The article reviews the state of knowledge on the biosynthesis of cardenolides in the genus Digitalis. It summarizes studies with labelled and unlabelled precursors leading to the formulation of the putative cardenolide pathway. Alternative pathways of cardenolide biosynthesis are discussed as well. Special emphasis is laid on enzymes involved in either pregnane metabolism or the modification of cardenolides.About 20 enzymes which are probably involved in cardenolide formation have been described "downstream" of cholesterol, including various reductases, oxido-reductases, glycosyl transferases and glycosidases as well as acyl transferases, acyl esterases and P450 enzymes. Evidence is accumulating that cardenolides are not assembled on one straight conveyor belt but instead are formed via a complex multidimensional metabolic grid. For example "fucose-type" cardenolides and "digitoxosetype" cardenolides seem to form via different biosynthetic branches and the "norcholanic acid pathway" identified recently seems to be operative only in the formation of fucosetype cardenolides.

Shoot cultures were established from axillary buds (11 strains) or seeds (1 strain) of individual Digitalis lanata Ehrh. plants and propagated partially submerged in liquid medium. Five of these shoot culture strains were characterized with regard to their growth and cardenolide content. The cultures were observed for more than one year and found to be relatively stable with regard to their growth and cardenolide spectrum and yield. The strains examined differed in terms of their total cardenolide yield, which ranged from about 30 nmol g DW-1 to almost 1000 nmol g DW-1. Cardenolide content was correlated with leaf size and development. Depending on the strain investigated up to ten different cardenolides could be detected by HPLC. The main cardenolides were identified by comparing HPLC and TLC results with those of authentic samples and chemical degradation as being the mono- and diglycosides glucodigifucoside, glucoverodoxin, odorobioside G, and odoroside H; minor amounts of digitalinum verum and glucoevatromonoside were also found. In addition, the tetrasaccharides lanatoside A and C were present. The shoots were cardenolide-free when cultivated in the dark for more than 30 weeks, but regained their characteristic cardenolide profile when transferred back to light. For the dark cultivation of chlorophyll-free cultures a medium containing 3.5% glucose was found to be optimal.

5-Pregnen-3beta-ol-20-one (pregnenolone), 4-pregnene-3,20-dione (progesterone), 5-pregnene-3beta,21-diol-20-one (21-hydroxypregnenolone), 4-pregnen-21 -ol-3,20-dione (cortexone), 5beta-pregnane-3,20-dione, 5alpha-pregnane-3,20-dione, 5beta-pregnan-3alpha-ol-20-one, 5beta-pregnan-3beta-ol-20-one, 5beta-pregnane-3beta,14beta, 21-triol-20-one 3-acetate, 23-nor-5-cholenic acid-3beta,20xi-diol, and 23-nor-3,20(22) E-choladienic acid-3beta-ol were administered to photomixotrophic shoot cultures of Digitalis lanata Ehrh. capable of synthesizing cardenolides, as well as to cardenolide-free tissue cultures, such as auxotrophic, dark-grown shoot cultures and cell suspension cultures of the same plant species. None of the pregnane precursors was qualified to restore cardenolide biosynthesis in the cardenolide-free tissues. The cardenolide content of light-grown shoot cultures, on the other hand, increased by 161%, 240%, 30%, 430% and 80% when 100 mg l(-1) of 21-hydroxypregnenolone, 5beta-pregnane-3,20-dione, 5beta-pregnan-3beta-ol-20-one, 5beta-pregnane-3beta,14beta,21-triol-20-one, 23-nor-5,20 (22) E-choladienic acid-3beta-ol, respectively, were administered. Pregnenolone, progesterone, cortexone, 5alpha-pregnanes, 5beta -pregnan-21-ols, and 23-nor-5-cholenic acid-3beta,20xi-diol, on the other hand, had no visible effect. Two different types of cardenolides (termed fucose-type cardenolides and digitoxose-type cardenolides) were identified which may be formed via different biosynthetic routes. The "norcholanic acid pathway" seems to be operative in D. lanata shoot cultures only in the formation of fucose-type cardenolides.

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Putative intermediates of cardenolide biosynthesis, namely progesterone, pregnenolone, 5β-pregnane-3,20-dione or 5β-pregnan-3β-ol-20-one, were administered to light- or darkgrown shoot cultures of Digitalis lanata. The unsaturated com pounds were reduced to their respective 5 a-pregnanes, 5β-pregnane-3,20-dione was reduced to 5β-pregnan-3α-ol-20-one and 5β-pregnan-3β-ol-20-one was isomerized to the respective 3α-pregnane. Suspension cultures of Digitalis lanata, on the other hand, accumulated both the 3α- and the 3β-isom er of 5β-pregnan-3-ol-20-one when incubated in the presence of 5β-pregnane- 3.20-dione. When 5β-pregnan-3α-ol-20-one was administered the cultured cells accumulated large amounts of the 3β-isomer together with small amounts of 5β-pregnane-3,20-dione, which may be regarded as an intermediate during the isomerization reaction. Cell-free, buffered extracts from light-grown shoots were shown to reduce 5β-pregnane- 3.20-dione almost exclusively to 5β-pregnan-3α-ol-20-one when 0.05 m MgCl2 were present in the incubation mixture. Under these conditions the formation of 5β-pregnan-3β-ol-20-one was inhibited. The enzyme activity could be recovered from m em brane-free supernatants. Optimum enzyme activity occurred at pH 7.0 and 42 °C. The energy of activation was 56.2 kJ/mol and the enzyme reaction was found to be NADPH -dependent. SH reagents were essential for enzyme activity. The enzyme seems to be specific for 5β-pregnan-3-ones since neither 5 a-pregnane-3-ones nor Δ4/Δ5-pregnenes were reduced. The NADPH : 5β-pregnane 3α-hydroxysteroid-5β-oxidoreductase described here may play a role in the regulation of cardenolide biosynthesis by removing precursors, such as 5β-pregnane-3,20-dione, from the pathway