PMID(sorted ascending)
2,4-d and alkaloid accumulation in periwinkle cell suspensions.omission of 2,4-d from culture medium during one subculture of catharanthus roseus cells, strain c20, resulted in an increased alkaloid accumulation, without effect on growth. alkaloid accumulation, rather than growth, seemed to be more sensitive to 2,4-d. 2,4-d inhibited alkaloid accumulation essentially during growth phase, but its inhibitory effect during this period was partially reversible. as this reversibility was underlined only during the stationary phase, this suggested that this actio ...19947849107
application of high-performance liquid chromatography to the determination of vinblastine in catharanthus roseus.a method for the determination of vinblastine in catharanthus roseus by hplc is described. a crude alkaloid extract, obtained by extraction of leaves with toluene, 2% citric or tartaric acid and benzene, was separated by tlc. the vinblastine fraction was cut out and vinblastine was eluted from the sorbent. the amount of vinblastine was determined by hplc with peak-height measurement. the standard deviation is 0.2 microgram/ml. the detection limit is 0.05 ng of vinblastine in a sample applied to ...19947866534
cdnas for s-adenosyl-l-methionine decarboxylase from catharanthus roseus, heterologous expression, identification of the proenzyme-processing site, evidence for the presence of both subunits in the active enzyme, and a conserved region in the 5' mrna leader.s-adenosyl-l-methionine decarboxylases (adometdc) are pyruvoyl-dependent enzymes producing the aminopropyl group for spermidine biosynthesis, and this reaction is the rate-limiting step in polyamine biosynthesis. we characterized cdnas from catharanthus roseus (madagascar periwinkle) and investigated the enzyme after heterologous expression. the largest cdna (1842 bp) had an 5' leader of 469 bp and encoded a protein of 357 residues and 30-35% identity with mammalian adometdc. the proenzyme expre ...19957883014
strictosidine synthase from catharanthus roseus: purification and characterization of multiple forms.multiple (six) forms of strictosidine synthase from catharanthus roseus cell suspension cultures were purified and characterized. a purification protocol is presented composed of hydrophobic-interaction, gel-permeation and ion-exchange chromatography and chromatofocusing. four of six isoforms were purified to apparent homogeneity, whereas two others were nearly homogeneous. all strictosidine synthase isoforms were found to be glycoproteins. the isoforms were also found in leaves and roots of the ...19957887913
suspension culture of catharanthus roseus crown gall cell induced by agrobacterium comparison with calli from leaf or stem, catharanthus roseus crown gall cell cultured on ms basic medium was superior in growth, total indole alkaloids and ajmalicine contents. the effects of illumination, cultural temperature, sucrose level of the medium and exogenous l-trp on the growth, total indole alkaloids and ajmalicine contents of c. roseus crown gall cell cultures were studied. the results will provide a theoretical basis for the attempt of using suspension cultures of c. roseus crow ...19947893941
mechanisms of the proliferation and differentiation of plant cells in cell culture systems.plant cell functions have been investigated in various cell culture systems. in this review, we summarize results obtained from investigations of gene expression during the cell cycle in synchronized cultures of catharanthus roseus during somatic embryogenesis in suspension cultures of daucus carota, during organogenesis in tissue cultures of arabidopsis thaliana and during the transdifferentiation of isolated mesophyll cells to tracheary elements in single-cell cultures of zinnia elegans.19947981037
hsp90 homologue from madagascar periwinkle (catharanthus roseus): cdna sequence, regulation of protein expression and location in the endoplasmic reticulum.we describe cdnas for a hsp90 homologue from catharanthus roseus and studies on the regulation of expression. the largest cdna (2670 bp) coded for a protein of 817 amino acids with a calculated size of 93,491 da and a pi of 4.61. it contained a eucaryotic secretory signal, the endoplasmic reticulum (er) targeting and retention signal (lys-asp-glu-leu), and the hsp90 protein family signature with one conservative exchange (asn-lys-asp-ile-phe-leu instead of asn-lys-glu-ile-phe-leu). rna blots rev ...19938106014
nucleotide sequence of the tryptophan decarboxylase gene of catharanthus roseus and expression of tdc-gusa gene fusions in nicotiana tabacum.the enzyme tryptophan decarboxylase (tdc; ec converts tryptophan into tryptamine. in catharanthus roseus and other plants capable of producing terpenoid indole alkaloids (tias) tdc links primary metabolism to the secondary metabolic pathway involved in the biosynthesis of these compounds. the accumulation of tdc mrna in c. roseus cells is developmentally regulated and transcriptionally influenced by elicitors (induction) and auxins (repression). here we report that tdc is encoded by a ...19948159173
cdna for a 14-kilodalton polypeptide from madagascar periwinkle (catharanthus roseus). eight conserved cysteines in six related proteins from different plants suggest common functional elements. 19948165254
cdna for s-adenosyl-l-homocysteine hydrolase from catharanthus roseus. 19948165255
meristem-specific gene expression directed by the promoter of the s-phase-specific gene, cyc07, in transgenic arabidopsis.a genomic clone for the cyc07 gene, which is expressed specifically at the s phase during the cell cycle in synchronous cultures of periwinkle (catharanthus roseus) cells, was isolated. determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. genomic southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. expression of related genes was detected in a wide range of oth ...19948204824
isolation and characterization of a cdna clone from catharanthus roseus encoding nadph:cytochrome p-450 reductase, an enzyme essential for reactions catalysed by cytochrome p-450 mono-oxygenases in plants.the membrane-bound flavoprotein nadph:cytochrome p-450 (cytochrome c) reductase, that functions in electron transfer to cytochrome p-450 monooxygenases, was purified from a cell suspension culture of the higher plant catharanthus roseus. anti-serum raised against the purified protein was found to inhibit nadph:cytochrome c reductase activity as well as the activities of the cytochrome p-450 enzymes geraniol 10-hydroxylase and trans-cinnamate 4-hydroxylase, which are involved in alkaloid biosynth ...19938220474
purification and partial characterization of microsomal nadh-cytochrome b5 reductase from higher plant catharanthus roseus.a simple three step procedure was used to purify microsomal nadh-cytochrome b5 (ferricyanide) reductase to homogeneity from the higher plant c. roseus. the microsomal bound reductase was solubilized using zwitterionic detergent-chaps. the solubilized reductase was subjected to affinity chromatography on octylamino sepharose 4b, blue 2-sepharose cl-6b and nad(+)-agarose. the homogeneous enzyme has an apparent molecular weight of 33,000 as estimated by sds-page. the purified enzyme catalyzes the r ...19938267585
isolation and characterization of a cdna clone for plant nuclear antigen 21d7 associated with cell division.a cdna clone was isolated from a carrot (daucus carota l.) cdna expression library using monoclonal antibody 21d7, which recognizes a nuclear antigen associated with cell division in plant cells. to show that the isolated cdna encodes the 21d7 antigen, a polyclonal antiserum was raised against a recombinant fusion protein specified by the cdna. both the polyclonal antiserum and the monoclonal antibody 21d7 recognized the same plant protein on immunoblots, in immunoprecipitation experiments, and ...19938310059
a chimaeric tryptophan decarboxylase gene as a novel selectable marker in plant cells.a novel selection system for plant genetic transformation was developed based on the enzyme tryptophan decarboxylase (tdc; ec from catharanthus roseus. this enzyme converts the toxic tryptophan analogue 4-methyl tryptophan (4-mt) into the non-toxic compound 4-methyl tryptamine. expression of tdc in transgenic plants that have no endogenous tdc-activity allows selection on 4-mt. a vector was constructed containing a tdc cdna clone under control of the constitutively expressed cauliflowe ...19938358036
exploration of the frontiers of tradomedical practices: basis for development of alternative medical healthcare services in developing countries.the study is a brief exploration of the functions and roles of the traditional healers in the total health care delivery system as a basis for tapping the salient features of this age old art: for the purpose of refining, and establishing it as an alternative medical health-care service. the investigation is considered relevant particularly in the developing countries where, in addition to the dearth of orthodox medical services, institutions and personnel, it is relatively cheaper, socio-cultur ...19938410912
purification and characterization of anthranilate synthase from catharanthus roseus.anthranilate synthase (ec has been purified from cell cultures of catharanthus roseus by poly(ethylene glycol) precipitation/fractionation and subsequent separation by anion exchange on q-sepharose, orange a dye chromatography, mono q anion-exchange chromatography and superose 6 gel filtration. by analogy to anthranilate synthases from other sources it does look like the enzyme is a tetramer composed of two large and two small subunits, with molecular mass 67 and 25.5 +/- 0.5 kda, resp ...19938444181
purification, characterization, and kinetic analysis of a 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis from catharanthus roseus.a 2-oxoglutarate-dependent dioxygenase (ec which catalyzes the hydroxylation at position 4 of the indole alkaloid, desacetoxyvindoline has been purified to near homogeneity from catharanthus roseus. the purification procedure combined conventional chromatographic methods and cosubstrate affinity chromatography on alpha-ketoglutarate-sepharose. the specific activity of the 4-hydroxylase was enriched over 2000-fold compared to the crude homogenate with a recovery of 1.6%. the molecular ...19938449913
isolation of cytochrome p-450 cdna clones from the higher plant catharanthus roseus by a pcr strategy.cytochrome p-450 monooxygenases are membrane-bound enzymes involved in a wide range of biosynthetic pathways in plants. an efficient pcr strategy for isolating cytochrome p-450 cdna clones from plant cdna libraries is described. a set of degenerate primers for pcr amplification was designed to recognize nucleotide sequences specifying the highly conserved haembinding region of cytochrome p-450 proteins. using this primer set and a non-specific primer, complementary to either the poly(a) tail of ...19938507838
overexpression of a tryptophan decarboxylase cdna in catharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production.the enzyme tryptophan decarboxylase (tdc) (ec catalyses a key step in the biosynthesis of terpenoid indole alkaloids in c. roseus by converting tryptophan into tryptamine. hardly any tdc mrna could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic t-dna of agrobacterium tumefaciens. supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. to investigate this possibility, chimaeric ...19958589734
uv-b- and oxidative stress-induced increase in nicotinamide and trigonelline and inhibition of defensive metabolism induction by poly(adp-ribose)polymerase inhibitor in plant tissue.nicotinamide and trigonelline contents increased in catharanthus roseus tissue culture after exposure to 2,2'-azobis(2-amidinopropane)dihydrochloride (aaph) or vanadylsulfate and in pisum sativum leaves after exposure to uv-b radiation. vanadylsulfate increased phenylalanine ammonia-lyase (pal) activity and the content of reduced and oxidized gluthathione in c. roseus tissue culture. the increases in pal activity caused by 2 mm aaph or 0.2mm vanadylsulfate were prevented by 0.1 mm 3-aminobenzami ...19968603735
characterization of a cdna encoding a proline-rich 14 kda protein in developing cortical cells of the roots of bean (phaseolus vulgaris) seedlings.a cdna clone, corresponding to mrnas preferentially expressed in the roots of bean (phaseolus vulgaris l.) seedlings, was isolated. this clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kda, designated pvr5 (phaseolus vulgaris root 5). the amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins ...19968639755
cytochrome p-450 in plant/insect interactions: geraniol 10-hydroxylase and the biosynthesis of iridoid monoterpenoids.the interactions between plant secondary metabolites (particularly monoterpenes) and insects are discussed. such metabolites are likely to have influenced the evolution of cyt p450-linked detoxification systems in animals, through animal/plant coevolution. the biosynthesis of many classes of plant secondary metabolites involves cyt p450 enzymes. of these, one of the best characterised is the geraniol/nerol 10-hydroxylase which catalyses a key step in the biosynthesis of the iridoid class of plan ...19958820862
novel type of receptor-like protein kinase from a higher plant (catharanthus roseus). cdna, gene, intramolecular autophosphorylation, and identification of a threonine important for auto- and substrate phosphorylation.we characterize crrlk1, a novel type of receptor-like kinase (rlk), from the plant catharanthus roseus (madagascar periwinkle). the protein (90.2 kda) deduced from the complete genomic and cdna sequences is a rlk by predicting a n-terminal signal peptide, a large extracytoplasmic domain, a membrane-spanning hydrophobic region followed by a transfer-stop signal, and a c-terminal cytoplasmic protein kinase with all 11 conserved subdomains. it is a novel rlk type because the predicted extracytoplas ...19968900145
enzymology of indole alkaloid biosynthesis in catharanthus roseus.indole alkaloids in catharanthus roseus have been in focus because of their medicinal value. these alkaloids consist of an indole moiety provided by tryptamine and a terpenoid portion provided by the secologanin. the most important catharanthus alkaloids are vinblastine (vlb), vincristine (vcr) and ajmalicine. vlb and vcr are clinically useful anticancer agents whereas ajmalicine is used for the treatment of circulatory diseases. vcr and vlb are the most expensive because of their low abundance ...19968936815
three differentially expressed s-adenosylmethionine synthetases from catharanthus roseus: molecular and functional characterization.we describe the molecular and functional characterization of three closely related s-adenosyl-l-methionine synthetase (sams) isoenzymes from catharanthus roseus (madagascar periwinkle). the genes are differentially expressed in cell cultures during growth of the culture and after application of various stresses (elicitor, nutritional down-shift, increased nacl). seedlings revealed organ-specific expression and differential gene regulation after salt stress. a relationship analysis indicated that ...19979037140
comparison of the activities of camv 35s and fmv 34s promoter derivatives in catharanthus roseus cells transiently and stably transformed by particle bombardment.activities of several camv 35s and fmv 34s promoter derivatives fused to the gusa reporter gene were compared in suspension-cultured catharanthus roseus cells that were transiently and stably transformed using particle bombardment. our data demonstrate that the 35s and a deletion derivative of the 34s promoter combined with particle bombardment form useful tools for genetic engineering of c. roseus cells. our results disagree on several points with activities of 35s and 34s promoter derivatives ...19979106518
studies on biosynthesis of brassinosteroids.biosynthesis of steroidal plant hormones, brassinosteroids, was studied using the cell culture system of catharanthus roseus. feeding labeled compounds of possible intermediates to the cultured cells, followed by analyzing the metabolites by gas chromatography-mass spectrometry disclosed the pathways from a plant sterol, campesterol, to brassinolide. there are two pathways, named the early c6-oxidation pathway and late c6-oxidation pathway, both of which would be operating in a wide variety of p ...19979178548
cell-cycle-regulated transcription of a- and b-type plant cyclin genes in synchronous cultures.synchronously dividing cell cultures of catharanthus roseus were used to isolate cdnas for two mitotic cyclins, named cys and cym. the deduced protein sequence of cys is similar to that of a-type cyclins, and cym belongs to the group of b-type cyclins. in a fashion similar to the pattern of expression seen for a-type and b-type cyclins in mammalian cells, cys is expressed before cym in c. roseus cells during the cell cycle. cys mrna accumulated at the onset of s phase and disappeared early in th ...19979193070
an important developmental role for oligosaccharides during early embryogenesis of cyprinid fish.derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations. here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates. we characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp embryos in the late gastrulation stage by incorporation of radiolabeled n-acetyl-d-[u14c]glucosamine and by hplc in combination with enzymatic conversion using the bradyrhizobium nod ...19979223299
molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in catharanthus roseus (l.) g. don.a 2-oxoglutarate-dependent dioxygenase (ec which catalyzes the 4-hydroxylation of desacetoxyvindoline was purified to homogeneity. three oligopeptides isolated from a tryptic digest of the purified protein were microsequenced and one oligopeptide showed significant homology to hyoscyamine 6 beta-hydroxylase from hyoscyamus niger. a 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a catharanthus roseus cdna library and three clones, cd4h-1 to -3, wer ...19979290645
theophylline metabolism in higher plants.metabolism of [8-(14)c]theophylline was investigated in leaf segments from camellia sinensis (tea), camellia irrawadiensis, ilex paraguariensis (maté) and avena sativa, root segments of vigna mungo seedlings and cell suspension cultures of catharanthus roseus. there was extensive uptake and metabolism of [8-(14)c]theophylline by leaves of tea and camellia irrawadiensis and, to a lesser extent, maté. these purine alkaloid-containing species converted [8-(14)c]theophylline into 3-methylxanthine, x ...19979305805
terpenoid biosynthesis from 1-deoxy-d-xylulose in higher plants by intramolecular skeletal rearrangement.the incorporation of [1-13c]- and [2,3,4,5-13c4]1-deoxy-d-xylulose into beta-carotene, lutein, phytol, and sitosterol in a cell culture of catharanthus roseus was analyzed by nmr spectroscopy. the labeling patterns of the isoprene precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, were obtained from the terpenes by a retrobiosynthetic approach. 13c enrichment and 13c13c coupling patterns showed conclusively that 1-deoxy-d-xylulose and not mevalonate is the predominant isopren ...19979380681
determination of inorganic phosphate by coupling thymidine phosphorylase and reversed-phase high-performance liquid chromatography: application to tonoplast pyrophosphatase activity.we developed an hplc method for the measurement of inorganic phosphate using thymidine phosphorylase (ec this enzyme catalyzes the phosphorolysis of thymidine to 2-deoxyribose 1-phosphate and thymine. thymine release was measured at 265 nm after separation by reverse-phase hplc. the assay was sensitive enough to detect as little as 10 pmol of pi. the response to the phosphate concentration was linear from 1 to 100 microm. the value of this method was demonstrated in an analysis of the ...19979417785
a promoter region that controls basal and elicitor-inducible expression levels of the nadph:cytochrome p450 reductase gene (cpr) from catharanthus roseus binds nuclear factor gt-1.nadph:cytochrome p450 reductase (cpr) is essential for the activation of cytochrome p450 enzymes, which are involved in a wide variety of metabolic pathways in plants, including those related to defence responses. in the subtropical plant catharanthus roseus several cytochrome p450 enzymes operate in the biosynthesis of defence-related terpenoid indole alkaloids (tias). in agreement with the importance of cpr in defence, cpr mrna levels in c. roseus were found to be enhanced by fungal elicitor p ...19979435792
evidence of protein-tyrosine kinase activity in catharanthus roseus roots transformed by agrobacterium rhizogenes.homogenate fractions (soluble and particulate) from transformed roots of catharanthus roseus (l.) g. don showed several phosphorylated proteins when incubated with gamma-[32p]atp. the phosphorylation in the proteins of 55, 40, 25, 18 and 10 kda in the particulate fraction and 63 kda in the soluble fraction was resistant to alkali treatment. several proteins in both fractions gave a positive signal with monoclonal antiphosphotyrosine antibodies. in-situ phosphorylation in both fractions showed se ...19989443385
direct fluorometry of phase-extracted tryptamine-based fast quantitative assay of l-tryptophan decarboxylase from catharanthus roseus assay for the enzyme l-tryptophan decarboxylase (tdc; ec is described. it is based on direct fluorometry of the enzymatic reaction product (tryptamine) selectively recovered in ethyl acetate from the reaction mixture. catalytically formed tryptamine from tryptophan in the incubation mixture is selectively (free from tryptophan) physically separated as ethyl acetate solution under basic (ph > or = 11) conditions and subjected to direct fluorescence measurement in the organic solvent ...19989448840
[characterization of a protein related to bet v 1, the major birch pollen antigen, from cells of the madagascar periwinkle. universal presence of proteins of this type among higher plants].in this work we present the characterisation of a gene that codes for a protein of 17 kda, in in vitro cultures of a plant with ornamental and pharmaceutical properties, the madagascan periwinkle, (catharanthus roseus [l] g. don). this protein is very close to the principal allergen of birch and also to allergens isolated from or demonstrated in some foods such as celery, parsley, apple tree, peas, asparagus and potato, but it has no allergenic characteristics.19979453736
expression of extensin genes is dependent on the stage of the cell cycle and cell proliferation in suspension-cultured catharanthus roseus isolate cdnas expressed at a specific phase of the cell cycle in a higher plant, we performed differential screening of a cdna library prepared from the s-phase cells of synchronized cultures of catharanthus roseus. sequence analysis shows that two of the identified cdnas, cyc15 and cyc17, encode extensins that represent a family of cell wall hydroxyproline-rich glycoproteins. protein sequences deduced from the two cdnas contain the characteristic pentapeptide repeat sequence, ser-pro-pro-pro ...19989484475
influence of precursor availability on alkaloid accumulation by transgenic cell line of catharanthus roseuswe have used a transgenic cell line of catharanthus roseus (l.) g. don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. line s10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (str) gene. various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of s10. our results indica ...19989490777
a novel cis-acting element in promoters of plant b-type cyclin genes activates m phase-specific transcription.plant b-type cyclin genes are expressed late in the g2 and m phases of the cell cycle. previously, we showed that the promoter of a catharanthus roseus b-type cyclin, cym, could direct m phase-specific transcription of a beta-glucuronidase reporter gene in synchronously dividing by2 tobacco cells. in this study, we determined the regulatory elements contained within the cym promoter by using a luciferase reporter gene. mutational analysis showed that a 9-bp element is essential for m phase-speci ...19989501108
glyphosate is an inhibitor of plant cytochrome p450: functional expression of thlaspi arvensae cytochrome p45071b1/reductase fusion protein in escherichia coli.glyphosate (roundup) is an herbicide used extensively worldwide which acts as an inhibitor of 5'enolpyruvylshikimate-3-phosphate synthase and for which transgenic herbicide resistant plants have been developed. here we report for the first time that glyphosate is an inhibitor of cytochrome p450 using a functional expression system for thlaspi arvensae cyp71b1 in escherichia coli. cyp71b1 was fused to the soluble domain of a plant cytochrome p450 reductase (cpr) from catharanthus roseus. cyp71b1 ...19989514851
[new antitumor derivatives of vinblastine].the alkaloids of catharanthus roseus vincristine 1 and vinblastine 2 are widely used in the chemotherapy of cancer. their nitro-derivatives have also antitumour activity [2]. the dinitro-derivatives 4, 6-9 were prepared for pharmacological investigation. some new hydroxymethyl derivatives 12-14, 17-18, 23-24 of the antitumour indole-indoline alkaloids vinblastine 2 and leurosine 5 were synthetised in two different ways a) by the selective reduction of 2, 5 or deacetoxy-vinblastine 16, b) by the ...19989592933
effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by catharanthus roseus hairy root cultures.late exponential phase hairy root cultures of catharanthus roseus were elicited with pectinase and jasmonic acid. the effects of elicitor concentration and exposure time on growth and levels of several compounds in the indole alkaloid biosynthetic pathway were monitored. pectinase decreased the fresh weight to dry weight ratio of the roots, while addition of jasmonic acid had no significant effect. selective effects on indole alkaloid yields were observed upon addition of elicitors. an increase ...20129622525
effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of catharanthus roseus.cells of catharanthus roseus (l.) g. don were genetically engineered to over-express the enzymes strictosidine synthase (str; ec and tryptophan decarboxylase (tdc; ec, which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (tias). the cultures established after agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. cultures transgenic for str consistently showed tenfold higher str activ ...19989640666
purification and characterization of alpha-3',4'-anhydrovinblastine synthase (peroxidase-like) from catharanthus roseus (l.) g. h2o2-dependent enzyme capable of coupling catharanthine and vindoline into alpha-3',4'-anhydrovinblastine (avlb) was purified to apparent homogeneity from catharanthus roseus leaves. the enzyme shows a specific avlb synthase activity of 1.8 nkat/mg, and a molecular weight of 45.40 kda (sds-page). in addition to avlb synthase activity, the purified enzyme shows peroxidase activity, and the vis spectrum of the protein presents maxima at 404, 501 and 633 nm, indicating that it is a high spin fer ...19989654153
brassinosteroids in arabidopsis thaliana.from the seeds and siliques of arabidopsis thaliana, six brassinosteroids, brassinolide, castasterone, typhasterol, 6-deoxocastasterone, 6-deoxotyphasterol and 6-deoxoteasterone, were identified by gc-mass spectrometry or gc-selected ion monitoring. as the occurrence of castasterone, typhasterol, 6-deoxocastasterone and 6-deoxotyphasterol in the shoots of a. thaliana has already been reported, this study provides evidence for the occurrence of the above four brassinosteroids in different organs, ...19989664702
effect of different inhibitors on phospholipase c activity in catharanthus roseus transformed roots.we have previously reported that catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase c (plc) activities, one soluble and one membrane associated. in this paper, the effect of neomycin and several divalent cations was analyzed, both in the soluble and the membrane-associated plc activity in c. roseus transformed roots. in this system, neomycin, an aminoglycoside antibiotic, inhibited plc in a concentration-dependent fashion. the neomycin ...19989674018
in vitro evaluation of medicinal plant extracts against pestalotiopsis mangiferae.a serious leaf-spot disease of mangifera indica was noted during the last 10 years in satpura plateau of india. on the basis of characteristic symptoms and cultural characters, the pathogen was identified as pestalotiopsis mangiferae which is hitherto not reported from satpura plateau of india. screening of 17-medicinal plants against the test pathogen revealed 14 antimycotic whereas 3-plants, viz., argemone mexicana, caesalpinia bonducella, and casia fistula acclerated the growth of the pathoge ...19969676046
developmental and light regulation of desacetoxyvindoline 4-hydroxylase in catharanthus roseus (l.) g. don. . evidence of a multilevel regulatory mechanismthe expression of desacetoxyvindoline 4-hydroxylase (d4h), which catalyzes the second to the last reaction in vindoline biosynthesis in catharanthus roseus, appears to be under complex, multilevel developmental and light regulation. developmental studies with etiolated and light-treated seedlings suggested that although light had variable effects on the levels of d4h transcripts, those of d4h protein and enzyme activity could be increased, depending on seedling development, up to 9- and 8-fold, ...19989701591
the iridoid glucoside secologanin is derived from the novel triose phosphate/pyruvate pathway in a catharanthus roseus cell culture.secologanin is the iridoid building block of the majority of the terpenoid indole alkaloids. in the biosynthesis of secologanin, mevalonate was considered to be the exclusive precursor of isopentenyl diphosphate. after [1-(13)c]glucose feeding to a cell culture of catharanthus roseus, its incorporation into secologanin was studied by 13c nmr spectroscopy. the data showed that the novel triose phosphate/pyruvate and not the mevalonate pathway was the major route for the biosynthesis of secologani ...19989742965
jasmonate modulates development- and light-regulated alkaloid biosynthesis in catharanthus roseus.methyl jasmonate, a chemical inducer of secondary metabolism, has been shown to promote vindoline biosynthesis in developing seedlings, as a result of induction of tryptophan decarboxylase (tdc) and desacetylvindoline 4-hydroxylase (d4h). the present studies suggest that jasmonate-based induction of tdc and d4h activities involves modulation of transcriptional, post-transcriptional and post-translational controls. the effects of jasmonate on both enzymes were transient with maximum tdc activity ...19989747537
phytohormone regulation of isoperoxidases in catharanthus roseus suspension cultures.peroxidase (pod) activity was investigated in catharanthus roseus cell suspensions cultured under different hormonal conditions. depletion of 2,4-dichlorophenoxyacetic acid (2,4-d) from the culture medium enhanced pod activity in cells and spent medium. addition of phytohormones, in particular the auxin 2,4-d, reduced pod activity in medium and cellular compartments and enhanced ionically cell-wall bound pod. the differential modulation of pod is due to hormone effects on synthesis and/or accumu ...19989842727
expression of two consecutive genes of a secondary metabolic pathway in transgenic tobacco: molecular diversity influences levels of expression and product accumulation.we have created a population of transgenic tobacco plants carrying cdnas encoding two consecutive enzymes from early stages in monoterpenoid alkaloid biosynthesis in catharanthus roseus. the cdnas, encoding tryptophan decarboxylase (tdc) and strictosidine synthase (str1) together with a selectable marker gene, were introduced on a single transforming plasmid into tobacco leaves by particle bombardment. analysis of 150 independent transgenic plants at the dna and rna levels demonstrated a range o ...19989862494
purification and cdna cloning of isochorismate synthase from elicited cell cultures of catharanthus roseus.isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. it is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ics; ec we have purified ics to homogeneity from elicited catharanthus roseus cell cultures. two isoforms with an apparent molecular mass of 64 kd were purified and characterized. the km values for chorismate were 558 and 319 micr ...19999952467
dimethylallyl pyrophosphate is not the committed precursor of isopentenyl pyrophosphate during terpenoid biosynthesis from 1-deoxyxylulose in higher plants.cell cultures of catharanthus roseus were supplied with [2-13c, 3-2h]-deoxyxylulose or [2-13c,4-2h]1-deoxyxylulose. lutein and chlorophylls were isolated from the cell mass, and hydrolysis of the chlorophyll mixtures afforded phytol. isotope labeling patterns of phytol and lutein were determined by 2h nmr and 1h,2h-decoupled 13c nmr. from the data it must be concluded that the deuterium atom in position 3 of deoxyxylulose was incorporated into both isopentenyl pyrophosphate (ipp) and dimethylall ...19999990020
elicitor-responsive promoter regions in the tryptophan decarboxylase gene from catharanthus roseus.the tryptophan decarboxylase (tdc) gene from catharanthus roseus (madagascar periwinkle) encodes a key enzyme in biosynthesis of terpenoid indole alkaloids. the expression of the tdc gene is transcriptionally induced by fungal elicitors. tdc upstream sequences from -1818 to +198 relative to the transcriptional start site were functionally analysed to identify cis-acting elements that determine basal expression or respond to elicitor. in a loss-of-function analysis promoter derivatives with 5' or ...199910080715
alkaloid accumulation in catharanthus roseus suspension cultures. 199910081005
structural and kinetic properties of adenylyl sulfate reductase from catharanthus roseus cell cultures.a cdna encoding a plant-type aps reductase was isolated from an axenic cell suspension culture of catharanthus roseus (genbank/embl-databank accession number u63784). the open reading frame of 1392 bp (termed par) encoded for a protein (mr=51394) consisting of a n-terminal transit peptide, a paps reductase-like core and a c-terminal extension with homology to the thioredoxin-like domain of protein disulfide isomerase. the aps reductase precursor was imported into pea chloroplasts in vitro and pr ...199910082930
transient studies of light-adapted cultures of hairy roots of catharanthus roseus: growth and indole alkaloid accumulation.cultures of c. roseus transgenic ("hairy") root clones lbe-6-1 and lbe-4-2 were adapted with periodic daily illumination to investigate the effect of light on growth and nutrient utilization, and the accumulation of the indole alkaloids. light-adapted roots appeared green and had radially thickened morphology compared with dark-grown controls. their growth rates were higher than dark-grown controls, with 45% lower doubling times: lbe-6-1, 3.6 days; lbe-4-2, 2.8 days. relative to dark-grown contr ...199810099477
quantification of metabolites in the indole alkaloid pathways of catharanthus roseus: implications for metabolic engineeringin this article, we present a review of the current state of metabolic engineering in catharanthus roseus. a significant amount of research has contributed to characterization of several individual steps in the biosynthetic pathway of medicinally valuable alkaloids. however, knowledge of the regulation of these pathways is still sparse. using hairy root cultures, we studied the responses of alkaloid metabolism to environmental stimulation such as light and elicitation. through precursor feeding ...199810191413
involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthetic genes in catharanthus roseustwo key genes in terpenoid indole alkaloid biosynthesis, tdc and str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured catharanthus roseus cells. we have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. in addition to activating tdc and str gene expression, the elicitor also induc ...199910198087
microplate enzyme-coupled assays of mevalonate and phosphomevalonate kinase from catharanthus roseus suspension cultured cells.a microplate assay for mevalonate and 5-phosphomevalonate kinase activities has been developed using an enzyme-coupled system of pyruvate kinase and lactate dehydrogenase. mevalonate and 5-phosphomevalonate kinase activities were measured in crude and partially purified enzyme preparations from catharanthus roseus suspension-cultured plant cells. the assay was validated with respect to protein and substrate concentration. mevalonate and 5-phosphomevalonate kinase showed michaelis-menten kinetics ...199910221996
multicellular compartmentation of catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediatein situ rna hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in madagascar periwinkle (catharanthus roseus). tryptophan decarboxylase (tdc) and strictosidine synthase (str1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxylase (d4h) and deacetylvindoline 4-o-acetyltransferase (dat), which are involved in the terminal steps of vindoline biosynthes ...199910330473
the promoter of the strictosidine synthase gene from periwinkle confers elicitor-inducible expression in transgenic tobacco and binds nuclear factors gt-1 and gbf.strictosidine synthase (str) is a key enzyme in the biosynthesis of terpenoid indole alkaloids. this class of secondary metabolites harbours several pharmaceutically important compounds used, among other applications, in cancer treatment. terpenoid indole alkaloid biosynthesis and expression of biosynthetic genes including str1 is induced by fungal elicitors. to identify elicitor-responsive regulatory promoter elements and trans-acting factors, the single-copy str1 gene was isolated from the sub ...199910380815
nuclear factors gt-1 and 3af1 interact with multiple sequences within the promoter of the tdc gene from madagascar periwinkle: gt-1 is involved in uv light-induced expression.plant secondary metabolites of the terpenoid indole alkaloid (tia) class comprise several compounds with pharmaceutical applications. a key step in the tia biosynthetic pathway is catalysed by the enzyme tryptophan decarboxylase (tdc), which channels the primary metabolite tryptophan into tia metabolism. in catharanthus roseus (madagascar periwinkle), the tdc gene is expressed throughout plant development. moreover, tdc gene expression is induced by external stress signals, such as fungal elicit ...199910394897
a g-box element from the catharanthus roseus strictosidine synthase (str) gene promoter confers seed-specific expression in transgenic tobacco plants.the enzyme encoded by the strictosidine synthase (str) gene from catharanthus roseus catalyses a key step in the biosynthesis of the pharmaceutically important terpenoid indole alkaloids. str cdna and genomic clones have already been isolated, allowing us to study the regulation of str gene expression. here we focus on the role of a putative cis-acting element, cacgtg, in the str promoter. this sequence is known as a g-box, and functions as a transcription-regulating sequence in a number of othe ...199910394900
arabidopsis det2 is defective in the conversion of (24r)-24-methylcholest-4-en-3-one to (24r)-24-methyl-5alpha-cholestan-3-one in brassinosteroid biosynthesis.previously, we have shown that the arabidopsis det2 (deetiolated2) mutant is defective in the biosynthesis of brassinosteroids (br) and that det2 (a steroid 5alpha-reductase) acts early in the proposed br biosynthetic pathway. in this paper we present further biochemical characterization of det2. we have undertaken metabolic experiments with 2h-labeled substrates of intermediates involved in the formation of campestanol from campesterol, and quantitative analysis of intermediates in arabidopsis ...199910398719
purification of xyloglucan endotransglycosylase based on affinity sorption of the active glycosyl-enzyme intermediate complex to cellulose.xyloglucan endotransglycosylase (xet) catalyzes the cleavage of xyloglucan (xg) molecules by a transglycosylation mechanism involving two steps: (a) endocleavage of the beta-(1,4)-linked polyglucosyl backbone of the xyloglucan molecule with formation of a glycosyl-enzyme intermediate; (b) transfer of the glycosyl residue from the intermediate to the c-4 position of the nonreducing end glucosyl unit of another molecule of xg or an xg-derived oligosaccharide with liberation of the enzyme (z. sulov ...199910419819
kinetic analysis of phospholipase c from catharanthus roseus transformed roots using different assaysthe properties of phospholipase c (plc) partially purified from catharanthus roseus transformed roots were analyzed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles, and phospholipid monolayers spread at an air-water interface. using [(33)p]phosphatidylinositol 4,5-bisphosphate (pip(2)) of high specific radioactivity, plc activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol ph ...199910444091
a novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene str interacts with a jasmonate- and elicitor-inducible ap2-domain transcription factor, orca2.jasmonate (ja) is an important plant stress hormone that induces various plant defense responses, including the biosynthesis of protective secondary metabolites. the induction of the secondary metabolite biosynthetic gene strictosidine synthase (str) in catharanthus roseus (periwinkle) cells by elicitor requires ja as a second messenger. a 42 bp region in the str promoter is both necessary and sufficient for ja- and elicitor-responsive expression. this region is unlike other previously identifie ...199910449411
isolation of indole alkaloids from catharanthus roseus by centrifugal partition chromatography in the ph-zone refining mode.centrifugal partition chromatography (cpc) in the ph-zone refining mode allowed a preparative and efficient isolation of vindoline, vindolinine, catharanthine and vincaleukoblastine from a crude mixture of catharanthus roseus alkaloids. the separation protocol was tested with a synthetic mixture of vindoline, catharanthine and vincaleukoblastine. the fraction content was analyzed and the results compared with theoretical chromatograms obtained by numerical simulation. the increase in injected sa ...199910457440
flavonoid hydroxylase from catharanthus roseus: cdna, heterologous expression, enzyme properties and cell-type specific expression in plants.we investigated the p450 dependent flavonoid hydroxylase from the ornamental plant catharanthus roseus. cdnas were obtained by heterologous screening with the cyp75 hf1 cdna from petunia hybrida. the c. roseus protein shared 68-78% identity with other cyp75s, and genomic blots suggested one or two genes. the protein was expressed in escherichia coli as translational fusion with the p450 reductase from c. roseus. enzyme assays showed that it was a flavonoid 3', 5'-hydroxylase, but 3'-hydroxylated ...199910476065
light-induced cytochrome p450-dependent enzyme in indole alkaloid biosynthesis: tabersonine 16-hydroxylase.vinblastine and vincristine are two medically important bisindole alkaloids from catharanthus roseus (madagascar periwinkle). attempts at production in cell cultures failed because a part of the complex pathway was not active, i.e. from tabersonine to vindoline. it starts with tabersonine 16-hydroxylase (t16h), a cytochrome p450-dependent enzyme. we now show that t16h is induced in the suspension culture by light and we report the cloning of the cdna. the enzyme was expressed in escherichia coli ...199910481044
a simple structured model for maintenance, biomass formation, and ajmalicine production by nondividing catharanthus roseus cells.the stoichiometry of maintenance and carbohydrate storage as well as ajmalicine production kinetics of non-dividing catharanthus roseus cells in the second stage of a two-stage batch process were investigated. for the mathematical description of these processes, a simple structured model with 5 parameters is proposed. in the model the biomass is divided in two compartments: active biomass and storage carbohydrates. in induction medium (standard medium without phosphate, nitrogen and hormones), b ...199910577468
the effect of inoculum density and conditioned medium on the production of ajmalicine and catharanthine from immobilized catharanthus roseus cells.the effect of the cell-inoculum size and the addition of conditioned medium on ajmalicine and catharanthine production were studied using immobilized catharanthus roseus cells. higher specific-uptake rates of ammonium, nitrate, and sugars were observed in the low-inoculum-density cultures (50 g fw/l) compared to the high-inoculum-density cultures (100 g fw/l). alkaloid production was not correlated with the exhaustion of a particular nutrient from the medium. the high-inoculum-density cultures p ...200010581436
gene disruption through homologous recombination in spiroplasma citri: an scm1-disrupted motility mutant is determine whether homologous recombination could be used to inactivate selected genes in spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1. an internal scm1 gene fragment was inserted into plasmid pkt1, which replicates in escherichia coli but not in s. citri, and into the s. citri oric plasmid pbot1, which replicates in spiroplasma cells as well as in e. coli. electrotransformation of s. citri with the nonreplicative, recombinant plasmid pktm1 yielded no t ...199910601200
identification of uv-b light-responsive regions in the promoter of the tryptophan decarboxylase gene from catharanthus roseus.the tryptophan decarboxylase (tdc) gene encodes a key enzyme in the biosynthesis of terpenoid indole alkaloids (tias) in catharanthus roseus. tias absorb ultraviolet light (uv) and putative functions in plants include a role as uv protectants. in support of this possible function we demonstrate here that uv light induces accumulation of several tias as well as expression of the tdc gene in c. roseus. in addition, in tobacco a tdc-gusa construct was found to be specifically induced by uv-b light. ...199910608659
a comparative evaluation of some blood sugar lowering agents of plant origin.a comparison of blood sugar lowering activity of four important medicinal plants (azadirachta indica, gymnema sylvestre, catharanthus roseus and ocimum sanctum) were carried out against normal and streptozotocin-induced diabetic rat models. the plant extracts decreased the blood sugar level in varying degrees. blood sugar lowering unit (blu) of activity of each leaf extract and tolbutamide was calculated by ed50 values. statistical analysis revealed significant (p < 0.05) variation among the tre ...199910617074
subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from catharanthus roseus.electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from catharanthus roseus (cmv-cr). crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. rows of cmv-cr particles were f ...199910643647
molecular cloning and analysis of strictosidine beta-d-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in catharanthus roseus.strictosidine beta-d-glucosidase (sgd) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (tias) by converting strictosidine to cathenamine. the biosynthetic pathway toward strictosidine is thought to be similar in all tia-producing plants. somewhere downstream of strictosidine formation, however, the biosynthesis diverges to give rise to the different tias found. sgd may play a role in creating this biosynthetic diversity. we have studied sgd at both the molecular and enzym ...200010652285
biosynthesis of brassinosteroids in cultured cells of catharanthus roseus.precursor administration experiments with 2h-labeled 6-oxocampestanol, 6-deoxocastasterone and 6alpha-hydroxycastasterone in cultured cells of catharanthus roseus were performed and the metabolites were analyzed by gc-ms. [2h6]cathasterone was identified as a metabolite of [2h6]6-oxocampestanol, whereas [2h6]6alpha-hydroxycastasterone and [2h6]castasterone were identified as metabolites of [2h6]6-deoxocastasterone, and [2h6]castasterone was identified as a metabolite of [2h6]6alpha-hydroxycastas ...200010724179
effects of buffered media upon growth and alkaloid production of catharanthus roseus hairy roots.the influence of buffered media upon the growth and alkaloid productivity of catharanthus roseus hairy root culture was examined. as expected, the buffers minimized shifts in the ph of the media and had slightly negative effects upon growth. the growth of the hairy roots remained optimal in unbuffered media. the specific yield of lochnericine was significantly lower in response to the addition of buffers, while tabersonine was significantly higher. in contrast, the specific yields of ajmalicine, ...200010772463
determination of metabolic rate-limitations by precursor feeding in catharanthus roseus hairy root cultures.precursors from the terpenoid and tryptophan branches were fed to catharanthus roseus to determine which of the two branches limits metabolic flux to indole alkaloids. the feeding of tryptophan at 17 days of the culture cycle produced auxin-like effects. addition of low levels of auxin or tryptophan resulted in significant increases in flux to the indole alkaloids. conversely, feeding higher levels of auxin or tryptophan resulted in increased branching and thickening of the hairy root cultures. ...200010812182
regulation of cytoplasmic ph under extreme acid conditions in suspension cultured cells of catharanthus roseus: a possible role of inorganic phosphate.changes in cytoplasmic ph of suspension-cultured cells of catharanthus roseus under extreme acid conditions were measured with the ph-dependent fluorescence dye; 2',7'-bis-(2-carboxyethyl)-5 (and-6) carboxyfluorescein (-acetoxymethylester) (bcecf). when cells were treated with 1 mm hcl (ph 3 solution), the cytoplasmic ph first decreased then returned to the original level. treatment with 10 mm hcl (ph 2 solution) acidified the cytoplasm to a greater extent, and the acidification continued at a c ...200010845455
purification and characterization of mevalonate kinase from suspension-cultured cells of catharanthus roseus (l.) g. don.mevalonate kinase was purified to homogeneity from catharanthus roseus (l.) g. don suspension-cultured cells. the purified enzyme had an m(r) of 104,600 and a subunit size of about 41,500. kinetic studies indicated an ordered sequential mechanism of action, in which mevalonate was the first substrate to bind and adp was the last product to leave the enzyme. true values for the kinetic constants were determined for mevalonate, with k(ma) = 76 microm and k(ia) = 74 microm, and for atp, with k(mb) ...200010860546
orca3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism.biosynthesis of many classes of secondary metabolites in plants is induced by the stress hormone jasmonate. the gene for orca3, a jasmonate-responsive apetala2 (ap2)-domain transcription factor from catharanthus roseus, was isolated by transferred dna activation tagging. orca3 overexpression resulted in enhanced expression of several metabolite biosynthetic genes and, consequently, in increased accumulation of terpenoid indole alkaloids. regulation of metabolite biosynthetic genes by jasmonate-r ...200010894776
in vivo 31p and multilabel 13c nmr measurements for evaluation of plant metabolic pathways.reliable measurements of intracellular metabolites are useful for effective plant metabolic engineering. this study explored the application of in situ 31p and 13c nmr spectroscopy for long-term measurements of intracellular ph and concentrations of several metabolites in glycolysis, glucan synthesis, and central carbon metabolic pathways in plant tissues. an nmr perfusion reactor system was designed to allow catharanthus roseus hairy root cultures to grow for 3-6 weeks, during which time nmr sp ...199910935751
histopathology and polyphenol content in plants infected by phytoplasmas.the alterations of cell walls and the localization of several compounds such as polyphenols, suberin, lignin, in plum and apple plants infected with plum leptonecrosis (pln) and apple proliferation (ap) phytoplasmas respectively, were investigated. catharanthus roseus plants, infected with ap or pln were also studied. the 4,6-diamidino-2-phenylindole (dapi) test and transmission electron microscopy showed the presence of phytoplasmas in all infected plants. specific histological stainings for cu ...200010969878
phosphate uptake across the tonoplast of intact vacuoles isolated from suspension-cultured cells of catharanthus roseus (l.) g. don.transport of inorganic orthophosphate (pi) across the tonoplast membrane was studied using intact vacuoles isolated from suspension-cultured cells of catharanthus roseus. orthophosphate uptake was strongly stimulated in the presence of mg-atp and mg-pyrophosphate and inhibited by bafilomycin and concanamycin which are potent inhibitors of the vacuolar h+-atpase. these results indicated that the build-up of an electrochemical gradient by the h - pumps was essential for the uptake of pi. potassium ...200010987558
plant methionine synthase: new insights into properties and expression.we investigated the enzyme methionine synthase (msy) in catharanthus roseus. the properties were characterized with purified protein isolated either from plant cell cultures or after heterologous expression in escherichia coli. the protein was a monomer and accepted both the triglutamate (ch3-h4pteglu3, apparent km = 80 microm) and the monoglutamate (ch3-h4pteglu1, apparent km = 350 microm) of methyl-5,6,7,8-tetrahydropteroate as methyl donor, with a ratio of approximately 90:1 in favor of the t ...200011030427
the ternary transformation system: constitutive virg on a compatible plasmid dramatically increases agrobacterium-mediated plant transformation.this paper describes a so-called ternary transformation system for plant cells. we demonstrate that agrobacterium tumefaciens strain lba4404 supplemented with a constitutive virg mutant gene (virgn54d) on a compatible plasmid is capable of very efficient t-dna transfer to a diverse range of plant species. for the plant species catharanthus roseus it is shown that increased t-dna transfer results in increased stable transformation frequencies. analysis of stably transformed c. roseus cell lines s ...200011052201
28-norcastasterone is biosynthesized from castasterone.metabolic experiments with deuterium-labeled castasterone in seedlings of arabidopsis thaliana, oryza saliva and lycopersicon esculentum, and cultured cells of catharanthus roseus were performed, and the metabolites were analyzed by gc-ms. in all the plant species examined, [2h3]28-norcastasterone was identified as a metabolite of [26,28-2h6]castasterone, indicating that castasterone is the biosynthetic origin of 28-norcastasterone. moreover, the natural occurrence of 28-norcastasterone and 28-n ...200011065283
[studies on the biosynthesis of sterol side chain in higher plants].campesterol (3) and dihydrobrassicasterol (4), typical c28-sterols in higher plants, are biosynthesized from a steroidal 24-ene precursor (desmosterol 1) via 24-methylenecholesterol (2) and 24-methyldesmosterol (5). a typical plant c29-sterol, sitosterol (6), is produced from 24-methylenecholesterol via isofucosterol (7) and 24-ethyldesmosterol (8). the biosynthetic mechanism, focussing stereochemical features, of these side-chain transformations has been studied in detail by feeding regio- and ...200011082699
cloning and expression of cdnas encoding two enzymes of the mep pathway in catharanthus roseus.two periwinkle cdnas (crdxr and crmecs) encoding enzymes of the non-mevalonate terpenoid pathway were characterized using reverse transcription-pcr strategy based on the design of degenerated oligonucleotides. the deduced amino acid sequence of crdxr is homologue to 1-deoxy-d-xylulose 5-phosphate reductoisomerases. crmecs represents the first plant cdna encoding a protein similar to the 2c-methyl-d-erythritol 2,4-cyclodiphosphate synthase from escherichia coli. expression of crdxr and crmecs gen ...200011118631
light activation of vindoline biosynthesis does not require cytomorphogenesis in catharanthus roseus seedlings.upon illumination, the cotyledons of catharanthus roseus seedlings readily synthesise vindoline from late biosynthetic intermediates, which accumulate in etiolated seedlings. the cellular localisation of tryptophan decarboxylase (tdc) and desacetoxyvindoline 4-hydroxylase (d4h), which catalyse the first and penultimate reactions of vindoline biosynthesis, was identified by immunocytochemistry in developing seedlings. the expression of tdc was restricted to the upper epidermis of cotyledons, wher ...200011130662
indole alkaloid biosynthesis in catharanthus roseus: new enzyme activities and identification of cytochrome p450 cyp72a1 as secologanin synthase.the molecular characterization of cyp72a1 from catharanthus roseus (madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. we now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis-specific. it thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that cyp72a1 may be involved in their biosynthesis. the early reactions in that pathway, i.e ...200011135113
molecular and biochemical analysis of a madagascar periwinkle root-specific minovincinine-19-hydroxy-o-acetyltransferase.the terminal steps in the biosynthesis of the monoterpenoid indole alkaloids vindoline and minovincinine are catalyzed by separate acetyl coenzyme a-dependent o-acetyltransferases in madagascar periwinkle (catharanthus roseus g. don). two genes were isolated that had 63% nucleic acid identity and whose deduced amino acid sequences were 78% identical. active enzymes that were expressed as recombinant his-tagged proteins in escherichia coli were named minovincinine-19-o-acetyltransferase (mat) and ...200111154328
the jasmonate-inducible ap2/erf-domain transcription factor orca3 activates gene expression via interaction with a jasmonate-responsive promoter element.the ap2/erf-domain transcription factor orca3 is a master regulator of primary and secondary metabolism in catharanthus roseus (periwinkle). here we demonstrate that orca3 specifically binds to and activates gene expression via a previously characterized jasmonate- and elicitor-responsive element (jere) in the promoter of the terpenoid indole alkaloid biosynthetic gene strictosidine synthase (str). functional characterization of different domains in the orca3 protein in yeast and plant cells rev ...200111169181
a catharanthus roseus bpf-1 homologue interacts with an elicitor-responsive region of the secondary metabolite biosynthetic gene str and is induced by elicitor via a ja-independent signal transduction pathway.plants respond to pathogen attack by induction of various defence responses, including the biosynthesis of protective secondary metabolites. in catharanthus roseus, the elicitor-induced expression of the terpenoid indole alkaloid biosynthetic gene strictosidine synthase (str) is mediated via the plant stress hormonejasmonate. in the promoters of several defence-related genes, cis-acting elements have been identified that are important for transcriptional regulation upon stress signals. here we s ...200011198427
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