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Martin Luckner - One of the best experts on this subject based on the ideXlab platform.
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Haploid plants regenerated from androgenic cell cultures of Digitalis Lanata.
Planta medica, 2000Co-Authors: B Diettrich, S. Ernst, Martin LucknerAbstract:Androgenic callus was obtained from cold treated anthers and pollen of Digitalis Lanata. The callus was mixoploid and contained haploid, diploid and tetraploid cells as shown by impulse cytophotometry. Haploid cell lines were selected by colony cloning. They were unstable and selection had to be repeated every 1-2 months. Mixoploid shoot cultures were derived from embryogenic haploid cell lines via somatic embryos. Haploid shoots were selected by explanting shoot tips. The shoots showed wide variability in cardenolide content and profile. Rooting of the haploid shoots resulted in haploid plants. These plants were smaller in size than diploid plants. Often the flowers were morphologically abnormal and showed male sterility due to crippled anthers.
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Cloning and functional expression in Escherichia coli of a cDNA encoding cardenolide 16'-O-glucohydrolase from Digitalis Lanata Ehrh.
Plant & cell physiology, 2000Co-Authors: Johann J. Framm, Martin Luckner, Angela Peterson, Carola Thoeringer, Antje Pangert, Ellen Hornung, Ivo Feussner, Peter LindemannAbstract:A clone of cardenolide 16'-O-glucohydrolase cDNA (CGH I) was obtained from Digitalis Lanata which encodes a protein of 642 amino acids (calculated molecular mass 73.2 kDa). The amino acid sequence derived from CGH I showed high homology to a widely distributed family of beta-glucohydrolases (glycosyl hydrolases family 1). The recombinant CGH I protein produced in Escherichia coli had CGH I activity. CGH I mRNA was detected in leaves, flowers, stems and fruits of D. Lanata.
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uptake of cardenolides from phloem sap into crown galls of Digitalis Lanata
Phytochemistry, 1998Co-Authors: Wolfram Pinkwart, B Diettrich, Martin LucknerAbstract:Abstract Crown galls developing on leaves and stems of Digitalis Lanata plants grown in the greenhouse contained digitalinum verum, glucodigifucoside, deacetyllanatoside C and verodoxin as the main cardenolides. Crown galls formed on leaves of axenic plants accumulated digitalinum verum, strospeside, lanatoside C and glucodigifucoside. These cardenolide profiles differed from those of the tumour-bearing leaves and stems, but resembled that of phloem sap. Crown gall tissue cultivated in vitro did not form cardenolides. However, crown galls in situ and crown gall tissue cultivated in vitro took up cardenolides. Digitalinum verum, a cardenolide characteristic for the phloem sap, was transported to, and accumulated in, the crown galls if administered to tumour-bearing leaves. It may therefore be speculated that the crown galls are sinks for cardenolides transported in phloem sap. The cardenolide-modifying enzymes acetyl-coenzyme A: digitoxin 15′- O -acetyltransferase, lanatoside 16′- O -acetylesterase, UDP-glucose: digitoxin 16′- O -glucosyltransferase and cardenolide 16′- O -glucohydrolase were present in crown gall tissue.
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21 di dehydro deacetyllanatoside c a biotransformation product of deacetyllanatoside c from senescent shoot cultures of Digitalis Lanata
Phytochemistry, 1997Co-Authors: M. Rhenius, B Diettrich, Andrea Porzel, Martin LucknerAbstract:Feeding deacetyllanatoside C to senescent shoot cultures of Digitalis Lanata resulted in the formation of a new product, which was isolated by semi-preparative HPLC. The molecular structure was elucidated by means of HPLC-mass spectrometry and NMR as 21'-di-dehydro-deacetyllanatoside C.
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21′-di-dehydro-deacetyllanatoside C, a biotransformation product of deacetyllanatoside C from senescent shoot cultures of Digitalis Lanata
Phytochemistry, 1997Co-Authors: M. Rhenius, B Diettrich, Andrea Porzel, Martin LucknerAbstract:Feeding deacetyllanatoside C to senescent shoot cultures of Digitalis Lanata resulted in the formation of a new product, which was isolated by semi-preparative HPLC. The molecular structure was elucidated by means of HPLC-mass spectrometry and NMR as 21'-di-dehydro-deacetyllanatoside C.
Wolfgang Kreis - One of the best experts on this subject based on the ideXlab platform.
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The catalytic mechanism of the 3-ketosteroid isomerase of Digitalis Lanata involves an intramolecular proton transfer and the activity is not associated with the 3β-hydroxysteroid dehydrogenase activity
Tetrahedron Letters, 2016Co-Authors: Nadine Meitinger, Fernão Castro Braga, José Dias De Souza Filho, Rodrigo Maia De Pádua, Jennifer Munkert, Harald Maid, Walter Bauer, Wolfgang KreisAbstract:Abstract The isomerization of the Δ 5 -3-ketosteroid isoprogesterone into the Δ 4 -3-ketosteroid progesterone has been examined with recombinant 3β-hydroxysteroid dehydrogenase from Digitalis Lanata (r Dl 3βHSD), partially purified 3-ketosteroid isomerase from Digitalis Lanata ( Dl 3KSI) and under non-enzymatic conditions in deuterium oxide (D 2 O). Studies indicate that the isomerization catalyzed by the Dl 3KSI proceeds without significant isotope exchange between the medium and the steroid and thus involves an intramolecular proton transfer consistent with the mechanism of the bacterial 3-ketosteroid isomerase of Pseudomonas testosteroni . For the r Dl 3βHSD as well as under non-enzymatic conditions deuterium was incorporated from the incubation buffer during isomerization. Together with a comparison of the rate of isomerization under the different conditions, it was demonstrated that r Dl 3βHSD does not possess 3-ketosteroid isomerase activity.
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Purification of Δ5-3-ketosteroid isomerase from Digitalis Lanata
Phytochemistry, 2014Co-Authors: Nadine Meitinger, Daniel Geiger, Thierry W. Augusto, Rodrigo Maia De Pádua, Wolfgang KreisAbstract:Abstract The isomerization of 5-pregnene-3,20-dione into 4-pregnene-3,20-dione was investigated to shed further light on cardenolide biosynthesis and to characterize the enzymes involved in cardenolide formation. It was shown that the Δ 5 -3-ketosteroid isomerase of Digitalis Lanata , which catalyzes this isomerization, is an individual enzyme and not, as previously thought, associated with Δ 5 -3β-hydroxysteroid dehydrogenase. The enzyme was purified by fractionated ammonium sulfate precipitation, hydrophobic interaction chromatography and gel filtration. The purification protocol resulted in a 68.1-fold enriched specific enzyme activity with a yield of 2.2%. After an additional chromatofocusing step the 3KSI activity appeared as a single protein band at 17 kDa in SDS–PAGE. Plant 3KSI displayed similar properties to microbial 3-ketosteroid isomerases.
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Extracellular polysaccharides produced by suspension-cultured cells from Digitalis Lanata.
Planta medica, 1997Co-Authors: Andreas Hensel, Jörg Schmidgall, Wolfgang KreisAbstract:Extracellular polysaccharides (ECP) were isolated in yields of up to 4 mg/ml from the culture media of suspension-cultured cells from Digitalis Lanata Ehrh. ECP content was increasing continuously over the first ten days of cultivation and then stayed constant until day 20. ECP were fractionated by ion-exchange chromatography into two neutral and one acidic fractions. Further fractionation was achieved by gel-permeation chromatography (GPC). One neutral fraction was separated into two distinct fractions with average molecular weights of 160 and 70 kDa, respectively. The second neutral fraction was hetero-disperse in GPC with average molecular masses of 112, 32, and 8 kDa. Polysaccharides of all neutral fractions consisted of glucose, xylose, galactose, and arabinose. Methylation analysis indicated these fractions to contain xyloglucans besides minor amounts of highly branched arabinogalactans. Xyloglucans were, using endo-beta-(1-->4)glucanase, fragmented into subunits which were identified mainly as tri- and pentasaccharides. The acidic fraction eluated as a single peak during gel-permeation chromatography with an average molecular weight of 56 kDa. Analysis of carbohydrate composition and linkage analysis indicated that this polysaccharide is an acidic arabinogalactan. 2,6-Dideoxysugars, the typical carbohydrate components of cardiac glycosides in Digitalis Lanata, were not detected in ECP.
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Influence of precursors and inhibitors of the sterol pathway on sterol and cardenolide metabolism in Digitalis Lanata EHRH
Plant Physiology and Biochemistry, 1997Co-Authors: F. Milek, Ernst Reinhard, Wolfgang KreisAbstract:The influence of 25-azacycloartanol, an inhibitor of SAM:cycloartenol 24-methyl-transferase, and cyproconazole, an experimental triazole known to inhibit ergosterol biosynthesis in fungi, on cardenolide and phytosterol accumulation was studied in different Digitalis Lanata Ehrh. tissue cultures. The influence of various steroids was also examined. Cardenolide-producing as well as cardenolide-free tissue cultures contained free cholesterol, sitosterol, campesterol and stigmasterol. Stigmasterol was the predominant sterol in cardenolide-producing cultures, whereas sitosterol dominated in cardenolide-free tissues. Suspension-cultured cells had the highest sterol content of the materials analyzed. Cardenolides and 24-alkyl sterols decreased after administration of 25-azacycloartanol, whereas the pool of free cholesterol increased. Cyproconazol influenced mainly the pools of 24-alkyl sterols and cardenolides; cholesterol contents did not change significantly. The specificity and toxicity of the inhibitors applied were evaluated. Neither the 12β-hydroxylation of β-methyldigitoxin was influenced, nor was the stimulating effect of 21-hydroxypregnenolone on cardenolide formation inhibited. The results presented support the view that 24-alkyl sterols rather than cholesterol are the favoured sterol precursors of the cardenolides in Digitalis Lanata.
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Variation of cardenolides with growth in a Digitalis Lanata Brazilian cultivar
Phytochemistry, 1997Co-Authors: Fernão Castro Braga, Wolfgang Kreis, Rubens Almeida Récio, Alaíde Braga De OliveiraAbstract:The content of the main cardenolides in Digitalis Lanata harvested in Brazil was determined by HPLC, in two different stages of growth. The analysed plants, totaling 49, presented great variation in the contents of lanatoside C and digoxin (jointly quantified), lanatoside A, lanatoside B, glucoevatromonoside, odorobioside G, glucogitoroside, glucoverodoxine, glucodigifucoside and digitalinum verum. The sum of the analysed cardenolides in the 12-month-old plants was higher than the determined concentrations in plants collected later (18 months) (P < 0.0001). Lanatoside C and digoxin were the major cardenolides in both groups, followed by glucodigifucoside. Lanatoside A content decreased in the older plants (P < 0.0001), whereas lanatoside C showed an opposite trend (P < 0.05). The estimated average dry leaf content of lanatoside C and digoxin was 1820 ± 900 nmol g−1 in the younger plants, and 2040 ± 840 nmol g−1 in the older plants. Some individual plants have shown a leaf content above 3500 nmol g−1 of these cardenolides and will be selected for improvement of the cultivar.
Ernst Reinhard - One of the best experts on this subject based on the ideXlab platform.
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Influence of precursors and inhibitors of the sterol pathway on sterol and cardenolide metabolism in Digitalis Lanata EHRH
Plant Physiology and Biochemistry, 1997Co-Authors: F. Milek, Ernst Reinhard, Wolfgang KreisAbstract:The influence of 25-azacycloartanol, an inhibitor of SAM:cycloartenol 24-methyl-transferase, and cyproconazole, an experimental triazole known to inhibit ergosterol biosynthesis in fungi, on cardenolide and phytosterol accumulation was studied in different Digitalis Lanata Ehrh. tissue cultures. The influence of various steroids was also examined. Cardenolide-producing as well as cardenolide-free tissue cultures contained free cholesterol, sitosterol, campesterol and stigmasterol. Stigmasterol was the predominant sterol in cardenolide-producing cultures, whereas sitosterol dominated in cardenolide-free tissues. Suspension-cultured cells had the highest sterol content of the materials analyzed. Cardenolides and 24-alkyl sterols decreased after administration of 25-azacycloartanol, whereas the pool of free cholesterol increased. Cyproconazol influenced mainly the pools of 24-alkyl sterols and cardenolides; cholesterol contents did not change significantly. The specificity and toxicity of the inhibitors applied were evaluated. Neither the 12β-hydroxylation of β-methyldigitoxin was influenced, nor was the stimulating effect of 21-hydroxypregnenolone on cardenolide formation inhibited. The results presented support the view that 24-alkyl sterols rather than cholesterol are the favoured sterol precursors of the cardenolides in Digitalis Lanata.
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3 alpha-Hydroxysteroid-5 beta-oxidoreductase in tissue cultures of Digitalis Lanata.
Zeitschrift fur Naturforschung. C Journal of biosciences, 1993Co-Authors: Ursel Stuhlemmer, Wolfgang Kreis, Werner Haussmann, Frank Milek, Ernst ReinhardAbstract:Putative intermediates of cardenolide biosynthesis, namely progesterone, pregnenolone, 5 beta-pregnane-3,20-dione or 5 beta-pregnan-3 beta-ol-20-one, were administered to light- or dark-grown shoot cultures of Digitalis Lanata. The unsaturated compounds were reduced to their respective 5 alpha-pregnanes, 5 beta-pregnane-3,20-dione was reduced to 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregnan-3 beta-ol-20-one was isomerized to the respective 3 alpha-pregnane. Suspension cultures of Digitalis Lanata, on the other hand, accumulated both the 3 alpha- and the 3 beta-isomer of 5 beta-pregnan-3-ol-20-one when incubated in the presence of 5 beta-pregnane-3,20-dione. When 5 beta-pregnan-3 alpha-ol-20-one was administered the cultured cells accumulated large amounts of the 3 beta-isomer together with small amounts of 5 beta-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 5 beta-pregnane-3,20-dione almost exclusively to 5 beta-pregnan-3 alpha-ol-20-one when 0.05 M MgCl2 were present in the incubation mixture. Under these conditions the formation of 5 beta-pregnan-3 beta-ol-20-one was inhibited. The enzyme activity could be recovered from membrane-free supernatants. Optimum enzyme activity occurred at pH 7.0 and 42 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)
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Cardiac glycosides in partly submerged shoots of Digitalis Lanata.
Planta medica, 1993Co-Authors: Ursel Stuhlemmer, Wolfgang Kreis, Marina Eisenbeiss, Ernst ReinhardAbstract: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.
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Storage of cardiac glycosides in vacuoles of Digitalis Lanata mesophyll cells
Phytochemistry, 1992Co-Authors: Hubert Hoelz, Wolfgang Kreis, Birgit Haug, Ernst ReinhardAbstract:Abstract The intracellular distribution of cardenolides in mesophyll cells of Digitalis Lanata was investigated. Plantlets were grown under axenic conditions. Protoplasts were isolated from sliced leaves and lysed to yield intact vacuoles which were subsequently purified by flotation centrifugation. The cardenolide contents of mesophyll cells, protoplasts and vacuoles were analysed by HPLC. Primary glycosides, e.g. the lanatosides A and C, were found to be stored exclusively in the vacuoles. Although present in quite high amounts in leaf tissue, secondary glycosides, such as α-acetyldigoxin or α-acetyldigitoxin, could not be detected in vacuoles isolated from mesophyll cells. It is concluded that in Digitalis leaves cardenolides are stored solely as primary glycosides, whereas secondary glycosides found in leaf extracts are either biosynthetic intermediates or degradation products.
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Cryopreservation of Digitalis Lanata Ehrh. cell cultures: Preculture and freeze tolerance
Plant Cell Tissue and Organ Culture, 1991Co-Authors: Eva M. Göldner, Ursula Seitz, Ernst ReinhardAbstract:Cryopreservation experiments were performed with Digitalis Lanata cell cultures. The main stress was laid on the behaviour of the cells during the preculture period and the capacity of various preculture additives to induce freeze tolerance. The following compounds were used as preculture additives: trehalose, mannitol, sucrose, melibiose, proline, and sorbitol. They are listed in the order of their respective efficiency. Using trehalose, high post-thaw viability rates were achieved and the cells resumed growth after a short lag period. Melibiose was used as a preculture additive for the first time. Its suitability was in the range of that of sucrose. Proline and sorbitol were not able to induce freeze tolerance in Digitalis cells. Cell viability showed a considerable decrease at the beginning of the preculture period. This reduction was found to be transient in the presence of trehalose, mannitol, sucrose, and melibiose. The damaging effects of proline and sorbitol were too severe to be compensated for by the cells. The PAL activity increased markedly in the presence of proline, whereas the trehalose-treated and the control cells behaved nearly identical to one another.
B Diettrich - One of the best experts on this subject based on the ideXlab platform.
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Haploid plants regenerated from androgenic cell cultures of Digitalis Lanata.
Planta medica, 2000Co-Authors: B Diettrich, S. Ernst, Martin LucknerAbstract:Androgenic callus was obtained from cold treated anthers and pollen of Digitalis Lanata. The callus was mixoploid and contained haploid, diploid and tetraploid cells as shown by impulse cytophotometry. Haploid cell lines were selected by colony cloning. They were unstable and selection had to be repeated every 1-2 months. Mixoploid shoot cultures were derived from embryogenic haploid cell lines via somatic embryos. Haploid shoots were selected by explanting shoot tips. The shoots showed wide variability in cardenolide content and profile. Rooting of the haploid shoots resulted in haploid plants. These plants were smaller in size than diploid plants. Often the flowers were morphologically abnormal and showed male sterility due to crippled anthers.
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Isolation and characterization of two acyl-CoA-binding proteins from proembryogenic masses of Digitalis Lanata Ehrh.
Planta, 2000Co-Authors: Martin Metzner, Karl Peter Ruecknagel, Jens Knudsen, Gerhard Kuellertz, Frieder Mueller-uri, B DiettrichAbstract:Two acyl-CoA-binding-protein (ACBP) isoforms were isolated from proembryogenic masses of Digitalis Lanata Ehrh. by column chromatography and preparative HPLC. The ACBPs had molecular masses of 9926 and 9997 Da, respectively. Partial sequence data indicated high similarity to each other and to ACBPs of other plant species such as Ricinus communis, Brassica napus and Arabidopsis thaliana. The isolated ACBPs bound palmitoyl-CoA with high affinity as determined by isoelectric-point shift.
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uptake of cardenolides from phloem sap into crown galls of Digitalis Lanata
Phytochemistry, 1998Co-Authors: Wolfram Pinkwart, B Diettrich, Martin LucknerAbstract:Abstract Crown galls developing on leaves and stems of Digitalis Lanata plants grown in the greenhouse contained digitalinum verum, glucodigifucoside, deacetyllanatoside C and verodoxin as the main cardenolides. Crown galls formed on leaves of axenic plants accumulated digitalinum verum, strospeside, lanatoside C and glucodigifucoside. These cardenolide profiles differed from those of the tumour-bearing leaves and stems, but resembled that of phloem sap. Crown gall tissue cultivated in vitro did not form cardenolides. However, crown galls in situ and crown gall tissue cultivated in vitro took up cardenolides. Digitalinum verum, a cardenolide characteristic for the phloem sap, was transported to, and accumulated in, the crown galls if administered to tumour-bearing leaves. It may therefore be speculated that the crown galls are sinks for cardenolides transported in phloem sap. The cardenolide-modifying enzymes acetyl-coenzyme A: digitoxin 15′- O -acetyltransferase, lanatoside 16′- O -acetylesterase, UDP-glucose: digitoxin 16′- O -glucosyltransferase and cardenolide 16′- O -glucohydrolase were present in crown gall tissue.
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21 di dehydro deacetyllanatoside c a biotransformation product of deacetyllanatoside c from senescent shoot cultures of Digitalis Lanata
Phytochemistry, 1997Co-Authors: M. Rhenius, B Diettrich, Andrea Porzel, Martin LucknerAbstract:Feeding deacetyllanatoside C to senescent shoot cultures of Digitalis Lanata resulted in the formation of a new product, which was isolated by semi-preparative HPLC. The molecular structure was elucidated by means of HPLC-mass spectrometry and NMR as 21'-di-dehydro-deacetyllanatoside C.
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21′-di-dehydro-deacetyllanatoside C, a biotransformation product of deacetyllanatoside C from senescent shoot cultures of Digitalis Lanata
Phytochemistry, 1997Co-Authors: M. Rhenius, B Diettrich, Andrea Porzel, Martin LucknerAbstract:Feeding deacetyllanatoside C to senescent shoot cultures of Digitalis Lanata resulted in the formation of a new product, which was isolated by semi-preparative HPLC. The molecular structure was elucidated by means of HPLC-mass spectrometry and NMR as 21'-di-dehydro-deacetyllanatoside C.
M. Rhenius - One of the best experts on this subject based on the ideXlab platform.
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21 di dehydro deacetyllanatoside c a biotransformation product of deacetyllanatoside c from senescent shoot cultures of Digitalis Lanata
Phytochemistry, 1997Co-Authors: M. Rhenius, B Diettrich, Andrea Porzel, Martin LucknerAbstract:Feeding deacetyllanatoside C to senescent shoot cultures of Digitalis Lanata resulted in the formation of a new product, which was isolated by semi-preparative HPLC. The molecular structure was elucidated by means of HPLC-mass spectrometry and NMR as 21'-di-dehydro-deacetyllanatoside C.
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21′-di-dehydro-deacetyllanatoside C, a biotransformation product of deacetyllanatoside C from senescent shoot cultures of Digitalis Lanata
Phytochemistry, 1997Co-Authors: M. Rhenius, B Diettrich, Andrea Porzel, Martin LucknerAbstract:Feeding deacetyllanatoside C to senescent shoot cultures of Digitalis Lanata resulted in the formation of a new product, which was isolated by semi-preparative HPLC. The molecular structure was elucidated by means of HPLC-mass spectrometry and NMR as 21'-di-dehydro-deacetyllanatoside C.