Jean-Claude Chénieux

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.

1-Deoxy-D-xylulose 5-phosphate synthase from periwinkle: cDNA identification and induced gene expression in terpenoid indole alkaloid-producing cells.

Abstract Terpenoid indole alkaloids (TIAs) arise from the indole and the monoterpene pathways. The latter route derives from isopentenyl diphosphate (IPP). We report on the isolation and characterization of a cDNA ( crdxs ) encoding for 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) in Catharanthus roseus suspension cultures. The enzyme catalyses the formation of the precursor of the non-mevalonate pathway leading to IPP biosynthesis. Expression in Escherichia coli of the truncated DXPS lacking the putative plastid transit peptide revealed that this pseudomature protein was active. crdxs mRNA were detected only in TIA producing-cells and the accumulation of transcripts was found to be associated with TIA production. This result corroborates recent studies obtained with a labelled precursor, which have given evidence that the non-mevalonate pathway is involved in the biosynthesis of the precursor of TIAs secologanin.

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.

Metabolic changes and alkaloid production in habituated and non-habituated cells of Catharanthus roseus grown in hormone-free medium. Comparing hormone-deprived non-habituated cells with habituated cells

Summary Habituated cells of Catharanthus roseus (Apocynaceae) selected from an auxin-dependent line accumulated alkaloid in higher amounts than did the non-habituated cells grown in their maintenance medium. Ajmalicine and serpentine contents could be enhanced in both cell lines by feeding the indole alkaloid precursor secologanin or low concentrations of IAA or NAA. However the production remained low in the habituated cells as compared to that of non-autotrophic cells grown in a hormone-free production medium. By comparing the evolution of pH values, the levels of some cellular components and the activities of strictosidine synthase and tryptophan decarboxylase in habituated cells and non-habituated cells grown either in their maintenance medium (with auxin) or in a hormone-free medium, we present evidence that enhancement of alkaloid production in auxin depleted cells might be due to a combination of several factors including a better coordination between tryptamine and secologanine supplies and an increase of vacuolar accumulation capacities.

Alcaloïdes dihydrofuroquinoleiques de quelques rutaceae: Isolement, structure, proprietes biologiques

Abstract Six dihydrofuroquinoline quaternary bases have been isolated from Choisya ternata , Ptelea trifoliata and Ruta graveolens . Their structures have been determined by spectrometry and for some, the absolute configuration is reported. Some of the six isolated alkaloids have a prominent cytotoxic action on plant and animal tumor cells, and an inhibitory effect on Gram-positive bacteria.

Histidine-containing phosphotransfer domain extinction by RNA interference turns off a cytokinin signalling circuitry in Catharanthus roseus suspension cells

We previously reported that cytokinins (CK) induce the fast and specific transcription of CrRR1, a gene encoding a type A response regulator in Catharanthus roseus cell cultures. Here, we characterized the CrHPt1 gene that encodes a histidine‐containing phosphotransfer domain. CrHPt1 was silenced through RNA interference (RNAi) to test its possible implication in the CK signalling pathway. In transgenic lines stably transformed with an intron‐spliced construct, the degradation of CrHPt1 transcripts abolishes the CK inductive effect on CrRR1 transcription. These result give a new in vivo functional argument for the crucial role of HPt proteins in the CK signalling pathway leading to the expression of the genes encoding type A response regulators. They also show that RNAi is a powerful strategy to turn off the CK signalling circuitry.

Embryogenesis from microspores of Ginkgo biloba L., a medicinal woody species

SummaryThe present work establishes that isolated microspores of Ginkgo biloba L. cultured at densities of 1.5 to 5·104 per milliliter in Bourgin and Nitsch (1967) liquid medium are able to divide, both in the presence and in the absence of exogenous growth regulators, and to germinate by growing a pollen tube. In all experiments the microspores exhibited various modes of division leading to embryo formation in the liquid medium. Four weeks later, the microspores which had been previously submitted to various electrical stresses showed pro-embryo development earlier than those which had not. After ten weeks the number of embryos was found to be 300 to 5300 ml−1 following the experiments. When the embryos exhibited a slower growth in liquid medium, they were transferred onto various solid media for maturation. Two months later, embryos had proliferated visibly.

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.

CYTOKININ AND AUXIN-INDUCED REGULATION OF PROTEIN SYNTHESIS AND POLY(A)+ RNA ACCUMULATION IN CATHARANTHUS ROSEUS CELL CULTURES

Summary 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent C. roseus cells were groven for three days in a 2,4-D-free medium, then treated with 4.5 μM 2,4-D, 5 μM zeatin or 4.5 μM 2,4-D + 5 μM zeatin. Hormonetreated cells were labelled in vivo with [ 35 S]-methionine and polypeptides were analyzed by two-dimensional polyacrylamide gel electrophoresis. Translation products of poly (A) + RNAs isolated from cells treated with hormones were also taken as indicators of differential gene expression. Hormone treatments did not achieve dramatic changes in polypeptide patterns and changes in in vitro polypeptide synthesis were fewer than those encountered in vivo . However, accumulation of specific RNAs coding for 18 and 28 kDa polypeptides was demonstrated under conditions of alkaloid production in the cells (i.e., when cells veere grown in 2,4-D-free, zeatin-containing medium). Their molecular masses and isoelectric points are identical to those of two polypeptides whose in vivo synthesis are similarly regulated by 2,4-D and for zeatin. These polypeptides are candidates for a direct or indirect regulatory role in alkaloid synthesis in C. roseus cells, and particularly the polypeptide of 28 kDa whose in vivo and in vitro synthesis are repressed by 2,4D, yet enhanced by zeatin. Another polypeptide of 16 kDa might be derived from the 18 kDa polypeptide in a post-translational process.

The relationship between the accumulation of a 28 kd polypeptide and that of indole alkaloids in Catharanthus roseus cell suspension cultures

Summary We previously found that the accumulation of the indole alkaloid ajmalicine and that of a polypeptide of 28kD were induced by 2,4-dichlorophenoxyacetic acid removal in periwinkle ( Catharanthus roseus ) cell cultures and further increased by zeatin addition to the culture medium. In this work, we compared the effect of various plant growth regulators and stresses on the accumulation patterns of both compounds. In a first series of experiments, the periwinkle cells were subcultured in auxin-free medium for five passages and their contents in ajmalicine and 28 kD polypeptide analysed. The maximal biomass progressively decreased along subcultures; the 28 kD polypeptide and ajmalicine accumulated concomitantly in the cells (treated with or without zeatin) up to the third passage but fully disappeared afterwards. In a second series of experiments, treatment with gibberellic acid deeply inhibited, whereas NaCl treatment (given together with or without zeatin) increased the accumulation of both ajmalicine and the 28 kD polypeptide. Moreover, growing the cells in hypoxia for 2 days inhibited the accumulation of both compounds. These concomitant changes in the accumulation pattern of ajmalicine and the 28 kD polypeptide suggest that the latter can play a (direct or indirect) regulatory role in the indole alkaloid pathway of periwinkle cells.