, Volume 187, Issue 1, pp 83–88

Subcellular localization of luteolin glucuronides and related enzymes in rye mesophyll

  • Stephan Anhalt
  • Gottfried Weissenböck


Vacuoles were isolated by osmotic rupture of mesophyll protoplasts from the primary leaves of 4-d- and 7-d-old plants of rye (Secale cereale L.). Their content of two flavones, luteolin 7-O-[β-d-glucuronosyl-(1→2)β-d-glucuronide] (R2) and luteolin 7-O-[β-d-glucuronosy 1 (1→2) β-d-glucuronide]-4′-O-β-d-glucuronide (R1), as well as that of three specific flavone-glucuronosyltransferases involved in their biosynthesis and of a specific β-glucuronidase was determined in comparison to the parent protoplasts. The two flavonoids were found to be entirely located in the vacuolar fraction, together with 70% of the activity of UDP-glucuronate: luteolin 7-O-diglucuronide-4′-O-glucuronosyl-transferase (LDT; EC 2.4.1.), the third enzyme of the sequence of three transferases in the anabolic pathway. The activities of the first and second anabolic enzymes, UDP-glucuronate: luteolin 7-O-glucuronosyltransferase (LGT; EC 2.4.1.) and UDP-glucuronate: luteolin 7-O-glucuronide-glucuronosyltransferase (LMT; EC 2.4.1.) could not be found in the vacuolar fraction in appreciable amounts. The specific β-glucuronidase (EC 3.2.1.), catalyzing the deglucuronidation of luteolin triglucuronide to luteolin diglucuronide, was present with 90% of its activity in the digestion medium after isolation of mesophyll protoplasts, indicating an apoplastic localization of this enzyme. The data presented indicate a directed anabolic and subsequent catabolic pathway for the luteolin glucuronides in the mesophyll cells of rye primary leaves. This includes two cytosolic and a last vacuolar step of glucuronidation of luteolin, and the vacuolar storage of the luteolin triglucuronide. We propose the transport of the latter into the cell wall, after which the triglucuronide is deglucuronidated, this being the first step for further turnover.

Key words

β-Glucuronidase Flavone-glucuronosyltransferase Luteolin glucuronide (localization) Secale (enzyme localization) Vacuole 



UDP-glucuronate: luteolin 7-O-di-glucuronide-4′-O-glucuronosyltransferase


UDP-glucuronate: luteolin 7-O-glucuronosyltransferase


UDP-glucuronate: luteolin 7-O-glucuronide-glucuronosyltransferase


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bergmeyer, H.U. (1974) Methoden der enzymatischen Analyse, vol. I, pp. 673–681, Verlag Chemie, Weinheim/Bergstr., FRGGoogle Scholar
  2. Boller, T., Kende, H. (1979) Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol. 63, 1123–1132Google Scholar
  3. Boudet, A.M., Canut, H., Alibert, G. (1981) Isolation and characterization of vacuoles from Melilotus alba mesophyll. Plant Physiol. 68, 1354–1358Google Scholar
  4. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Analyt. Biochem. 72, 248–254Google Scholar
  5. Bray, E.A., Zeevaart, J.A.D. (1985) The compartmentation of abscisic acid and β-d-glucopyranosyl abscisate in mesophyll cells. Plant Physiol. 79, 719–722Google Scholar
  6. Bruinsma, J. (1961) A comment on the spectrophotometric determination of chlorophyll. Biochim. Biophys. Acta 52, 576–578Google Scholar
  7. Busch, E., Strack, D., Weissenböck, G. (1986) Cyanidin 3-gentiobioside from primary leaves of rye (Secale cereale L.). Z. Naturforsch. 41c, 485–486Google Scholar
  8. Butcher, H.C., Wagner, G.J., Siegelman, H.W. (1977) Localization of acid hydrolases in protoplasts. Plant Physiol. 59, 1098–1103Google Scholar
  9. Dellamonica, G., Meurer, B., Strack, D., Weissenböck, G., Chopin, J. (1983) Two isovitexin 2″-O-glycosides from primary leaves of Secale cereale. Phytochemistry 22, 2627–2628Google Scholar
  10. Drawert, H. (1968) Vitalfärbung und Vitalfluorochromierung pflanzlicher Zellen und Gewebe. In: Protoplasmatologia, vol. II D3., pp. 427–428, Springer, Wien, HeidelbergGoogle Scholar
  11. Gaff, D.F., Okong'O-Ogola, O. (1971) The use of non-permeating pigments for testing the survival of cells. J. Exp. Bot. 22, 756–758Google Scholar
  12. Heck, U., Martinoia, E., Matile, Ph. (1981) Subcellular localization of acid proteinase in barley mesophyll protoplasts. Planta 151, 198–200Google Scholar
  13. Hodges, T.K., Leonard, R.T. (1974) Purification of a plasma membrane-bound adenosine triphosphatase from plant roots. Methods Enzymol. 32, 392–406Google Scholar
  14. Hopp, W., Hinderer, W., Petersen, M., Seitz, H.U. (1985) Anthocyanin-containing vacuoles isolated from protoplasts of Daucus carota cell cultures. In: The physiological properties of plant protoplasts, pp. 122–132, Pilet, P.E. ed. Springer, Berlin, Heidelberg, New York, TokyoGoogle Scholar
  15. Knogge, W., Weissenback, G. (1986) Tissue distribution of secondary phenolic biosynthesis in developing primary leaves of Avena sativa L. Planta 167, 196–205Google Scholar
  16. Kreis, W., Reinhard, E. (1985) Rapid isolation of vacuoles from suspension-cultured Digitalis lanata cells. J. Plant Physiol. 121, 385–390Google Scholar
  17. Le-Quoc, K., Le-Quoc, D., Pugin, A. (1987) An efficient method for plant vacuole isolation using digitonin for plasmalemma lysis. J. Plant Physiol. 126, 329–335Google Scholar
  18. Marty, F., Branton, D., Leigh, R.A. (1980) Plant vacuoles. In: The biochemistry of plants, vol. 1, pp. 625–658, Stumpf, P., Conn, E.E., eds. Academic Press, New York, LondonGoogle Scholar
  19. Matern, U., Reichenbach, C., Heller, W. (1986) Efficient uptake of flavonoids into parsley (Petroselinum hortense) vacuoles requires acylated glycosides. Planta 167, 183–189Google Scholar
  20. Matile, Ph. (1975) The lytic compartment of plant cells. In: Cell biology monographs, vol. 1, pp. 1–175. Springer, Berlin, Heidelberg, New York, TokyoGoogle Scholar
  21. Matile, Ph. (1984) Das toxische Kompartiment der Pflanzenzelle. Naturwissenschaften 71, 18–24Google Scholar
  22. Nichimura, M., Beevers, H. (1978) Hydrolases in vacuoles from castor bean endosperm. Plant Physiol. 62, 44–48Google Scholar
  23. Oba, K., Conn, E.E., Canut, H., Boudet, A.M. (1981) Subcellular localization of 2-(β-d-glycosyloxy)-cinnamic acids and the related β-glucosidase in leaves of Melilotus alba Desr. Plant Physiol. 68, 1359–1363Google Scholar
  24. Saunders, J.A., Conn, E.E. (1978) Presence of the cyanogenic glucoside dhurrin in isolated vacuoles from Sorghum. Plant Physiol. 61, 154–157Google Scholar
  25. Schnabl, H., Kottmeier, C. (1984) Determination of malate levels during the swelling of vacuoles isolated from guard-cell protoplasts. Planta 161, 27–31Google Scholar
  26. Schulz, M., Weissenböck, G. (1986) Isolation and separation of epidermal and mesophyll protoplasts from rye primary leaves — tissue specific characteristics of secondary phenolic product accumulation. Z. Naturforsch. 41c, 22–27Google Scholar
  27. Schulz, M., Weissenback, G. (1987) Partial purification and characterization of a luteolin-triglucuronide-specific β-glucuronidase from rye primary leaves (Secale cereale). Phytochemistry 26, 933–937Google Scholar
  28. Schulz, M., Weissenback, G. (1988a) Dynamics of the tissue-specific metabolism of luteolin glucuronides in the mesophyll of rye primary leaves (Secale cereale). Z. Naturforsch. 43c, 187–193Google Scholar
  29. Schulz, M., Weissenböck, G. (1988b) Three specific UDP-glucuronate: flavone-glucuronosyl-transferases from primary leaves of Secale cereale. Phytochemistry 27, 1261–1267Google Scholar
  30. Schulz, M., Strack, D., Weissenböck, G., Markham, G.R., Dellamonica, G., Chopin, J. (1985) Two luteolin O-glucuronides from primary leaves of Secale cereale. Phytochemistry 24, 343–345Google Scholar
  31. Sharma, V., Strack, D. (1985) Vacuolar localization of 1-sinapoylglucose: l-malate sinapoyltransferase in protoplasts from cotyledons of Raphanus sativus. Planta 163, 563–568Google Scholar
  32. Strack, D., Meurer, B., Weissenböck, G. (1982) Tissue-specific kinetics of flavonoid accumulation in primary leaves of rye (Secale cereale). Z. Pflanzenphysiol. 108, 131–141Google Scholar
  33. Wagner, G.J., Siegelman, H.W. (1975) Large-scale isolation of intact vacuoles and isolation of chloroplasts from protoplasts of mature plant tissue. Science 190, 1298–1299Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Stephan Anhalt
    • 1
  • Gottfried Weissenböck
    • 1
  1. 1.Botanisches Institut der Universität zu KölnKöln 41Germany

Personalised recommendations