Abstract
In contrast to undifferentiated cell suspension cultures of Digitalis lanata, photomixotrophic shoot cultures of Digitalis purpurea accumulate cardiac glycosides in substantial concentrations. They are used to investigate enzymes of the cardenolide pathway. All cardenolides are 5β-configurated. The progesterone 5β-reductase and the 3β-hydroxysteroid-5β-oxidoreductase are present in shoot cultures but not in undifferentiated cell cultures. These enzymes provide precursors for cardenolides, whereas the presence of the progesterone 5α-reductase, also present in shoot cultures, is discussed with regard to its role in phytosterol biosynthesis and may be attributed to the general steroid pathway. The progesterone 5α-reductase had an activity maximum during the early growth period seven days after onset of cultivation, whereas the corresponding progesterone 5β-reductase activity was highest on day 11. The maximum cardenolide accumulation was after 24 days. The enzyme activities present in crude extracts from shoot cultures were characterized with regard to their requirements for NADPH and NADH, pH-optimum, temperature optimum, affinity to their substrates and their localization in the cell. The progesterone 5β-reductase was purified 769-fold.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsPreview
Unable to display preview. Download preview PDF.
Abbreviations
- DW:
-
dry weight
- FW:
-
fresh weight
- PVP:
-
polyvinylpyrrolidone
References
Ansorg W (1985) Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J. Biochem. Biophys. Meth. 11: 13–20
Bradford MM (1976) A rapid and sensitive method forthe quantificationof microgram quantities of protein utilizing the principleof protein-dye-binding. Anal. Biochem. 72:248–254
Campbell JS & Karavolas HJ (1990) Purificationof the NADP:5α-dihydroprogesterone 3α-hydroxysteroid oxidoreductase fromfemale rat pituitary cytosol. J. SteroidBiochem. Mol. Biol.37:215–222
Corchete MP, Sanchez JM, Cacho M, Moran M & Fernandez-Tarrago J (1990) Cardenolide content in suspensioncell culturesderived from root and leaf callus of Digitalis thapsiL. J. PlantPhysiol. 137: 196–200
Diettrich B, Mertinat H & Luckner M (1990)Formation of Digitalislanata clone lines by shoot tip culture. Planta Med. 56: 53–58
Dubois M, Gilles KA, Hamilton JK, Rebers PA &Smith F (1956)Colorimetric methodfor determination of sugars and related substances.Anal. Chem. 28: 350–356
Gärtner DE, Wendroth S & Seitz HU (1990) A stereospecific enzymeof the putativebiosynthetic pathway of cardenolides. Characterization of a progesterone 5β-reductase fromleaves of Digitalispurpurea L. FEBS Lett. 271: 239–242
Gärtner DE & Seitz HU (1993) Enzyme activities incardenolide-accumulating, mixotrophic shoot cultures of Digitalis purpureaL. J. Plant Physiol. 141: 269–275
Graves JMH & Smith WH (1967)Transformation of pregnenoloneand progesterone bycultured plant cells. Nature 214: 1248
Hagimori M, Matsumoto T & ObiY (1982) Studies on the production of Digitaliscardenolides by plant tissue culture. II Effectof light and plant growth substances ondigitoxin formation byundifferentiated cell andshoot-forming cultures of Digitalis purpurea L. grown inliquid media. Plant Physiol. 69: 653–656
Hagimori M, Matsumoto T & Obi Y (1983) Effectsof mineral salts,initial pH and precursors on digitoxinformation by shoot-formingcultures of DigitalispurpureaL. grown in liquid media. Agric.Biol. Chem. 47: 565–571
Hirotani M & Furuya T (1977)Restoration of cardenolide-synthesisin redifferentiatedshoots from callus cultures of Digitalis purpurea. Phytochemistry16: 610–611
Laemmli UK (1970) Cleavage of structural proteinsduring theassembly of the head of bacteriophage T4.Nature 227: 680–685
Lichtenthaler HK (1987) Chlorophylls andcarotinoids: Pigments ofphotosyntheticbiomembranes. In: Packer L & Douce R (Eds)Methods inenzymology, Vol 148 (pp 350–382). Academic Press,San Diego, New York, Berkeley
Lui JHC & Staba EJ (1979)Effects of precursors on serially propagated Digitalis lanataleaf and root cultures.Phytochemistry 18:1913–1916
McGuire JS & Tomkins GM (1959)The multiplicity and specificityof Δ4-3-ketosteroid hydrogenase (5α).Arch. Biochem. Biophys.82: 476–477
Morikawa T & Tamaoki BJ (1990) Purification and characterization of 3α-hydroxysteroid dehydrogenase from chicken hepaticcytosol. J. Steroid Biochem. Mol. Biol.37: 569–574
Okuda A & Okuda K (1984) Purification andcharacterization ofΔ4-3-ketosteroid 5β-reductase.J. Biol. Chem. 259: 7519–7524
Penning TM, Mukharji I, Barrows S & Talalay P(1984) Purificationand properties of a3α-hydroxysteroid dehydrogenase ofrat livercytosol and its inhibition by anti-inflammatory drugs. Biochem.J. 222: 601–611
Pilgrim H (1972) ‘Cholesterol side-chain cleavingenzyme’ Aktivitätin Keimlingen und in vitrokultivierten Geweben von Digitalispurpurea. Phytochemistry 11: 1725–1728
Rücker W, Jentzsch K & Wichtl M (1976) Wurzeldifferenzierungund Glykosidbildung bei in vitrokultivierten Blattexplantatenvon Digitalis purpureaL. Z. Pflanzenphysiol. 80: 323–335
Seidel S, Kreis W & Reinhard E (1990) Δ5-3β-Hydroxysteroiddehydrogenase/ Δ5-Δ4-ketosteroidisomerase (3β-HSD), a possible enzymeof cardiac glycoside biosynthesis in cell culturesand plants of Digitalis lanataEHRH.Plant Cell Rep. 8: 621–624
Tschesche R, Hombach R, Scholten H & Peters M (1970) Neue Beiträge zur Biogenese der Cardenolide in Digitalis lanata. Phytochemistry 9:1505–1515
Warneck HM & Seitz HU (1990) 3β-Hydroxysteroid oxidoreductaseinSuspension culturesof DigitalislanataEHRH.Z. Naturforsch.45c: 963–972
Wendroth S & Seitz HU (1990) Characterisation andlocalisation ofprogesterone 5α-reductase from cell cultures offoxglove (Digitalis lanata EHRH). Biochem. J. 266: 41–46
Author information
Authors and Affiliations
Editor information
Rights and permissions
Copyright information
© 1994 Springer Science+Business Media Dordrecht
About this chapter
Cite this chapter
Seitz, H.U., Gärtner, D.E. (1994). Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L. In: Schripsema, J., Verpoorte, R. (eds) Primary and Secondary Metabolism of Plants and Cell Cultures III. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-0237-7_30
Download citation
DOI: https://doi.org/10.1007/978-94-011-0237-7_30
Publisher Name: Springer, Dordrecht
Print ISBN: 978-94-010-4106-5
Online ISBN: 978-94-011-0237-7
eBook Packages: Springer Book Archive