non-transformed seedling roots and the six clones ofrolB transformed hairy roots. However, the levels of specific IAA binding considerably varied among different hairy root clones and between transformed and non-transformed roots. The levels of the detectable polypeptide in immunoblotting with an antibody against maize 22-kD auxin binding protein subunit were in good agreement to the levels that were detected in auxin binding assays. Differences in the indole-3-acetic acid levels were found between transformed and non-transformed roots and also between different transformed hairy root clones. A negative correlation was observed between free intracellular IAA levels and its specific binding to the plasma membrane-bound auxin binding proteins. A latency study indicated that the binding site for auxin may be located on the exterior face of the plasma membrane

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

ABP:

auxin binding protein

GS-SIM-MS:

gas chromatography-selected ionmonitoring-mass spectroscopy

IAA:

indole-3-acetic acid

PM:

plasma membrane

References

  • Barbier-Brygoo, H., Ephritikhine, G., Klambt, D., Ghislan, M., Guem, J.: Functional evidence for an auxin receptor at the plasmalemma of tobacco mesophyll protoplasts. -Proc. nat. Acad. Sci. USA86: 891–895, 1989.

    Article  PubMed  CAS  Google Scholar 

  • Barbier-Brygoo, H., Ephritikhine, G., Klambt, D., Maurel, C., Palme, K., Schell, J., Guern, J.: Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts. -Plant J.1: 83–93, 1991.

    Article  CAS  Google Scholar 

  • Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. -Anal. Biochem.72: 248–254, 1976.

    Article  PubMed  CAS  Google Scholar 

  • Cardarelli, M., Mariotti, D., Pomponi, M., Spano, L., Capone, I., Costantino, P.:Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype. -Mol. gen. Genet.209: 475- 480, 1987.

    Article  PubMed  CAS  Google Scholar 

  • Davies, P.J.: Plant Hormones and their Role in Plant Growth and Development. - Martinus Nijhoff Publishers, Dordrecht 1991.

    Google Scholar 

  • Delbarre, A., Muller, P., Imhoff, V., Barbier-Brygoo, H., Maurel, C., Leblanc, N., Perrot-Rechenmann, C., Guern, J.: TherolB gene ofAgrobacterium rhizogenes does not increase the auxin sensitivity of tobacco protoplasts by modifying the intracellular auxin concentration. - Plant Physiol.105: 563–569, 1994.

    PubMed  CAS  Google Scholar 

  • Ephritikhine, G., Barbier-Brygoo, H., Muller, J.F., Guem, J.: Auxin effect on the transmembrane potential difference of wild-type and mutant tobacco protoplasts exhibiting a differential sensitivity to auxin. - Plant Physiol.83: 801–804, 1987.

    PubMed  CAS  Google Scholar 

  • Estruch, J.J., Schell, J., Spena, A.: The protein encoded by therolB plant oncogene hydrolyses indole glucosides. - EMBO J.10: 3125–3128, 1991.

    PubMed  CAS  Google Scholar 

  • Filippini, F., Schiavo, F.L., Terzi, M., Costantino, F., Trovato, M.: The plant oncogenerolB alters binding of auxin to plant cell membranes. - Plant Cell Physiol.35: 767–771, 1994.

    CAS  Google Scholar 

  • Gartland, K.M.A., McInnes, E.,Hall, J.F., Mulligan, B.J., Morgan, A.J., Elliott, M.C., Davey, M.R.: Effects ofRi plasmidrol gene expression on the IAA content of transformed roots ofSolarium dulcamara L. - Plant Growth Regul.10: 235–241, 1991.

    Article  CAS  Google Scholar 

  • Hagen, G.: Molecular approaches to understanding auxin action. - New Biol.1: 19–23, 1989.

    PubMed  CAS  Google Scholar 

  • Hodges, T.K., Leonard, R.T.: Purification of a plasma-membrane bound adenosine triphosphatase from plant roots. -Methods Enzymol.32: 392–406, 1974.

    PubMed  CAS  Google Scholar 

  • Huffman, G.A., White, F.W., Gordon, M.P., Nester, E.W.: Hairy-root-inducing plasmid: physical map and homology to tumor-inducing plasmids. - J. Bacteriol.157: 269–276, 1984.

    PubMed  CAS  Google Scholar 

  • Inohara, N., Shimomura, S., Fukui, T., Futai, M.: Auxin-binding protein located in the endoplasmic reticulum of maize shoots: molecular cloning and complete primary structure. - Proc. nat. Acad. Sci. USA86: 3564–3568, 1989.

    Article  PubMed  CAS  Google Scholar 

  • Jones, A.M., Prasad, P.V.: Auxin-binding proteins and their possible roles in auxin-mediated plant cell growth. -BioAssays14: 43–48, 1992.

    Article  CAS  Google Scholar 

  • Jones, A.M., Herman, E.M.: KDEL-containing auxin-binding protein is secreted to the plasma membrane and cell wall. -Plant Physiol.101: 595–606, 1993.

    PubMed  CAS  Google Scholar 

  • Jones, A.M., Lamerson, P.L., Venis, M.A.: Comparison of site I auxin binding and a 22-kilodalton protein in maize. - Planta179: 409–413, 1989.

    Article  CAS  Google Scholar 

  • Julliard, J., Sotta, B., Pelletier, G., Miginiac, E.: Enhancement of naphthaleneacetic acid-induced rhizogenesis in TL-DNA-transformedBrassica nctpus without significant modification of auxin levels and auxin sensitivity. - Plant Physiol.100: 1277–1282, 1992.

    PubMed  CAS  Google Scholar 

  • Larsson, C., Widell, S., Kjellbom, P.: Preparation of high-purity plasma membranes. - Methods Enzymol.148: 558–568, 1987.

    CAS  Google Scholar 

  • Löbler, M., Klämbt, D.: Auxin-binding protein from coleoptile membranes of corn. I. Purification by immnological methods. - J. biol. Chem.260: 9848–9853, 1985a.

    PubMed  Google Scholar 

  • Löbler, M., Klämbt, D.: Auxin-binding protein form coleoptile membranes of corn. II. Localization of a putative auxin receptor. - J. biol. Chem.260: 9854–9859, 1985b.

    PubMed  Google Scholar 

  • Maurel, C., Barbier-Brygoo, H., Spena, A., Tempe, J., Guern, J.: Singlerol genes from theAgrobacterium rhizogenes TL-DNA alter some of the cellular responses to auxin inNicotiana tabacum. - Plant Physiol.97: 212–216, 1991.

    PubMed  CAS  Google Scholar 

  • Munro, S., Pelham, H.R.B.: A C-terminal signal prevents secretion of luminal ER proteins. - Cell48: 899–907, 1987.

    Article  PubMed  CAS  Google Scholar 

  • Napier, R.M., Venis, M.: From auxin-binding protein to plant hormone receptor. -Trends Biochem. Sci.16: 72–75, 1991.

    Article  PubMed  Google Scholar 

  • Napier, R.M., Venis, M.A., Bolton, M.A., Richardson, L.I., Butcher, G.W.: Preparation and characterization of monoclonal and polyclonal antibodies to maize membrane auxin-binding protein. - Planta176: 519–525, 1988.

    Article  CAS  Google Scholar 

  • Nilsson, O., Crozier, A., Schmulling, T., Sandberg, G., Olsson, O.: Indole-3-acetic acid homeostasis in transgenic tobacco plants expressing theAgrobacterium rhizogenes rolB gene. - Plant J.3: 681- 689, 1993.

    Article  CAS  Google Scholar 

  • Ohnishi, T., Gall, R.S., Mayer, M.L.: An improved assay of inorganic phosphate in the presence of extra-labile phosphate compounds: application to the ATPase assay in the presence of phosphocreatine. -Anal. Biochem.69: 261–267, 1975.

    Article  PubMed  CAS  Google Scholar 

  • Palme, K., Hesse, T., Moore, I., Campos, N., Fildwisch, J., Garbers, C., Hesse, F., Schell, J.: Hormonal modulation of plant growth: the role of auxin perception. - Mechanism Develop.33: 97- 106, 1991.

    Article  CAS  Google Scholar 

  • Scatchard, G.: The attraction of proteins for small molecules and ions. - Ann. N.Y. Acad. Sci.57: 660–672, 1949.

    Article  Google Scholar 

  • Schaerer, S., Pilet, P.E.: Quantification of indole-3-acetic acid in untransformed andAgrobacterium rhizogenes-transformed pea roots using gas chromatography mass spectroscopy. - Planta189: 55- 59, 1993.

    Article  CAS  Google Scholar 

  • Schmülling, T., Schell, J., Spena, A.: Single genes fromAgrobacterium rhizogenes influence plant development. - EMBO J.7: 2621–2629, 1988.

    PubMed  Google Scholar 

  • Schmulling, T., Fladung, M., Grossmann, K., Schell, J.: Hormonal content and sensitivity of transgenic tobacco and potato plants expressing singlerol genes ofAgrobacterium rhizogenes. - Plant J.3:371–382, 1993.

    Google Scholar 

  • Shen, W.H., Petit, A., Guern, J., Tempe, J.: Hairy roots are more sensitive to auxin than normal roots. - Proc. nat. Acad. Sci. USA85: 3417–3421, 1988.

    Article  PubMed  CAS  Google Scholar 

  • Shimomura, S., Sotobayashi, T., Futai, M., Fukui, T. Purification and properties of an auxin-binding protein from maize shoot membranes. - J. Biochem.99: 1513–1524, 1986.

    PubMed  CAS  Google Scholar 

  • Shimomura, S., Inohara, N., Fukui, T., Futai, M.: Different properties of two types of auxin-binding sites in membranes from maize coleoptiles. - Planta175: 558–566, 1988.

    Article  CAS  Google Scholar 

  • Sitbon, F., Hennion, S., Sundberg, B., Little, C.H.A., Olsson, O., Sandberg, G.: Transgenic tobacco plants co-expressing theAgrobacterium tumefaciens iaaM andiaaH genes display altered growth and indoleacetic acid metabolism. - Plant Physiol.99: 1062–1069, 1992.

    PubMed  CAS  Google Scholar 

  • Spano, L., Mariotti, D., Cardarelli, M., Branca, C. Costantino, P.: Morphogenesis and auxin sensitivity of transgenic tobacco with different complements of Ri T-DNA. - Plant Physiol.87: 479–483, 1988.

    PubMed  CAS  Google Scholar 

  • Spena, A., Schmülling, T. Koncz, C. Schell, J.: Independent and synergistic activity ofrolA, B andC loci in stimulating abnormal growth in plants. - EMBO J.6: 3891–3899, 1987.

    PubMed  CAS  Google Scholar 

  • Spena, A., Estruch J.J., Hansen, G., Langenkemper, K., Berger, S., Schell, J.: The rhizogenes tale: modification of plant growth and physiology by an enzymatic system of hydrolysis of phytohormone conjugates. - In: Nester, E.W., Verma D.P.S. (ed.): Advances in Molecular Genetics of Plant Microbe Interactions. Pp. 109–124. Kluwer Academic Publishers, Dordrecht 1993.

    Google Scholar 

  • Sundberg, B.: Influence of extraction solvent (buffer, methanol, acetone) and time on the quantification of indole-3-acetic acid in plants. - Physiol. Plant.78: 293–297, 1990.

    Article  CAS  Google Scholar 

  • Thomson, L.J., Xing, T., Hall, J.L., Williams, L.E.: Investigation of the calcium-transporting ATPases at the endoplasmic reticulum and plasma membrane of red beet (Beta vulgaris). - Plant Physiol.102: 553–564, 1993.

    PubMed  CAS  Google Scholar 

  • Tillman, U., Viola, G., Kayser, B., Siemeister, G., Hesse, T., Palme, K., Löbler, M., Klambt, D.: cDNA clones of the auxin-binding protein from corn coleoptiles (Zea mays L.): isolation and characterization by immunological methods. - EMBO J.8: 2463–2467, 1989.

    Google Scholar 

  • Venis, M.A., Thomas, E.W., Barbier-Brygoo, H., Ephritikhine, G., Guern, J.: Impermeant auxin analogues have auxin activity. - Planta182: 232–235, 1990.

    Article  CAS  Google Scholar 

  • White, F.F., Taylor, B.H., Huffman, G.A., Gordon, M.P., Nester, E.W.: Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid ofAgrobacterium rhizogenes. - J. Bacteriol.164: 33–44, 1985.

    PubMed  CAS  Google Scholar 

  • Williams, L.E., Schueler, S.B., Briskin, D.P.: Further characterization of the red beet plasma membrane Ca2+-ATPase using GTP as an alternative substrate. - Plant Physiol.92: 747–754, 1990.

    Article  PubMed  CAS  Google Scholar 

  • Xing, T., Williams, L.E., Nelson, S.J., East, J.M., Hall, I.L.: Immunological detection and localization of a calsequestrin-like protein in red beet and cucumber cells. - Protoplasma179: 158- 165, 1994.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Botany, University of Toronto, 25 Willcocks Street, M5S 3B2, Toronto, Ontario, Canada

    T. Xing & E. Blumwald

  2. Shanghai Institute of Plant Physiology, 300 Fonglin Rd, 200032, Shanghai, China

    D. -Y. Zhang

  3. Department of Biological Sciences, De Montfort University, LE7 9SU, Scraptoft, Leicester, UK

    J. F. Hall

Authors
  1. T. Xing
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E. Blumwald
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D. -Y. Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. F. Hall
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to T. Xing.

Additional information

Acknowledgments: We would like to thank Dr. R. M. Napier (Horticulture Research International, East Mailing, Kent, UK) for the generous gifts of antibodies.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xing, T., Blumwald, E., Zhang, D.Y. et al. Auxin levels and auxin binding protein availability inrolB transformedBeta vulgaris cells. Biol Plant 38, 351–362 (1996). https://doi.org/10.1007/BF02896662

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02896662

Additional key words

Search

Navigation