, Volume 195, Issue 1, pp 36–42 | Cite as

Compartmentation of cyanogenic glucosides and their degrading enzymes

  • Christiane Gruhnert
  • Böle Biehl
  • Dirk Selmar


Whereas high activities of β-glucosidase occur in homogenates of leaves of Hevea brasiliensis Muell.-Arg., this enzyme, which is capable of splitting the cyanogenic monoglucoside linamarin (linamarase), is not present in intact protoplasts prepared from the corresponding leaves. Thus, in leaves of H. brasiliensis the entire linamarase is located in the apoplasmic space. By analyzing the vacuoles obtained from leaf protoplasts isolated from mesophyll and epidermal layers of H. brasiliensis leaves, it was shown that the cyanogenic glucoside linamarin is localized exclusively in the central vacuole. Analyses of apoplasmic fluids from leaves of six other cyanogenic species showed that significant linamarase activity is present in the apoplasm of all plants tested. In contrast, no activity of any diglucosidase capable of hydrolyzing the cyanogenic diglucoside linustatin (linustatinase) could be detected in these apoplasmic fluids. As described earlier, any translocation of cyanogenic glucosides involves the interaction of monoglucosidic and diglucosidic cyanogens with the corresponding glycosidases (Selmar, 1993a, Planta 191, 191–199). Based on this, the data on the compartmentation of cyanogenic glucosides and their degrading enzymes in Hevea are discussed with respect to the complex metabolism and the transport of cyanogenic glucosides.

Key words

Apoplasm Compartmentation Cyanogenic glucosides Hevea Linamarin Linustatin 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Adamon, A., Jacoby, B. (1980) Assessment of cytoplasmatic contamination in isolated vacuole preparation. Plant Physiol. 65, 85–87Google Scholar
  2. Aldridge, W.N. (1944) A new method for the estimation of microquantities of cyanide and thiocyanide. Analyst. 69, 262–265Google Scholar
  3. Arnon, D.J. (1949) Copper enzymes in isolated chloroplasts. Phenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15Google Scholar
  4. Bergmeyer, H.U. (1970) Methoden der enzymatischen Analyse. Verlag Chemie, WeinheimGoogle Scholar
  5. Clegg, D.O., Conn, E.E., Janzen, D.H. (1979) Developmental fate of the cyanogenic glucoside linamarin in Costa Rica wild lima bean seeds. Nature 278, 343–344Google Scholar
  6. Conn, E.E. (1981) Biosynthesis of cyanogenic glycosides. In: Cyanide in biology, pp. 183–196, Vennesland, B., Conn, E.E., Knowles, C.J., Westley, J., Wissing, F., eds. Academic Press, LondonGoogle Scholar
  7. Conn, E.E. (1984) Compartmentation of secondary compounds. Annu. Proc. Phytochem. Soc. Europe 24, 1–28Google Scholar
  8. Czapek, F. (1921) Biochemie der Pflanzen. Fischer, JenaGoogle Scholar
  9. DeBruijn, G.H. (1973) The cyanogenic character of cassava (Manihot esculenta). In: Chronic cassava toxicity, pp. 43–48, Nestel, B., MacIntyre, R., eds. International Development Research Centre, Ottawa, CanadaGoogle Scholar
  10. Erickson, R.O. (1986) Symplastic growth and symplasmic transport. Plant Physiol. 82, 1153Google Scholar
  11. Frehner, M., Conn, E.E. (1987) The linamarin β-glucosidase in Costa Rica wild bean (Phaseolus lunatus L.) is apoplastic. Plant Physiol. 84, 1296–1300Google Scholar
  12. Frehner, M., Keller, F., Wiemken, A. (1984) Localization of fructan metabolism in the vacuoles isolated from protoplasts of Jerusalem artichoke tubers (Helianthus tuberosus L.). J. Plant Physiol. 116, 197–208Google Scholar
  13. Fritsch, H., Grisebach, H. (1975) Biosynthesis of cyanidin in cell cultures of Happlopappus gracilis. Phytochemistry 14, 2437–2441Google Scholar
  14. Grob, K., Matile, P. (1979) Vacuolar localization of glucosinolates in horseradish root cells. Plant Sci. Lett. 14, 327–335Google Scholar
  15. Grützmacher, H., Biehl, B., Czygan, F.-Ch., Selmar, D. (1990) Variations in HCN-potential in Dimorphotheca sinuata. Planta Med. 56, 610–611Google Scholar
  16. Hahlbrock, K., Conn, E.E. (1970) The biosynthesis of cyanogenic glycosides in higher plants: Purification and properties of a uridine diphosphate-glucose-ketone cyanohydrin β-glucosyltransferase from Linum usitatissimum L. J. Biol. Chem. 245, 917–922Google Scholar
  17. Halkier, B.A., Scheller, H.V., Møller, B.L. (1988) Cyanogenic glucosides: the biosynthetic pathway and the enzyme system involved. In: Cyanide compounds in biology, pp. 49–61, Everett, D., Harnett, S., eds. Wiley & Sons, ChichesterGoogle Scholar
  18. Heyn, A.N.J. (1969) Glucanase activity in coleoptiles of Avena. Arch. Biochem. Biophys. 132, 442–449Google Scholar
  19. Hrazdina, G., Wagner, G.J. (1985) Compartmentation of plant phenolic compounds; sites of synthesis and accumulation. In: Ann. Proc. Phytochem. Soc. Europe, vol. 25: The biochemistry of plant phenolics, pp. 119–133, Sumere, C.F., Lea, P.J., eds. Oxford University Press, OxfordGoogle Scholar
  20. Kakes, P. (1985) Linamarase and other β-glucosidases are present in the cell walls of Trifolium repens leaves. Planta 166, 156–160Google Scholar
  21. Kojima, M., Poulton, J.E., Thayer, S.S., Conn, E.E. (1979) Tissue distribution of dhurrin and enzymes involved in its metabolism in leaves of Sorghum bicolor. Plant Physiol. 94, 401–405Google Scholar
  22. Kuroki, G., Lizotte, P.A., Poulton, J.E. (1984) Catabolism of (R)-amygdalin and (R)-vicianin by partially purified β-glucosidases from Prunus serotina Ehrh. and Davallia trichomanoides. Z. Naturforsch. 39c, 232–239Google Scholar
  23. Kurzhals, Ch., Grützmacher, H., Selmar, D., Biehl, B. (1989) Linustatin, the linamarin-glucoside protected against cleavage by apoplastic linamarase. Planta Med. 55, 673Google Scholar
  24. Leegood, R.C., Walker, D.A. (1983) Chloroplasts. In: Isolation of membranes and organelles from plant cells, pp. 185–210, Hall, J.L., Moore, A.L., eds. Academic Press, LondonGoogle Scholar
  25. Lieberei, R. (1984) Cyanogenese und Resistenz. Habilitationsschrift, Technische Universität BraunschweigGoogle Scholar
  26. Marcinowski, S., Grisebach, H. (1978) Enzymology of lignification. Cell wall bound β-glucosidase for coniferin from spruce (Piceaabies). Eur. J. Biochem. 87, 37–44Google Scholar
  27. Marcinowski, S., Falk, H., Hammer, D.K., Hoyer, B., Grisebach, H. (1979) Appearance and localization of β-glucosidase hydrolyzing coniferin in spruce (Picea abies). Planta 144, 161–167Google Scholar
  28. Matile, Ph. (1975) The lytic compartment of plant cells. (Cell biology monographs, vol. 1). Springer, Wien New WorkGoogle Scholar
  29. Mkpong, O.E., Yan, H., Chism, G., Sayre, R.T. (1990) Purification, characterization and localization of linamarase in cassava. Plant Physiol. 93, 176–181Google Scholar
  30. Møller, B.L., Conn, E.E. (1980) The biosynthesis of cyanogenic glucosides in higher plants. Channeling of intermediates in dhurrin biosynthesis by a microsomal system from Sorghum bicolor. J. Biol. Chem. 255, 3049–3056Google Scholar
  31. Münch, E. (1930) Die Stoffbewegungen in der Pflanze. Fischer Verlag, JenaGoogle Scholar
  32. Noltman, E.A. (1964) Isolation of crystalline phosphoglucose-isomerase from rabbit muscle. J. Biol. Chem. 239, 1545–1550Google Scholar
  33. Oba, K., Canut, H., Boudet, A.M., Conn, E.E. (1981) Subcellular localization of 2-β-D-(glucosyloxy)-cinnamic acids and the related β-glucosidase in leaves of Melilotus alba. Plant Physiol. 68, 1359–1363Google Scholar
  34. Poulton, J.E. (1990) Cyanogenesis in plants. Plant Physiol. 94, 401–405Google Scholar
  35. Quail, P.H. (1979) Plant cell fractionation. Annu. Rev. Plant Physiol. 30, 425–484Google Scholar
  36. Reay, P.F., Conn, E.E. (1974) The purification and properties of a uridine diphosphate glucose: aldehyde cyanohydrin β-glucosyltransferase from Sorghum seedlings. J. Biol. Chem. 249, 5826–5830Google Scholar
  37. Robinson, M.E. (1930) Cyanogenesis in plants. Biol. Rev. 5, 126–141Google Scholar
  38. Saunders, J.A. (1979) Investigation of vacuoles isolated from tobacco. Plant Physiol. 64, 74–78Google Scholar
  39. Saunders, J.A., Conn, E.E. (1978) The presence of the cyanogenic glucoside dhurrin in isolated vacuoles from Sorghum. Plant Physiol. 61, 154–157Google Scholar
  40. Selmar, D. (1989) Cyanogenic glucosides — constituents of xylem and phloem sap? Plant Physiol. (Suppl.) 86, 153Google Scholar
  41. Selmar, D. (1993a) Transport of cyanogenic glucosides: Linustatin uptake by Hevea cotyledons. Planta 191, 191–199Google Scholar
  42. Selmar, D. (1993b) Apoplastic occurrence of cyanogenic β-glucosidases and consequences for the metabolism of cyanogenic glucosides. In: The biochemistry and molecular biology of β-glucosidases, pp. 191–204, A.Esen, ed., ACS, WashingtonGoogle Scholar
  43. Selmar, D., Lieberei, R., Biehl, B., Voigt, J. (1987a) Linamarase in Hevea — a nonspecific β-glucosidase. Plant Physiol. 83, 557–563Google Scholar
  44. Selmar, D., Lieberei, R., Biehl, B., Nahrstedt, A., Schmidtmann, V., Wray, V. (1987b) Occurrence of linustatin in Hevea brasiliensis. Phytochemistry 26, 2400–2401Google Scholar
  45. Selmar, D., Carvalho, F.J.P., Conn, E.E. (1987c) A colorimetric assay for α-hydroxynitrile lyase. Analyt. Biochem. 166, 208–211Google Scholar
  46. Selmar, D., Lieberei, R., Biehl, B. (1988) Mobilization and utilization of cyanogenic glycosides: the linustatin pathway. Plant Physiol. 86, 711–716Google Scholar
  47. Selmar, D., Lieberei, R., Biehl, B., Conn, E.E. (1989) α-Hydroxynitrile lyase in Hevea brasiliensis and its significance for rapid cyanogenesis. Plant Physiol. 75, 97–101Google Scholar
  48. Selmar, D., Grocholewski, S., Seigler, D.S. (1990) Cyanogenic lipids: Utilization during seedling development of Ungnadia speciosa. Plant Physiol. 93, 631–636Google Scholar
  49. Swain, E., Li, C.P., Poulton, J.E. (1992) Tissue and subcellular localization of enzymes catabolizing (R)-amygdalin in mature Prunus serotina seeds. Physiol. Plant. 100, 291–300Google Scholar
  50. Thayer, S.S., Conn, E.E. (1981) Subcellular localization of dhurrin β-glucosidase and hydroxynitrile lyase in the mesophyll cells of Sorghum leaf blades. Plant Physiol. 67, 617–622Google Scholar
  51. Wagner, G.J. (1979) Content and vacuole/extravacuole distribution of neutral sugars, free amino acids and anthocyanin in protoplasts. Plant Physiol. 64, 88–93Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Christiane Gruhnert
    • 1
  • Böle Biehl
    • 1
  • Dirk Selmar
    • 1
  1. 1.Botanisches Institut der Technischen Universität BraunschweigBraunschweigGermany

Personalised recommendations