ABSTRACT
A functionally Pin1-like peptidyl-prolyl cis/trans isomerase (PPIase(1)) was isolated from proembryogenic masses (PEMs) of Digitalis lanata according to its enzymatic activity. Partial sequence analysis of the purified enzyme (DlPar13) revealed sequence homology to members of the parvulin family of PPIases. Similar to human Pin1 and yeast Ess1, it exhibits catalytic activity toward substrates containing (Thr(P)/Ser(P))-Pro peptide bonds and comparable inhibition kinetics with juglone. Unlike Pin1-type enzymes it lacks the phosphoserine or phosphothreonine binding WW domain. Western blotting with anti-DlPar13 serum recognized the endogenous form in nucleic and cytosolic fractions of the plant cells. Since the PIN1 homologue ESS1 is an essential gene, complementation experiments in yeast were performed. When overexpressed in Saccharomyces cerevisiae DlPar13 is almost as effective as hPin1 in rescuing the temperature-sensitive phenotype caused by a mutation in ESS1. In contrast, the human parvulin hPar14 is not able to rescue the lethal phenotype of this yeast strain at nonpermissive temperatures. These results suggest a function for DlPar13 rather similar to parvulins of the Pin1-type.
Subject(s)
Peptidylprolyl Isomerase/chemistry , Peptidylprolyl Isomerase/physiology , Plant Proteins , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Blotting, Western , Cell Nucleus/metabolism , Cloning, Molecular , Cytosol/metabolism , DNA, Complementary/metabolism , Digitalis/enzymology , Enzyme Inhibitors/pharmacology , Escherichia coli/chemistry , Escherichia coli Proteins , Genetic Complementation Test , Humans , Kinetics , Molecular Sequence Data , Mutation , NIMA-Interacting Peptidylprolyl Isomerase , Naphthoquinones/pharmacology , Peptidylprolyl Isomerase/metabolism , Phenotype , Phosphoserine/metabolism , Phosphothreonine/metabolism , Plants, Medicinal , Plants, Toxic , Protein Structure, Tertiary , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae Proteins , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism , Substrate Specificity , Temperature , Time FactorsABSTRACT
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.
Subject(s)
DNA, Complementary/genetics , Digitalis/genetics , Escherichia coli/genetics , Glucosidases/genetics , Plant Proteins , Plants, Medicinal , Plants, Toxic , Amino Acid Sequence , Blotting, Northern , Cardenolides/metabolism , Cloning, Molecular , DNA, Complementary/chemistry , Digitalis/enzymology , Gene Expression Regulation, Enzymologic , Glucosidases/metabolism , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
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.
Subject(s)
Androgens/biosynthesis , Digitalis/growth & development , Haploidy , Plants, Medicinal , Plants, Toxic , Cells, Cultured , Digitalis/cytology , Digitalis/metabolismABSTRACT
Delta(5)-3beta-Etaydroxysteroid dehydrogenase (Delta(5)-3beta-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3beta-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Delta(5)-3beta-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Delta(5)-3beta-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed.
Subject(s)
3-Hydroxysteroid Dehydrogenases/chemistry , Digitalis/enzymology , Plants, Medicinal , Plants, Toxic , 3-Hydroxysteroid Dehydrogenases/isolation & purification , Amino Acid Sequence , Kinetics , Models, Chemical , Molecular Sequence Data , Pregnenolone/metabolism , Progesterone/metabolism , Sequence Homology, Amino Acid , Steroid Isomerases/chemistry , Steroids/metabolism , Substrate SpecificityABSTRACT
Using proembryonic masses (PEMs) of Digitalis lanata Erh., it was demonstrated that cold, hormonal or osmotic stress, which increased freezing tolerance during cryopreservation, induced an increasing level of two peptidyl-prolyl-cis/transisomerases (PPIases). The difference in pI (9.2 +/- 0.2 and 9.5 +/- 0.2, +/- SD; n = 3) allowed the separation of the two enzymes by free-flow isoelectrophoresis. Both were inhibited by cyclosporin A and thus belong to the cyclophilin family of PPIases. The enzymes differed slightly in their substrate specificity and their relative molecular masses of 18038 +/- 4 Da (D. lanataCyp18.0) and 18132 +/- 3 Da (D. lanataCyp18.1). Both cyclophilins were blocked N-terminally. Partial internal amino acid sequences from the two cyclophilins, with a length of 34 amino acids, displayed 82% sequence identity to each other. Pretreatment of PEMs with abscisic acid, sorbitol or a combination of both substances led to a 270 +/- 30% elevation of the total cytosolic cyclophilin concentration determined with a cyclophylin affinity sensor. During the first 4 d of pretreatment, the total PPIase activity was enhanced up to 230 +/- SD% compared with the control culture. The lag phase between maximal PPIase concentration after 4 d of pretreatment and maximal effect of freezing tolerance after 10 d of pretreatment indicated that increasing levels of cytosolic PPIases may be necessary to overcome the stress induced by hormones and osmotica during pretreatment but not to protect against freezing/thawing stress.
Subject(s)
Digitalis/metabolism , Freezing , Peptidylprolyl Isomerase/metabolism , Plants, Medicinal , Plants, Toxic , Adaptation, Physiological , Amino Acid Sequence , Digitalis/enzymology , Digitalis/physiology , Molecular Sequence Data , Peptidylprolyl Isomerase/chemistry , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
A three-step chromatographic procedure was developed for purification of cardenolide 16'-O-glucohydrolase (CGH) from Digitalis lanata Ehrh. leaves, including Phenyl-Sepharose hydrophobic interaction chromatography followed by SP-Sepharose cation exchange and Q-Sepharose anion-exchange chromatography. Starting with acetone dry powder the purification resulted in an 760-fold enrichment of CGH. Molecular weight, substrate specificity, pH optimum and temperature stability of CGH were determined. Antibodies against CGH were prepared in rabbits. The SDS gel electrophoresis of protein extracts from leaves of D. lanata and other D. species showed bands at 70 kDa and 36 kDa reacting with the antibodies. The 70-kDa protein is the main protein stained with CGH antibodies in freshly prepared extracts of D. lanata. It may represent undegraded CGH. The 36-kDa protein is enriched in aged CGH preparations. It is probably a degradation product. Proteins related to 70-kDa and 36-kDa bands also occur in crude protein preparations from leaves of D. heywoodii P. et M. Silva, D. mariana Boiss., D. purpurea L., and D. thapsi L. indicating that CGH is also present in these species. Purified CGH was digested with proteases V8 and Lys-C and the resulting fragments obtained were sequenced. One fragment had the typical amino-acid sequence of the catalytic center of family-1 glycosyl hydrolases (EC 3.2.1.x). Cardenolide 16'-O-glucohydrolase, like the other members of this enzyme family, appeared to have a glutamic acid residue directly involved in glycosidic bond cleavage as a nucleophile.
Subject(s)
Cardenolides/isolation & purification , Digitalis/enzymology , Glucosidases/isolation & purification , Plant Proteins , Plants, Medicinal , Plants, Toxic , Amino Acid Sequence , Animals , Carbohydrate Sequence , Cardenolides/chemistry , Cardenolides/metabolism , Glucosidases/chemistry , Glucosidases/metabolism , Molecular Sequence Data , Molecular Weight , Rabbits , Sequence AnalysisABSTRACT
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 mumol.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.
Subject(s)
Acetylesterase/isolation & purification , Digitalis/enzymology , Plants, Medicinal , Plants, Toxic , Acetylesterase/chemistry , Acetylesterase/metabolism , Amino Acid Sequence , Carbohydrate Sequence , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Kinetics , Molecular Sequence DataABSTRACT
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.
Subject(s)
Deslanoside/analogs & derivatives , Deslanoside/metabolism , Digitalis/metabolism , Plants, Medicinal , Plants, Toxic , Biotransformation , Carbohydrate Conformation , Carbohydrate Sequence , Cells, Cultured , Cellular Senescence , Chromatography, High Pressure Liquid , Digitalis/cytology , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Sequence Data , Oligosaccharides/chemistry , Oligosaccharides/isolation & purificationABSTRACT
Crown galls were induced by transformation of leaves, leaf discs, and shoots of the plant DIGITALIS LANATA with the AGROBACTERIUM TUMEFACIENS strains C58 pTi C58, B6S3 pTi B6S3, and A136 pTi A6NCtmr-338::Tn5. Integration of plasmid DNA in the genome of D. LANATA was demonstrated by hybridization experiments. The transformed cells synthesized opines and showed hormone-autotrophic growth. The crown galls formed on leaves of D. LANATA plants contained digitoxigenin derivatives (up to 0.8 muimol digitoxin equivalents g (-1) dry weight). Transformed cell lines derived from the crown galls built cardenolides IN VITRO (ca. 0.03 mumol digitoxin equivalents g (-1) dry weight). The rate of cardenolide biosynthesis IN VITRO did not decrease during a cultivation period of 12 months.
ABSTRACT
Somatic embryos of DIGITALIS LANATA STRAIN VII were successfully grown as batch cultures in gaslift fermenters. Embryo development and formation of cardenolides in the embryos resembled those of cultures grown in shake flasks. Both processes depended on the developmental stage of the embryos at the time point of inoculation, the homogeneity of the embryo structures, the density of the inoculum, the composition of the nutrient medium, the intensity of irradiation, and the composition of the gas mixtures used for agitation of the embryo suspension. The cultures contained 0.7&-1.0 (micro,mol digitoxin equivalents g (-1) dry wt. (2.6-6.5 micromol digitoxin equivalents l (-1)) after a cultivation period of 28 d.
ABSTRACT
Propagation of DIGITALIS LANATA by shoot tip culture made possible (a) the rapid multiplication of elite plants with the formation of plant clones and (b) the long-term cultivation of these plants which under normal growth conditions would die at the end of the second vegetation period. Optimum conditions were established for the regeneration of shoots from shoot tips, for daughter shoot formation and rooting as well as for the adaptation of the regenerated plants to the open ground. Gene banks of valuable clones were built by keeping shoots at 4 degrees C on media with high sucrose concentration (maximum period of storage 2 years) or by growing juvenile clone plants in the greenhouse at temperatures preventing the induction of flowering. The clone plants were in the juvenile state even if they were derived from flowering mother plants. They showed normal growth and development.
ABSTRACT
A method for the preservation in liquid nitrogen of shoot tips (meristems) of D. LANATA is described. It includes the following steps: (a) hardening of shoots by cultivation at 4 degrees C for 8 weeks, (b) treatment of the explanted shoot tips with cryoprotectors, e.g., 2 mol DMSO l (-1) for 2 h, (c) either ultrarapid cooling (ca. 4000 K min (-1)) of the shoot tips by submerging in liquid nitrogen or slow cooling (ca. 0.5 K min (-1)) of the shoot tips to -40 degrees C using a suitable freezer, (d) storage of the shoot tips at -196 degrees C in liquid nitrogen, (e) ultrarapid rewarming of the ultrarapidly cooled shoot tips by placing them directly into nutrient medium or rapid rewarming of the ampoules containing the slowly cooled shoot tips with water at 40 degrees C, and (f) recultivation of the shoot tips at the surface of a solidified nutrient medium containing 2.5 micromol BA 1 (-1). About 70% of the shoot tips survived this procedure and about 30% of the shoot tips regenerated shoots.
ABSTRACT
Suspension cultures of Digitalis lanata strain I were grown in a medium containing 3% mannitol. For cryopreservation cell suspensions were treated with a mixture of sucrose-glycerol (20%/20 V%), cooled slowly (about 1 degrees C/min) till -100 degrees C and then were transferred to liquid nitrogen. After storage in liquid nitrogen the cells were thawed rapidly in a water bath of 40 degrees C and spread on the surface of a solidified nutrient medium. After 7 days of regrowth the cells were suspended in liquid nutrient medium for further cultivation. About 50% of the cells survived freezing and thawing. However, also the apparently surviving cells showed signs of injury (membrane vesicles outside the plasmalemma, dilated ER cisternae and separation of the nuclear membranes). The cultures derived from the surviving cells had the same growth rate and biochemical activity relative to the transformation of cardenolides, e.g., digitoxin, as the parent cultures. The frequency distribution of the nuclear DNA content in the cell cultures was the same before and after cryopreservation. These results indicate that there is no selection of a special cell type during freezing and thawing.
ABSTRACT
By means of a digoxin-RIA it has been shown that all organs and tissues of Digitalis lanata contain cardenolides.