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Structural analysis of the cell walls regenerated by carrot protoplasts

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Abstract

A procedure was developed to isolate protoplasts rapidly from carrot (Daucus carota L. cv. Danvers) cells in liquid culture. High purity of cell-wall-degrading enzymes and ease of isolation each contributed to maintenance of viability and initiation of regeneration of the cell wall by a great majority of the protoplasts. We used this system to re-evaluate the chemical structure and physical properties of the incipient cell wall. Contrary to other reports, callose, a (1 → 3)β-d-glucan whose synthesis is associated with wounding, was not a component of the incipient wall of carrot protoplasts. Intentional wounding by rapid shaking or treatment with dimethyl sulfoxide initiated synthesis of callose, detected both by Aniline blue and Cellufluor fluorescence of dying cells and by an increase in (1 → 3)-linked glucan quantified in methylation analyses. Linkage analyses by gas-liquid chromatography of partially methylated alditol-acetate derivatives of polysaccharides of the incipient wall of protoplasts and various fractions of the cell walls of parent cells showed that protoplasts quickly initiated synthesis of the same pectic and hemicellulosic polymers as normal cells, but acid-resistant cellulose was formed slowly. Complete formation of the wall required 3 d in culture, and at least 5 d were required before the wall could withstand turgor. Pectic substances synthesized by protoplasts were less anionic than those of parent cells, and became more highly charged during wall regeneration. We propose that de-esterification of the carboxyl groups of pectin uronic-acid units permits formation of a gel that envelops the protoplast, and the rigid cellulose-hemicellulose frame-work forms along with this gel matrix.

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Abbreviations

DEAE:

Diethylaminoethyl

DMSO:

dimethyl sulfoxide

ECP:

extracellular polymers

EDTA:

ethylenediaminetetraacetic acid

HGA:

nomogalacturonan

RG:

rhamnogalacturonan

Tes:

N-tris(hydroxymethyl)methyl-2-amino-ethanesufonic acid

TFA:

trifluoroacetic acid

References

  1. Asamizu, T., Nishi, A. (1980) Regenerated cell wall of carrot protoplasts isolated from suspension-cultured cells. Physiol. Plant. 48, 207–212

  2. Asamizu, T., Tanaka, K., Takebe, I., Nishi, A. (1977) Change in molecular size of cellulose during regeneration of cell wall on carrot protoplasts. Physiol Plant. 40, 215–218

  3. Aspinall, G.O., Greenwood, C.T., Sturgeon, R.J. (1962) The degradation of xylans by alkali. J. Chem. Soc., 3667–3677

  4. Bacic, A., Harris, P.J., Stone, B.A. (1988) Structure and function of plant cell walls. In: The biochemistry of plants, vol. 14, pp. 297–371, Preiss, J., ed. Academic Press, New York

  5. Blakeney, A.B., Harris, P.J., Henry, R.J., Stone, B.A. (1983) A simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohydr. Res. 113, 291–299

  6. Blaschek, W., Haass, D., Koehler, H., Franz, G. (1981) Cell wall regeneration by Nicotiana tabacum protoplasts: chemical and biochemical aspects. Plant Sci. Lett. 22, 47–57

  7. Bradford, M.M. (1976) 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

  8. Carpita, N.C., Kanabus, J. (1987) Extraction of starch by dimethyl sulfoxide and quantitation by enzymatic assay. Anal. Biochem. 161, 132–139

  9. Carpita, N.C., Shea, E.M. (1988) Linkage structure of carbohydrates by gas chromatography-mass spectrometry of partially methylated alditol acetates. In: Analysis of carbohydrates by gas-liquid chromatography and mass spectrometry, pp. 156–215, Biermann, C., MacGinnis, G., eds. CRC Press, Boca Raton, Fla., USA

  10. Carpita, N.C., Whittern, D. (1986) A highly substituted glucuronoarabinoxylan from developing maize coleoptiles. Carbohydr. Res. 146, 129–140

  11. Clark, G. (1973) Staining procedures used by the Biological Staining Commission, edn. 3, p. 213

  12. Cooper, J.B., Chen, J.A., van Holst, G-J., Varner, J.E. (1987) Hydroxyproline-rich glycoproteins of plant cell walls. Trends Biochem. Sci. 12, 24–27

  13. Darvill, J.E., McNeil, M., Darvill, A.G., Albersheim, P. (1980) Structure of plant cell walls XI. Glucuronoarabinoxylan, a second hemicellulose in the primary cell walls of suspension-cultured sycamore cells. Plant Physiol. 66, 1135–1139

  14. Delmer, D.P. (1987) Cellulose biosynthesis. Annu. Rev. Plant Physiol. 38, 259–290

  15. Dische, Z. (1947) A new specific color reaction of hexuronic acids. J. Biol. Chem. 167, 189–198

  16. DuBois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356

  17. Fincher, G.B., Stone, B.A., Clarke, A.E. (1983) Arabinogalactan-proteins: Structure, biosynthesis, and function. Annu. Rev. Plant Physiol. 34, 47–70

  18. Fry, S.C. (1986) Cross-linking of matrix polymers in the growing cell walls of angiosperms. Annu. Rev. Plant Physiol. 37, 165–186

  19. Galambos, J.T. (1967) The reaction of carbazole with carbohydrates 1. Effect of borate and sulfamate on the carbazole color of sugars. Anal Biochem. 19, 119–132

  20. Gamborg, O.L. (1975) Callus and cell culture. In: Plant tissue culture methods, pp. 1–10, Gamborg, O.L., Wetter, L.R., eds. National Research Council of Canada, Ottawa, Ont.

  21. Gould, J.H., Palmer, R.L., Dugger, W.M. (1986) Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall. Plant Cell Tiss. 6, 47–59

  22. Hanke, D.E., Northcote, D.H. (1974) Cell wall formation by soybean callus protoplasts. J. Cell Sci. 14, 29–50

  23. Hayashi, T., Polonenko, D.R., Camirand, A., Maclachlan, G. (1986) Pea xyloglucan and cellulose IV. Assembly of β-glucans by pea protoplasts. Plant Physiol. 82, 301–306

  24. Herth, W., Meyer, Y. (1977) Ultrastructural and chemical analysis of the wall fibrils synthesized by tobacco mesophyll protoplasts. Biol. Cell. 30, 33–40

  25. Iraki, N.M., Bressan, R.A., Hasegawa, P.M. Carpita, N.C. (1989) Alterations of the physical and chemical structure of the primary cell wall of growth limited plant cells adapted to osmotic stress. Plant Physiol. (in press)

  26. Jarvis, M.C. (1984) Structure and properties of pectin gels in plant cell walls. Plant Cell Environ. 7, 153–164

  27. Kanabus, J., Bressan, R.A., Carpita, N.C. (1986) Carbon assimilation in carrot cells in liquid culture. Plant Physiol. 82, 363–368

  28. Kauss, H., Hassid, W.Z. (1967) Enzymic introduction of the methyl ester groups of pectin. J. Biol. Chem. 242, 3449–3453

  29. Klein, A.S., Montezinos, D., Delmer, D.P. (1981) Cellulose and 1,3-glucan synthesis during the early stages of wall regeneration in soybean protoplasts. Planta 152, 105–114

  30. Konno, H., Yamasaki, Y., Katoh, K. (1986) Enzymatic degradation of pectic substances and cell walls purified from carrot cell cultures. Phytochemistry 25, 623–627

  31. Kvernheim, A.L. (1987) Methylation analysis of polysaccharides with butyllithium in dimethylsulfoxide. Acta Chem. Scand., B41, 150–154

  32. Lamport, D.T.A., Epstein, L. (1983) A new model for the primary cell wall: A concatenated extensin-cellulose network. In: Current topics in plant biochemistry, vol. 2, pp. 73–83, Randall, D.D., Blevins, D.G., Larson, R.L., Rapp, B.J., eds. University of Missouri, Columbia, USA

  33. Maltby, D., Carpita, N.C., Montezinos, D., Kulow, C., Delmer, D.P. (1979) β-1,3-Glucan in developing cotton fibers. Structure, localization, and relationship of synthesis to that of secondary wall cellulose. Plant Physiol. 63, 1158–1164

  34. Moustacas, A-M-. Nari, J., Diamantidis, G., Noat, G., Crasnier, M., Borel, M., Ricard, J. (1986) Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 2. The role of pectin methyl esterase in the modulation of electrostatic effects in soybean cell walls. Eur. J. Biochem. 155, 191–197

  35. Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and biossays with tobacco tissue cultures. Physiol. Plant. 15, 473–497

  36. Pilet, P.E., Blaschek, W., Seen, A., Franz, G. (1984) Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides. Planta 161, 465–469

  37. Prat, R., Roland, J.-C. (1971) Etude ultrastructurale des premiers stades de néoformation d'une enveloppe par les protoplastes végétaux séparés mécaniquement de leur paroi. C.R. Acad. Sci. Paris 273D, 165–168

  38. Rees, D.A. (1977) Polysaccharide shapes. Outlines Studies in Botany Series. Chapman & Hall, London

  39. Shea, E.M. (1988) Structural analysis of walls regenerated by isolated protoplasts and plasmolyzed cells. Ph.D. thesis, Purdue University, West Lafayette, Ind., USA

  40. Shea, E.M., Carpita, N.C. (1988) Separation of partially methylated alditol acetates on SP-2330 and HP-1 vitreous silica capillary columns. J. Chromatogr. 445, 424–428

  41. Somogyi, M. (1952) Notes on sugar determination. J. Biol. Chem. 195, 19–23

  42. Stevens, B.J.H., Selvendran, R.R. (1984) Structural features of cell-wall polysaccharides of the carrot Daucus carota. Carbohydr. Res. 128, 321–333

  43. Sweet, D.P., Shapiro, R.H., Albersheim, P. (1975) Quantitative analysis by various g.1.c. response-factor theories for partially methylated and partially-ethylated alditol acetates. Carbohydr. Res. 40, 217–225

  44. Takeuchi, Y., Komamine, A. (1978) Composition of the cell wall formed by protoplasts isolated from cell suspension cultures of Vinca rosea. Planta 140, 227–232

  45. Taylor, J.A., West, D.W. (1980) The use of Evan's Blue stain to test the survival of plant cells after exposure to high salt and high osmotic pressure. J. Exp. Bot. 121, 571–576

  46. Updegraff, D.M. (1969) Semimicro determination of cellulose in biological materials. Anal. Biochem. 32, 420–424

  47. Van der Walk, H.C.P.M. (1984) Determination of proteases in isolated washed protoplasts: Inactivation of proteases in cell wall-degrading enzyme mixtures used in protoplast isolation. Plant Sci. Lett. 36, 201–204

  48. Willison, M., Klein, A. (1982) Cell-wall regeneration by protoplasts isolated from higher plants. In: Cellulose and other natural polymer systems, pp. 61–85, R.M. Brown, Jr., ed. Plenum Press, New York

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Author information

Correspondence to Nicholas C. Carpita.

Additional information

Journal paper No. 11,776 of the Purdue University Agriculture Experiment Station

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Shea, E.M., Gibeaut, D.M. & Carpita, N.C. Structural analysis of the cell walls regenerated by carrot protoplasts. Planta 179, 293–308 (1989). https://doi.org/10.1007/BF00391074

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Key words

  • Callose
  • Cell culture (cell-wall regeneration)
  • Cell wall (composition, regeneration)
  • Daucus (cell-wall regeneration)
  • Pectic substances
  • Protoplast (wall formation)