Molecular cloning and expression of a cDNA encoding a hybrid histidine kinase receptor in tropical periwinkle Catharanthus roseus.

Signalling pathways involving histidine kinase receptors (HKRs) are widely used by prokaryotes and fungi to regulate a large palette of biological processes. In plants, HKRs are known to be implicated in cytokinin, ethylene, and osmosensing transduction pathways. In this work, a full length cDNA named CRCIK was isolated from the tropical species CATHARANTHUS ROSEUS (L.) G. Don. It encodes a 1205 amino acid protein that belongs to the hybrid HKR family. The deduced amino acid sequence shows the highest homology with AtHK1, an osmosensing HKR in ARABIDOPSIS THALIANA. In return, CrCIK protein shares very low identity with the other 10 ARABIDOPSIS HKRs. Southern blot analysis indicates that the CRCIK corresponding gene is either present in multiple copies or has very close homologues in the genome of the tropical periwinkle. The gene is widely expressed in the plant. In C. ROSEUS C20D cell suspension, it is slightly induced after exposure to low temperature, pointing to a putative role in cold-shock signal transduction.

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 genes was up-regulated in periwinkle cells producing monoterpenoid indole alkaloids. Involvement of the 2C-methyl-D-erythritol 4-phosphate pathway in alkaloid biosynthesis is discussed.

Shemin pathway and peroxidase deficiency in a fully habituated and fully heterotrophic non-organogenic sugarbeet callus: an adaptative strategy or the consequence of modified hormonal balances and sensitivities in these cancerous cells? A review and reassessment.

There are many arguments for considering a specific fully habituated (auxin and cytokinin-independent) and fully heterotrophic non-organogenic (HNO) sugarbeet callus cell line as terminating a neoplastic progression, and thus to be made of cancerous cells. The similarities with animal tumour and cancer cells are recalled. All types of habituated tissues examined in the literature share at least three common biochemical characteristics: low apparent peroxidase activity, high content of polyamines (PAs) and low production of ethylene. However, results concerning their auxin and cytokinin levels are not consistent. Peroxidase synthesis in the achlorophyllous HNO callus appears to arise from aminolevulinic acid (ALA) synthesis through the Shemin pathway, commonly used by animals and fungi. This pathway is limited by disturbed nitrogen metabolism that diverts glutamate (directly used for ALA synthesis in green higher plants) from the Kreb's cycle into PA synthesis. There is no argument to suggest that the low ethylene production is caused by a competition with PAs for their common precursor, S-adenosylmethionine. The results we report here indicate modified anabolic and catabolic pathways of auxins and cytokinins but also the possibilities of unusual compounds playing similar roles (dehydrodiconiferyl alcohol glucosides, for instance). A higher turnover of PAs is shown in the HNO callus, which could suggest a role for H2O2 and gamma-aminobutyric acid, products or intermediates in the PA catabolic pathway, as secondary messengers. The habituated cells retain some sensitivity towards exogenous auxins and cytokinins. Their increased sensitivity to PAs and ethylene suggests modified hormonal balances for the control of these actively dividing cells.

Molecular characterization of a cytokinin-inducible periwinkle protein showing sequence homology with pathogenesis-related proteins and the Bet v 1 allergen family.

Cytokinin treatment of periwinkle callus cultures increased the accumulation of a protein, designated T1, in two-dimensional separated protein extracts. The first 30 NH2-terminal amino acids were determined by Edman degradation and showed significant sequence homology with intracellular pathogenesis-related (IPR) plant proteins and the Bet v 1 allergen family. The deduced amino acid sequence of cDNAs coding for T1, isolated by RT-PCR and 5' RACE-PCR, exhibited an average sequence identity of 40% with both IPR and Bet v 1-related allergens. T1 and all related proteins contained a p-loop motif typically found in nucleotide-binding proteins as the most conserved sequence feature. Northern blot analysis showed that cytokinin treatment of periwinkle callus induced T1 transcripts, whereas addition of 2,4-dichlorophenoxyacetic acid inhibited this accumulation. Hybridization of genomic periwinkle DNA with the T1 cDNA suggested that the protein is encoded by a single-copy gene. Immunoblot studies with a panel of Bet v 1-specific antibodies and sera from Bet v 1 allergic individuals identified T1 as a protein that is immunologically distinct from the Bet v 1 allergen family and has no allergenic properties.

[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]. / Caractérisation d'une protéine apparentée à Bet v 1, l'allergène majeur du pollen de Bouleau, dans des cellules de Pervenche de Madagascar. Présence ubiquitaire de ce groupe de protéines chez les végétaux supérieurs.

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.

Putative sites of cytokinin action during their enhancing effect on indole alkaloid accumulation in periwinkle cell suspensions.

The effects of cytokinins on the different branches of the indole alkaloid pathway were investigated in Catharanthus roseus cell cultures. Addition of zeatin to a 2,4-dichlorophenoxyacetic acid-containing medium decreased tryptamine levels and increased the bioconversion of secologanin to ajmalicine. Zeatin also enhanced the geraniol-10 hydroxylase activities and modified the indole alkaloid pattern. The results are discussed in the light of previous works showing that cytokinins have a positive effect on indole alkaloid accumulation in some lines of C. roseus.

Cytokinin-enhanced accumulation of indole alkaloids in Catharanthus roseus cell cultures - The factors affecting the cytokinin response.

Cytokinins were found to stimulate the alkaloid synthesis induced by removing auxin from the medium of a cell line of Catharanthus roseus. Diluting the mineral salts of the culture medium decreased the alkaloid production but increased the "sensitivity" of the cells. Addition of high levels of Ca(2+), Mg(2+) or Sr(2+) to B5 media in which the mineral salts were diluted to 5-40%, increased the alkaloid production. The latter effect is related only partially to enhanced osmotic potential.

Variability in tissue cultures of Choisya ternata. III comparing alkaloid production in cell lines obtained by various strategies.

Callus cultures of Choisya ternata have been prepared by different strategies: aggregate clones, subclones and protoclones obtained from well-established strains; protoclones obtained from mesophyll tissue; cultures transformed by Agrobacterium tumefaciens. All of them show high variability in their dihydrofuroquinoline alkaloid production. As compared to the alkaloid content of the whole plant, one alkaloid (platydesminium) could be obtained in higher amounts in some lines, but it was impossible to get high-balfourodinium accumulating lines. Moreover balfourodinium-producing capacities were lower in transformed cells as compared to those of normal cell lines.

Tissue culture of Chrysosplenium americanum and its potential for flavonoid production.

Chrysosplenium americanum (Saxifragaceae) accumulates a variety of partially O-methylated flavonol glucosides. Because of the semi-aquatic nature of this plant and its extensive contamination with endogenous organisms, the initiation of shoot and callus cultures could only be achieved after (a) using a special surface sterilization procedure, (b) production of new shoots from initial explants, and (c) selective elimination of organogenic structures during several subcultures. HPLC analysis of the cultured tissues established the presence of a number of flavonoids characteristic of the intact plant, though in lower and variable concentrations. However, shoot and callus cultures exhibited flavonoid profiles similar to those of the intact leaves and roots, respectively.

Time-Course Studies in Indole Alkaloid Accumulation and Changes in Tryptophan Decarboxylase and Strictosidine Synthase Activities: A Comparison in Three Strains of Catharanthus roseus Cells.

Three different strains of CATHARANTHUS ROSEUS cells were compared during one subculture with regard to tryptophan, tryptamine, ajmalicine, serpentine contents and tryptophane decarboxylase (TDC) (4) and Strictosidine synthase activities. The strains differed greatly in their accumulation of tryptamine and alkaloid. The TDC of all three strains showed the highest activity during the growth phase and declined sharply at the end of this phase. On the contrary, strictosidine synthase activity was the lowest during the growth phase and increased distinctly at the same time when the alkaloids were accumulating. By comparing the three strains with each other, no correlation was observed between the values of enzymatic activities and the contents of accumulated alkaloids.

Dihydrofuro [2,3-b] quinolinium alkaloids in cultured cells of Ptelea trifoliata L. : Isolation of a new alkaloid, ptelecultinium.

Two dihydrofuroquinoline alkaloids, ptelefolonium and another new alkaloid, ptelecultinium, have been isolated from callus strains of Ptelea trifoliata L.. Ptelecultinium was not known to occur in the mature plants. The structure of these two alkaloids were established by UV, MS and (1)H-NMR spectra.

Variability in tissue cultures of Choisya ternata. Alkaloid accumulation in protoclones and aggregate clones obtained from established strains.

Protoclones and aggregate clones have been prepared from 5 callus strains of C. ternata chosen for their dihydrofuroquinoline-accumulating capacities in a population of well-established strains. The results show that it is possible to obtain cell lines which accumulate higher alkaloid contents than the highest alkaloid-producing strain; the probability of obtaining a high-producing clone is greater if a high-producing strain is chosen as the parent strain for cloning. Compared to the alkaloid content of the whole plant, one alkaloid (platydesminium) could be obtained in greater amounts in some clones, but another alkaloid (balfourodinium) was always found in lesser quantities, even in the clones which accumulated most alkaloids. Aggregate clones were easier to obtain than protoclones and alkaloid production was rather unstable in all the clones. The protoplast-cloning procedure was not more efficient than the aggregate-cloning procedure for the selection of high-alkaloid producing lines.

Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media.

Catharanthus roseus cells were cultured in three types of media. These media were: a low sucrose subculture medium and two high sucrose media, each of which differed in their mineral and hormonal contents. The kinetics of tryptophan decarboxylase activity and the accumulations of tryptophan, tryptamine, ajmalicine and serpentine were different in each series but no correlation between maximum enzyme activity and alkaloid contents was observed. Ajmalicine and serpentine productions were unaffected by addition of Trp to the media, whereas addition of secologanin enhanced alkaloid production. The results seem to imply that the terpenoid pathway is the limiting factor in alkaloid production in C. roseus cells.

Effects of nutritional and hormonal factors on growth and production of anthraquinone glucosides in cell suspension cultures of Cinchona succirubra.

Cell suspension cultures of Cinchona succirubra were found to produce anthraquinone glucosides. The effects of nutritional and hormonal factors on growth and anthraquinone production were investigated in order to study the enzymecatalyzed glucosylation of these metabolites.

Alkaloid Production by Ochrosia elliptica Cell Suspension Cultures.

Ochrosia elliptica cell suspension cultures were obtained from callus cultures using an original «cell washing technique¼. These suspensions accumulated ellipticine, 9-methoxyellipticine, elliptinine, isoreserpiline, and reserpiline (a new alkaloid for this species) during the stationary growth phase. Alkaloid production can be enhanced by cloning small cell aggregates.

Influence of Sucrose on Levels of Ajmalicine, Serpentine, and Tryptamine in Catharanthus roseus Cells in vitro.

The fates of major nutritional elements and the synthesis of tryptophan, tryptamine and 2 alkaloid markers in C. ROSEUS cells, cultivated in media containing 20 and 60 g/l of sucrose, were studied. Sucrose effects were greatest, with these parameters, during the stationary growth phase. Intracellular ajmalicine and serpentine levels increased with sucrose concentration, whereas intracellular tryptamine (nitrogen precursor) levels were unaffected. The role of sucrose in the alkaloid pathway is discussed.

Biotransformation of ellipticine into 5-formyl ellipticine by Choisya ternata strains.

Sixteen Choisya ternata strains of same genotypic origin were examined for their capacities to biotransform ellipticine. Four strains were able to convert the drug into 5-formylellipticine with good yields. The reaction occurred only in agar medium and was different from ellipticine bioconversion by microorganisms or mammals.