Expression of 3β-HSD and P5βR, genes coding for Δ5–3β-hydroxysteroid dehydrogenase (3β-HSD) and progesterone 5β-reductase (P5βR), in leaves and cell cultures of Digitalis lanata EHRH.

Plants of the genus Digitalis produce cardiac glycosides that are used in the therapy of cardiac insufficiency in humans [1]. 3β-HSD and P5βR are both supposed to be important enzymes in the biosynthesis of these products [2, 3, 4]. Enzyme activity and expression of the respective genes encoding 3β-HSD and P5βR were demonstrated in cardenolide-accumulating leaves of Digitalis lanata but also in cardenolide-free permanent cell suspension cultures (K3OHD) initiated from D. lanata leaves. P5βR activity was 3.7 times higher in D. lanata leaves (7.8µkat/kg protein) than in K3OHD cells (2.1µkat/kg protein) (analysed by GC-MS). 3β-HSD activity in D. lanata leaves and K3OHD cells was detected by HPLC and it was found that the activity is about in the same range in leaves (13.9µkat/kg protein) and in suspension-cultured cells (11.3µkat/kg protein). Expression of the respective genes, namely AY585867.1 (P5βR gene) and DQ466890.1 (3β-HSD gene), was demonstrated by real-time qPCR analysis. The expression of the 3β-HSD gene in leaves (8.7 mRNA level) is nearly twice as high as in K3OHD cells (4.9 mRNA level). Analyses revealed that P5βR is most strongly expressed in D. lanata leaves (42 mRNA level). The relative mRNA level was about 3.2 times higher than in suspension cells (13.3 mRNA level) which nicely fits to the activity data. Since a new, inducible progesterone 5β-reductase was identified recently [5] the expression of the respective gene, P5βR2, in K3OHD cells and D. lanata leaves is now under examination and the new results will be presented and discussed.

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2. Kreis, W. et al. (1998) Planta Med 64:491–499.

3. Kreis W, Müller-Uri F (2009) Ann Rev Plant 40:304–363.

4. Gärtner, DE. Et al. (1990) FEBS Lett 271:239–242.

5. Pedro Peréz-Bermúdez et al. (2010) New Phytol 185:687–700.