Cardenolide glycoside production in Digitalis lanata

Leaves of Digitalis lanata metabolize progesterone more rapidly than pregnenolone when applied to the surface of the leaves. Their transformation into digitoxigenin, gitoxigenin, digoxigenin, digitoxin, gitoxin, digoxin and the corresponding cardenolides was demonstrated.     

Effect of light on cardenolide production by Digitalis lanata tissue cultures

The influence of light on the accumulation of cardenolides by Digitalis lanata S-1 was studied. Optimal radiant flux for chlorophyll accumulation w     

Mechanism of 14β hydroxylation in the biosynthesis of cardenolides: The role of 14β-cholest-5-en-3β-ol

The intermediary role of 14β-cholest-5-en-3β-ol in the biosynthesis of cardenolides in Digitalis lanata was excluded. This rules out one plausi     

Biosynthesis of cardenolides in Digitalis lanata

[8-³H]-Cholesterol was synthesized. A doubly labelled sample of [8-³H, 4-¹⁴C]-cholesterol was administered to Digitalis lanata plants and the cardenolides were isolated. Biosynthesized digitoxigenin and digoxigenin retained all the tritium. Barring the migration of the tritium in biosynthesis the results are interpreted as indicative that neither intermediates with δ7, δ⁷ or δ⁸⁸⁽¹⁴⁾ are participating in the elaboration of cardenolides.     

Glycosylation in cardenolide biosynthesis

The glycosylation and deglycosylation of cardiac glycosides was investigated using cell suspension cultures and shoot cultures, both established from Digitalis lanata EHRH. plants, as well as isolated enzymes. Shoots were capable of glucosylating digitoxigenin, evatromonoside, digiproside, glucodigitoxigenin and digitoxin. Suspension cultured Digitalis cells glucosylated all the substrates mentioned but digiproside, whereas the UDP-glucosedependent cardinolide glucosyltransferase isolated from that source did not accept digitoxigenin and digiproside as substrates. It is concluded that at least three different glucosyltransferases are involved in cardiac glycoside formation in Digitalis. Similar experiments carried out with glucosylated cardenolides which were administered to cultured cells, shoots and a cardenolide -glucosidase isolated from young leaves revealed that at least two different glucosidases occur in Digitalis lanata, albeit in different tissues or during different phases of development. The biotransformation of glucoevatromonoside was investigated using unlabelled compound and [¹⁴C-glucose]-glucoevatromonoside synthesized enzymatically. After 7 d of incubation almost no radioactivity could be recovered from the cardenolide fraction, indicating that the terminal glucose of glucoevatromonoside was now incorporated into volatile, hydrophilic and insoluble compounds. Since, on the other hand, large amounts of cardenolides were found in the experiments with unlabelled glucoevatromonoside it is assumed that steady state or pool size regulation is achieved by the coordinated action of a cardenolide glucosidase and a glucosyltransferase.Abbreviations Acdox D-acetyldigitoxose - dgen digoxigenin - dox D-digitoxose - dten digitoxigenin - dtl D-digitalose - fuc D-fucose - gten gitoxigenin - qun D-quinovose - CGH cardenolide 16-O-glucohydrolase - DFT UDP-fucose:digitoxigenin 3-O-fucosyltransferase - DGT UDP-glucose:Digitoxin 16-O-glucosyltransferase - DQT UDP-quinovose:digitoxigenin 3-O-quinovosyltransferase     

UDP-glucose:digitoxin 16′-O-glucosyltransferase from suspension-cultured Digitalis lanata cells

Suspension cultures from several cell lines of Digitalis lanata, as well as cultures from 6 other plant species were checked for their ability to form purpurea-glycoside A from digitoxin. An in-vitro assay for the UDP-glucose:digitoxin 16-O-glucosyltransferase (DGT, EC 2.4.1.-) has been established based on an HPLC method. The enzyme is located in the soluble fraction. Its pH optimum is at 7.4. No enzyme activity was found in either purified vacuole preparations or lysed vacuoles. Ascorbate (10 mM) increased the transferase activity about 4-fold. Of the sugar nucleotides tested, only UDP-glucose served as a glucosyl donor. Digitoxin, digoxin, -acetyldigitoxin, and -acetyldigoxin are substrates for the glucosyltransferase. The role of the DGT during the biotransformation of cardenolides in Digitalis lanata cell suspension cultures is discussed.Abbreviation DGT UDP-glucose:digitoxin 16-C-glucosyltransferase     

Beiträge zur biochemischen 14β-hydroxylierung von C21-steroiden zu cardenoliden

It has been demonstrated that 7-³H-Δ⁸⁽¹⁴⁾-5β-pregnen-3,20-dione (I) and 7α-³H-pregnenolone (II), but not 6-³H-Δ⁷-progesterone (III) were utilized for the biogenesis of digitoxigenin by plants of Digitalis lanata. These results are discussed in relation to the 14β-hydroxylation of the cardenolides.     

The VEP1 gene (At4g24220) encodes a short-chain dehydrogenase/reductase with 3-oxo-Δ-steroid 5β-reductase activity in Arabidopsis thaliana L.

The Arabidopsis thaliana VEP1 gene product shows about 70% sequence identity to Digitalis lanata progesterone 5β-reductase, an enzyme considered to catalyze a key step in the biosynthesis of cardiac glycosides. A. thaliana does not accumulate cardenolides but protein extracts prepared from its leaves were capable of reducing progesterone to 5β-pregnane-3,20-dione. A full-length cDNA clone encoding a Δ^4,5-steroid 5β-reductase (At5β-StR, EC 1.1.1.145/1.3.1.23), a member of the short-chain dehydrogenase/reductase (SDR) family, was isolated from A. thaliana leaves. A SphI/SalI At5β-StR gene fragment was cloned into the pQE vector system and transformed into Escherichia coli. The gene was functionally expressed and the recombinant His-tagged fusion protein was characterized. K_m values and specific activities for putative 3-oxo-Δ^4,5-steroid substrates such as progesterone, cortisol, cortexone and 4-androstene-3,17-dione, and for the co-substrate NADPH were determined. Progesterone was stereo-specifically reduced to 5β-pregnane-3,20-dione and none of the 3-oxo-Δ^5,6-steroids tested were accepted as a substrate. The gene encoding At5β-StR was strongly transcribed in stems and leaves. A three-dimensional model of At5β-StR highlights a close structural similarity to the related, previously described D. lanata progesterone 5β-reductase. This homology extends to the active site where single amino acid substitutions might be responsible for the increased catalytic efficiency of At5β-StR when compared to the activity of the recombinant form of the D. lanata enzyme.     

Kinetic model for biotransformation of digitoxin in plant cell suspension culture ofDigitalis lanata

Batch suspension cultures ofDigitalis lanata plant cell were performed to investigate the biotransformation of digitoxin.Digitalis lanata K3OHD plant cells were used to biotransform digitoxin into deacetyllanatoside C. A kinetic model was proposed to describe cell growth, substrate consumption, depletion of digitoxin, formation and depletion of digoxin and purpureaglycoside A, and formation of deacetyllanatoside C. The digoxin and purpureaglycoside A are intermediates of deacetyllanatoside C formation from digitoxin. Interactions between extracellular and intracellular compounds were considered. The proposed model could accurately predict cell growth, substrate consumption and product synthesis. And it can provide a useful framework for quantitative analysis of biotransformation in a plant cell culture system.     

Purification and characterisation of the cardenolide-specificβ-glucohydrolase CGH II from Digitalis lanata leaves

A cardenolide-hydrolysing β-D-glucosidase was isolated from young leaves of Digitalis lanata. Since this enzyme differs from the cardenolide glucohydrolase (CGH) described and characterised previously, it was termed cardenolide glucohydrolase II (CGH II). CGH II was detected in various Digitalis tissue cultures as well as in young leaves of D. lanata. The latter source was used as the starting material for the isolation and purification of CGH II. The specific enzyme activity reached about 15 pkat·mg^–1 protein in buffered leaf extracts. Optimal CGH II activity was seen at around pH 6.0 and 50 °C. CGH II was purified about 600-fold by anion exchange chromatography, size exclusion chromatography and hydroxyapatite chromatography. The apparent molecular mass of CGH II was 65 kDa as determined by SDS-PAGE. CGH II exhibited a high substrate specificity towards cardenolide disaccharides, especially to those with a 1-4-β-linked glucose-digitoxose moiety such as glucoevatromonoside. The K_m- and V_max-values for this particular substrate were calculated to be 101 μM and 19.8 nkat·mg^–1 protein, respectively.     

Cardenolides from Gomphocarpus sinaicus and Pergularia tomentosa (Apocynaceae: Asclepiadoideae) deter the feeding of Spodoptera littoralis

Methanol extracts of Gomphocarpus sinaicus, Pergularia tomentosa and Cynanchum acutum (Apocynaceae, sub-family Asclepiadoideae) deterred feeding of Spodoptera littoralis in a binary-choice bioassay. Analyses of extracts using high-performance liquid chromatography with photodiode array detection indicated that methanol extracts of P. tomentosa and G. sinaicus contained cardenolides, while these compounds were not detected in extracts of C. acutum. Activity-guided fractionation of the methanol extracts of G. sinaicus and P. tomentosa resulted in the isolation of six cardenolides: 7,8-dehydrocalotropin, calotropin and coroglaucigenin 3-(6-deoxy-β-allopyranoside)-19-acetate (frugoside 19-acetate) from G. sinaicus, and coroglaucigenin, 16α-acetoxycalotropin and calactin from P. tomentosa. The isolation of 16α-acetoxycalotropin was a new report from P. tomentosa. Each of the 6 cardenolides deterred feeding by S. littoralis, while two cardenolide standards, digoxin and digitoxin, did not affect feeding. Differences among cardenolides in their effect on feeding were associated with specific structural features. C. acutum is the only one of the three species tested that is known to support the development of S. littoralis, although the development of larvae was delayed. The observed feeding deterrent activity of the cardenolide-free methanol extract of C. acutum would suggest that compounds other than cardenolides are responsible for the deterrent activity. These compounds, although deterrent in a short-term feeding assay, might not prevent long term feeding, thus allowing the larvae to develop on the plant.     

Increased cardenolides production by elicitation of Digitalis lanata shoots cultured in temporary immersion systems

Digitalis lanata is an important source of cardenolides such as digoxin and lanatoside C, which have been widely applied in the treatment of cardiac insufficiencies. Elicitation is one of the most effective methods to enhance the biosynthesis of several secondary metabolites in medicinal plants. We studied the effect of elicitation with Chitoplant^?, Silioplant^? and methyl jasmonate on biomass and cardenolides accumulation in shoots of D. lanata cultivated in temporary immersion systems. Morphological response of the shoots was influenced by elicitors. A reduction in length and number of shoots was evident with all MJ concentrations. Regarding biomass production, Chitoplant^? (0.1?g?l^?1) was found to impact significantly on fresh and dry weight of the shoots. HPLC analysis revealed a higher content of lanatoside C compared to digoxin in all treatments. The highest accumulation of lanatoside C was achieved with Chitoplant^? (0.1?g?l^?1), which resulted in 316???g?g-DW^?1 and with Silioplant^? (0.01?g?l^?1; 310???g?g-DW^?1), which accounted for a 2.2-fold increase in lanatoside C content compared to non-elicited shoot cultures. Additionally, elicitation of D. lanata shoots in temporary immersion systems resulted in an oxidative stress characterized by hydrogen peroxide and malondialdehyde accumulation. These observations point to a connection between hydrogen peroxide generation, lipid peroxidation and cardenolide accumulation. The optimization of elicitor treatment and culture conditions for cardenolide production as well as the advantages of TIS for this purpose are discussed.     

Cardenolide biosynthesis in light- and dark-grown Digitalis lanata shoot cultures

Shoot cultures of the cardenolide-producing species Digitalis lanata Ehrh. accumulated up to 0.6 μmol cardenolides per g dry mass when cultivated under continuous white light. After transfer to permanent dark, the cardenolide content of cultured shoots gradually decreased and reached non-detectable levels after 12 weeks. After transfer back to light conditions, cardenolides started to accumulate and reached the levels of light-grown controls after 4 weeks. Radiolabelled pregnenolone and progesterone were incorporated into cardenolides in both green light-grown and white dark-grown shoots. It was thus established that cardenolides are synthesised de novo in chloroplast-free tissues without apparent cardenolide accumulation, indicating that these compounds are efficiently turned over in the dark and that tissue differentiation, but not intact chloroplasts, is essential for cardenolide formation. The time course of two late anabolic enzymes of cardenolide metabolism, acetyl-CoA:digitoxin 15′-O-acetyltransferase (DAT, EC 2.3.1.-) and UDP-glucose:digitoxin 16′-glucosyltransferase (DGT, EC 2.4.1.-) was established during transfer of shoots from light to dark and vice versa. Only DAT was affected and was not measurable any more under dark conditions. The DGT may not be down-regulated because of its important, maybe even vital, role as an enzyme providing the vacuolar storage forms of cardenolides. Two catabolic cardenolide-specific enzymes, lanatoside 15′-O-acetylesterase (LAE, EC 3.1.1.6.) and cardenolide 16′-O-glucohydrolase I (CGH I, EC 3.2.1.21), were also investigated and it was demonstrated that CGH I is inactive in dark-grown shoots. These observations indicate that CGH I is not involved in cardenolide degradation in situ, but may instead play a role in cardenolide remetabolisation and activation after wounding or in developmental programs.     

Plantlet regeneration from mesophyll protoplasts of Digitalis lanata Ehrh.

Summary Protoplasts were isolated from the mesophyll of Digitalis lanata enzymatically and cultured in a liquid regeneration medium (D2a). Protoplast division occurred at a rate of approximately 30%. Mature cell colonies were transferred onto agar medium (D2b)where they developed into cell clusters with a diameter of about 4–5 mm. After transfer onto MS medium, these calli differentiated leaves and shoots which could be rooted on MS medium containing a low hormone concentration.The main part of this work was carried out in the Max-PlanckInstitut für Züchtungsforschung, Cologne (FRG)     

Incorporation of 14CO2, into cardenolide and sapogenin steroids of Digitalis purpurea

The relative rates of biosynthesis of cardenolide and sapogenin steroids of Digitalis purpurea were estimated by their uptake of ¹⁴CO₂. The incorporation of label into both groups, although initially slow, indicated that biosynthesis occurs even at the end of the growing season. The sapogenins were produced more rapidly than the cardenolides at this stage of plant development. Within the group of sapogenins, digitogenin, the trihydroxy compound, was produced at a greater rate than the dihydroxy steroid gitogenin. In the case of the cardenolides, the trihydroxy gitoxigenin was produced at a slower rate than the dihydroxy digitoxigenin.     

Stability of Lanatoside C content in thein vitro propagatedDigitalis lanata clones

In vitro culture was established from shoot tips ofDigitalis lanata cotyledonous plants. The propagated plant material was rooted, transplanted into soil and grown under field conditions. Lanatoside C content was determined in a total of 20 clones and statistically evaluated by means of variance analysis of unequal-sized samples.In vitro clonal propagation ofD. lanata was found not to affect lanatoside C content. Drug level was dependent on a plant genotype.     

The Influence of n-[2-isopentenyl] adenine on shoot differentiation in digitalis purpurea l. Tissue cultures

The morphogenetic capacity of Digitalis purpurea L. leaf explants and callus cultures has been studied. The promoting growth of calli was induced by the presence of exogenously applied auxins and cytokinins in the ratio 2:1. Bud and shoot differenatiation occurred in leaf explants and callus cultures of the 2^nd subculture under the influence of 1.00 to 5.00 mg·1-¹ N⁶7hyphen;[2-Isopentenyl] adenine. The regeneration protocol described provides 7 to 10 plantlets per leaf explant. The results are compared with the effect of other cytokinins in Digitalis purpurea and a related Digitalis lanata species.     

Biotransformation of β-methyldigitoxin by cell cultures ofDigitalis lanata in airlift and stirred tank reactors

Summary Digitalis lanata cells were grown at dif-ferent dissolved-oxygen (DO) levels in 20-1 airlift reactors. A DO level of 30% saturation (using air for aeration) was found to be optimal for growth and the biotransformation ofβ-methyldigitoxin toβ-methyldigoxin. Product yield was further in-creased by using stirred tank reactors instead of the airlift reactor.     

Gene Control in Somatic Embryos of Digitalis lanata: Expression of the β-Glucuronidase Gene Fused to a Plastocyanin Promoter

Digitalis lanata was transformed by agrobacteria-mediated gene transfer with a chimeric reporter gene encoding for β-glucuronidase (CUS) from Escherichia coll under the control of the plastocyanin 3 (Pc3) promoter from Spinada oleracea (Pc3::uidA fusion gene). Transformed cell lines were regenerated to plants via somatic embryos. CUS activity was determined fluorometrically and histochemically. The Pc3::uidA fusion gene was expressed in the late globular and bipolar stages of somatic embryos. Expression started in globular embryos (stage-1-globules) in that part of the parenchymatic tissue which later on formed the cotyledons. No GUS activity was detectable in the parenchymatic tissue forming the root pole, in cells of the developing procambium or in epidermal cells. These tissues were free of GUS activity also in bipolar embryos. The parenchymatic cells of the cotyledons and the primary cortex of the hypocotyl of germinating embryos showed GUS activity, in contrast to the epidermal cells and the cells of the central cylinder.     

Purification and characterization of lanatoside 15′-O-acetylesterase from Digitalis lanata Ehrh.

Lanatoside 15′-O-acetylesterase (LAE) from in-vitro-cultivated cells of Digitalis lanata Ehrh. was isolated and partially sequenced. The enzyme was extracted with citrate buffer from acetone dry powder. It was purified in a two-step chromatographical procedure including Phenyl Sepharose hydrophobic interaction chromatography followed by CM Sepharose cation-exchange chromatography to more than 330 μmol · s⁻¹ · (g protein)⁻¹. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified protein showed a major band at 39 kDa. The protein was identified by correlation of band intensity on SDS-PAGE and enzyme activity of CM Sepharose column fractions. Size-exclusion chromatography on Sephacryl 200 revealed a single activity peak with an apparent molecular mass of about 85 kDa. Electrophoresis under nondenaturating conditions of purified LAE showed only one band with esterase activity. The intensity of this band was correlated with that of the 39-kDa band after SDS-PAGE. About 30% of the protein, including the N-terminus and several fragments obtained by Lys-C protease digestion, was sequenced. A fragment obtained by Lys-C digestion showed partial homology to other hydrolases and apoplasmic proteins. It included the probable location of an active-site histidine. The activity of LAE was high in non-morphogenic D. lanata cell strains selected for high activities in the chemical transformation of cardenolides, but rather low in the proembryogenic masses of the embryogenic cell strain VIII. It increased during the development of somatic embryos. The LAE activity in leaves of D. lanata plants was in the range 4–24 nmol · s⁻¹ · (g protein)⁻¹. Received: 25 March 1997 / Accepted: 3 July 1997