, Volume 206, Issue 1–3, pp 143–151 | Cite as

RGD-mediated membrane-matrix adhesion triggers agarose-induced embryoid formation in sunflower protoplasts

  • H. Barthou
  • M. Petitprez
  • C. Brière
  • A. Souvré
  • G. Alibert


Agarose embedding of sunflower (Helianthus annuus L.) hypocotyl protoplasts induces an asymmetric division pattern and subsequent polarized development leading to embryoid formation. We cultured protoplasts in media with different mannitol concentrations. Induction of plasmolysis of agarose-embedded protoplasts by increasing the mannitol concentration lowered the proportion of embryoids formed. This indicates that adhesion sites between the plasma membrane and the agarose matrix are involved in embryoid formation. The involvement of such adhesion sites was tested by incubating embedded protoplasts with RGD peptide. 1 μM RGD heptapeptide reduced embryoid formation by 50% as compared to the control DGR peptide. We also showed that RGD heptapeptide acts on the cytoskeleton by disrupting cortical microtubules. The results are discussed in terms of a model in which the anchorage of the protoplast membrane to the agarose matrix is mediated by RGD-binding proteins connected with microtubules, determining asymmetric division of the cell and polarized development.


Sunflower Protoplasts Somatic embryogenesis Peptide RGD Cell adhesion Microtubule 



extracellular matrix


Tris-buffered saline


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  1. Asano Y, Ito Y, Sugiura K, Fujiie A (1994) Improved protoplast culture of bentgrass (Agrostis L.) using a medium with increased agarose concentration. J Plant Physiol 143: 122–124Google Scholar
  2. Bachewich C, Heath IB (1993) Effects of cell wall-cytoskeleton linkage inhibition on growth and development inSaprolegnia ferax. Inoculum 44: 25Google Scholar
  3. Barbotin JN, Nava Saucedo JE, Bazinet C, Kersulec A, Thomasset B, Thomas D (1993) Immobilization of whole cells and somatic embryos: coating process and cell-matrix interactions. In: Redenbaugh K (ed) Synseeds: application of synthetic seeds to crop improvement. CRC Press, Boca Raton, pp 65–103Google Scholar
  4. Barthou H, Brière C, Caumont C, Petitprez M, Kallerhoff J, Borin C, Souvré A, Alibert G (1997) Effect of atmospheric pressure on sunflower (Helianthus annuus L.) protoplast division. Plant Cell Rep 16: 310–314Google Scholar
  5. Boucaut JC, Darribère T, Poole TJ, Aoyama H, Yamada KM, Thiery JP (1984) Biologically active synthetic peptide as probes of embryonic development: a competitive peptide inhibitor of fibronectin function inhibits gastrulation in amphibian embryos and neural crest cell migration in avian embryos. J Cell Biol 99: 1822–1830Google Scholar
  6. Caumont C, Petitprez M, Woynaroski S, Barthou H, Brière C, Kallerhoff J, Borin C, Souvré A, Alibert G (1997) Agarose embedding affects cell wall regeneration and microtubule organization in sunflower hypocotyl protoplasts. Physiol Plant 99: 129–134Google Scholar
  7. Chanabé C, Burrus M, Alibert G (1989) Factors affecting the improvement of colony formation from sunflower protoplasts. Plant Sci 64: 125–132Google Scholar
  8. — —, Bidney D, Alibert G (1991) Studies on plant regeneration from protoplasts in the genusHelianthus. Plant Cell Rep 9: 635–638Google Scholar
  9. Corrêa A, Staples RC, Hoch HC (1996) Inhibition of thigmostimulated cell differentiation with RGD-peptides inUromyces germlings. Protoplasma 194: 91–102Google Scholar
  10. Gens JS, Reuzeau C, Doolittle KW, McNally JG, Pickard BG (1996) Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts. Protoplasma 194: 215–230Google Scholar
  11. Haas TA, Plow EF (1994) Integrin-ligand interactions: a year in review. Curr Opin Cell Biol 6: 656–662Google Scholar
  12. Haubner R, Gratias R, Diefenbach B, Goodman SL, Jonczyk A, Kessler H (1996) Structural and functional aspects of RGD-containing cyclic pentapeptides as highly potent and selective integrin antagonists. J Am Chem Soc 118: 7461–7472Google Scholar
  13. Henry CA, Jordan JR, Kropf DL (1996) Localized membrane-wall adhesions inPelvetia zygotes. Protoplasma 190: 39–52Google Scholar
  14. Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69: 11–25Google Scholar
  15. Katembe WJ, Swatzell LJ, Makaroff CA, Kiss JZ (1997) Immunolocalization of integrin-like proteins inArabidopsis andChara. Physiol Plant 99: 7–14Google Scholar
  16. Klotz SA, Smith RL (1991) A fibronectin receptor onCandida albicans mediates adherence of the fungus to extracellular matrix. J Infect Dis 163: 604–610Google Scholar
  17. Lénée P, Chupeau Y (1986) Development of nitrogen assimilating enzymes during growth of cell derived from protoplasts of sunflower and tabacco. Plant Sci 59: 109–117Google Scholar
  18. Lloyd CW (1994) Why should stationary plant cells have such dynamic microtubules? Mol Biol Cell 5: 1277–1280Google Scholar
  19. Maniotis AJ, Chen CS, Ingber DE (1997) Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc Natl Acad Sci USA 94: 849–854Google Scholar
  20. Novotny AM, Forman M (1974) The relationships between changes in cell wall composition and the establishment of polarity inFucus embryos. Dev Biol 40: 162–173Google Scholar
  21. O'Neill CM, Mathias RJ (1993) Regeneration of plants from protoplasts ofArabidopsis thaliana L. cv. Columbia (C24), via direct embryogenesis. J Exp Bot 44: 1579–1585Google Scholar
  22. Oparka KJ (1994) Plasmolysis: new insights into an old process. New Phytol 126: 571–591Google Scholar
  23. Pavalko FM, Otey CA (1994) Role of adhesion molecule cytoplasmic domains in mediating interactions with the cytoskeleton. Proc Soc Exp Biol Med 205: 282–293Google Scholar
  24. Petitprez M, Brière C, Borin C, Kallerhoff J, Souvré A, Alibert G (1995) Characterisation of protoplasts from hypocotyls ofHelianthus annuus in relation to their tissular origin. Plant Cell Tissue Organ Cult 41: 33–40Google Scholar
  25. Pont-Lezica RF, McNally JG, Pickard BG (1993) Wall-to-membrane linkers in onion epidermis: some hypotheses. Plant Cell Environ 16: 111–123Google Scholar
  26. Quatrano RS, Brian L, Aldridge J, Schultz T (1991) Polar axis fixation inFucus zygotes: components of the cytoskeleton and extracellular matrix. Development Suppl 1: 11–16Google Scholar
  27. Reuzeau C, Pont-Lezica RF (1995) Comparing plant and animal extracellular matrix—cytoskeleton connections — are they alike? Protoplasma 186: 113–121Google Scholar
  28. Robinson CR, Sligar SG (1995) Hydrostatic and osmotic pressure as tools to study macromolecular recognition. Methods Enzymol 259: 395–427Google Scholar
  29. Ryu JH, Mizuno K, Takagi S, Nagai R (1997) Extracellular components implicated in the stationnary organization of the actin cytoskeleton in mesophyll cells ofVallisneria. Plant Cell Physiol 38: 420–432Google Scholar
  30. Shibaoka H (1993) Regulation by gibberellins of the orientation of cortical microtubules in plant cells. Aust J Plant Physiol 20: 461–470Google Scholar
  31. Schindler M, Meiners S, Cheresh DA (1989) RGD-dependent linkage between plant cell wall and plasma membrane: consequences for growth. J Cell Biol 108: 1955–1965Google Scholar
  32. Schnabl H, Youngman RJ (1985) Immobilisation of plant cell protoplasts inhibits enzymic lipid peroxidation. Plant Sci 40: 65–69Google Scholar
  33. Smidsrod O, Skjak-Braek G (1990) Alginate as immobilization matrix for cells. Trends Biotechnol 8: 71–78Google Scholar
  34. Traas J (1989) Le rôle du cytosquelette dans la détermination du plan de division. Bull Soc Bot Fr 136: 99–106Google Scholar
  35. Wayne R, Staves MP, Leopold AC (1992) The contribution of the extracellular matrix to gravisensing in characean cell. J Cell Sci 101: 611–623Google Scholar
  36. Woldringh CL (1994) Significance of plasmolysis spaces as markers for periseptal annuli and adhesion sites. Mol Microbiol 14: 597–607Google Scholar
  37. Wyatt SE, Carpita NC (1993) The plant cytoskeleton-cell-wall continuum. Trends Cell Biol 3: 413–417Google Scholar
  38. Yuan M, Shaw PJ, Warn RM, Lloyd CW (1994) Dynamic reorientation of cortical microtubules from transverse to longitudinal in living plant cells. Proc Nat Acad Sci USA 91: 6050–6053Google Scholar
  39. Zandomeni K, Schopfer P (1994) Mechanosensory microtubule reorientation in the epidermis of maize coleoptiles subjected to bending stress. Protoplasma 182: 96–101Google Scholar
  40. Zhu JK, Shi J, Singh U, Wyatt SE, Bressan RA, Hasegawa PM, Carpita NC (1993) Enrichment of vitronectin- and fibronectinlike proteins in NaCl-adapted plant cells and evidence for their involvement in plasma membrane-cell wall adhesion. Plant J 3: 637–646Google Scholar

Copyright information

© Springer-Verlag 1999

Authors and Affiliations

  • H. Barthou
    • 1
  • M. Petitprez
    • 1
  • C. Brière
    • 1
  • A. Souvré
    • 1
  • G. Alibert
    • 1
  1. 1.Laboratoire de Biotechnologie et Amélioration des PlantesEcole Nationale Supérieure AgronomiqueCastanet-Tolosan CedexFrance

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