Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus

Veau, Bertrand; Courtois, Martine; Oudin, Audrey; Chénieux, Jean-Claude; Rideau, Marc; Clastre, Marc

doi:10.1016/s0167-4781(00)00240-2

Cited by 121 publications

(94 citation statements)

References 29 publications

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“…There are ygbB/ispF homologues in Arabidopsis and Catharanthus roseus (CrMECS); the CrMECS transcript is up-regulated along with the DXR gene in cultured cells that produce monoterpene indole alkaloids (Veau et al, 2000). The final steps of the MEP pathway are unknown, but lead to isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) which are substrates for isoprenoid biosynthetic enzymes.…”

Section: Early Shared Steps In Aba Biosynthesismentioning

confidence: 99%

Abscisic Acid Biosynthesis and Response

Finkelstein¹,

Rock

2002

The Arabidopsis Book

190

141

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Section: Early Shared Steps In Aba Biosynthesismentioning

confidence: 99%

Abscisic Acid Biosynthesis and Response

Finkelstein¹,

Rock

2002

The Arabidopsis Book

190

141

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“…In the second step, DXP is converted into MEP and the reaction is catalyzed by a NADPH-dependent reductoisomerase (DXR; EC 1.1.1.267) specified by the DXR gene; the reaction catalyzed by DXR is actually the first committed step of the MEP pathway (Kuzuyama et al, 2000). The participation of DXR in the control of terpenoid accumulation in plants is also supported by experimental results (Veau et al, 2000;Mahmoud and Croteau, 2001;Carretero-Paulet et al, 2002). In Arabidopsis thaliana, DXR, encoded by a single gene, is a committed enzyme of the MEP pathway proven by the transgenic method and analysis of the metabolites (Carretero-Paulet et al, 2002); in antitumour-TIAproducing Catharanthus roseus, the MEP pathway provides isoprene blocks for building the monoterpenoid indole alkaloids and the expression of DXR gene isolated from C. roseus was up-regulated in parallel with the production of monoterpenoid indole alkaloids (Veau et al, 2000); overexpression of DXR in transgenic peppermint plants has led to an increase of essential oil monoterpenes in peppermint (Mahmoud and Croteau, 2001).…”

Section: Introductionmentioning

confidence: 64%

“…Following gradient temperature PCR amplification, an approximately 1000 bp product was specifically amplified at 58.4 ∞C that was subcloned and sequenced to generated a 974 bp nucleotide sequence. The BLAST search demonstrated that the 974 bp cDNA fragment showed homology with DXR genes from plant species such as Catharanthus roseus (Veau et al, 2000), Lycopersicon esculentum (RodriguezConcepcion et al, 2001) and Antirrhinum majus (Dudarev et al, 2005). These strongly suggested that the core fragment of RvDXR had been obtained, which provided necessary and enough sequence information for isolating the cDNA ends of RvDXR by RACE.…”

Section: Cloning Of the Full-length Rvdxr Cdnamentioning

confidence: 99%

A New 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase Gene Encoding the Committed-Step Enzyme in the MEP Pathway from Rauvolfia verticillata

Liao

Chen

et al. 2007

Zeitschrift Für Naturforschung C

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1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes a committed step of the methylerythritol phosphate (MEP) pathway for the biosynthesis of pharmaceutical terpenoid indole alkaloid (TIA) precursors. The full-length cDNA sequence was cloned and characterized from a TIA-producing species, Rauvolfia verticillata, using rapid amplification of cDNA ends (RACE) technique. The new cDNA was named as RvDXR and submitted to GenBank " to be assigned with an accession number (DQ779286). The fulllength cDNA of RvDXR was 1804 bp containing a 1425 bp open reading frame (ORF) encoding a polypeptide of 474 amino acids with a calculated molecular mass of 51.3 kDa and an isoelectric point of 5.88. Comparative and bioinformatic analyses revealed that RvDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPHbinding motif in its N-terminal region owned by all plant DXRs. The phylogenetic analysis revealed that DXRs had two groups including a plant and bacterial group; RvDXR belonged to angiosperm DXRs that were obtained from Synechocystis through gene transfer according to the phylogenetic analysis. The structural modeling of RvDXR showed that RvDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that RvDXR expressed in all tissues including roots, stems, leaves, fruits and followers but at different levels. The lowest transcription level was observed in followers and the highest transcription was found in fruits of R. verticillata; the transcription level of RvDXR was a little higher in roots and stems than in leaves. The cloning and characterization of RvDXR will be helpful to understand more about the role of DXR involved in R. verticillata TIA biosynthesis at the molecular level and provides a candidate gene for metabolic engineering of the TIAs pathway in R. verticillata.

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“…To develop a hypothesis for the possible function of the products of the glc operon structural genes, we constructed a thorough alignment of the predicted products of the four ORFs with known proteins from data banks, which revealed the following. ORF1 and ORF2 exhibited strong homology with IspD (4-diphosphocytidyl-2C-methyl-D-erythritol synthase) (16,37,38) and IspF (2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase) (22,51), respectively, which are enzymes involved in the nonmevalonate (DOXP) pathway for the biosynthesis of isoprenoids (Fig. 8a and b), a pathway found to be present in certain bacteria (including Z. mobilis), as well as in the plastids of plants and protozoans (17, 39, 40).…”

Section: Resultsmentioning

confidence: 99%

Transcriptional Analysis of a Gene Cluster Involved in Glucose Tolerance in Zymomonas mobilis : Evidence for an Osmoregulated Promoter

et al. 2005

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Exponentially growing cells of Zymomonas mobilis normally exhibit a lag period of up to 3 h when they are transferred from a liquid medium containing 2% glucose to a liquid medium containing 10% glucose. A mutant of Z. mobilis (CU1) exhibited a lag period of more than 20 h when it was grown under the same conditions, whereas it failed to grow on a solid medium containing 10% glucose. The glucose-defective phenotype of mutant CU1 was due to a spontaneous insertion in a putative gene (ORF4) identified as part of an operon (glc) which includes three additional putative genes (ORF1, ORF2, and ORF3) with no obvious involvement in the glucose tolerance mechanism. The common promoter controlling glc operon transcription, designated P glc , was found to be osmoregulated and stimulated by the putative product of ORF4 in an autoregulated fashion, as indicated by expression of the gfp reporter gene. Additionally, reverse transcriptase PCR analysis showed that the gene cluster produces a single mRNA, which verified the operon organization of this transcription unit. Further transcriptional analysis demonstrated that glc operon expression is regulated by the concentration of glucose, which supported the hypothesis that this operon is directly involved in the uncharacterized glucose tolerance mechanism of Z. mobilis.Zymomonas mobilis is a strictly fermentative gram-negative ethanologenic bacterium with industrial importance that produces ethanol from simple hexoses at high rates and yields (11). It also has an unusual tolerance to high concentrations of ethanol (up to 13%, wt/vol) and glucose (over 30% for most strains) (47, 48). Therefore, Z. mobilis, a typical saccharophilic organism, is ideal for studying glucose tolerance and osmoregulation mechanisms. Exponentially growing cells of Z. mobilis normally exhibit a lag period of up to 3 h when they are transferred from a liquid medium containing 0.11 M (2%) glucose to a liquid medium containing 0.55 M (10%) glucose. A mutant of Z. mobilis (CU1) (14) and a rifampin-resistant derivative of this strain (CU1Rif2) (1) exhibited a lag period of more than 20 h and were unable to grow on a solid medium containing 0.55 M glucose when they were grown under the same conditions.In an effort to better understand this unusual glucose tolerance trait, we described in a previous paper isolation of a DNA fragment (4.5 kb) which complemented the glucose-defective phenotype of Z. mobilis mutant strains CU1 and CU1Rif2 (12). This fragment consists of four open reading frames (ORFs) coding for four putative polypeptides that are 167, 167, 145, and 220 amino acids long. These ORFs exhibit the typical Z. mobilis codon usage and have individual Shine-Dalgarno consensus sites under the control of a common Ϫ35 and Ϫ10 promoter element (Fig. 1). Interestingly, a protease-sensitive diffusible factor in the medium of a wild-type culture grown in medium containing 10% glucose could correct the defect in the CU1 mutant and its derivative (12).In the present work we focused on complete genetic analysis of ...

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Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus

Cited by 121 publications

References 29 publications

Abscisic Acid Biosynthesis and Response

Abscisic Acid Biosynthesis and Response

A New 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase Gene Encoding the Committed-Step Enzyme in the MEP Pathway from Rauvolfia verticillata

Transcriptional Analysis of a Gene Cluster Involved in Glucose Tolerance in Zymomonas mobilis : Evidence for an Osmoregulated Promoter

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