1. Molecular cloning and functional characterization of Catharanthus roseus hydroxymethylbutenyl 4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway.
- Author
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Ginis O, Courdavault V, Melin C, Lanoue A, Giglioli-Guivarc'h N, St-Pierre B, Courtois M, and Oudin A
- Subjects
- 5' Flanking Region genetics, Acetates pharmacology, Base Sequence, Biosynthetic Pathways drug effects, Catharanthus cytology, Catharanthus drug effects, Cloning, Molecular, Cyclopentanes pharmacology, Cytokinins pharmacology, Enzymes metabolism, Ethylenes pharmacology, Gene Expression Profiling, Gene Expression Regulation, Plant drug effects, Glucuronidase metabolism, Molecular Sequence Data, Nucleotide Motifs genetics, Organ Specificity drug effects, Organ Specificity genetics, Oxylipins pharmacology, Plant Leaves drug effects, Plant Leaves enzymology, Plant Leaves genetics, Plants, Genetically Modified, Secologanin Tryptamine Alkaloids metabolism, Sequence Analysis, DNA, Sequence Deletion genetics, Suspensions, Nicotiana drug effects, Nicotiana genetics, Transcription, Genetic drug effects, Catharanthus enzymology, Catharanthus genetics, Enzymes genetics, Erythritol analogs & derivatives, Erythritol metabolism, Genes, Plant genetics, Promoter Regions, Genetic, Sugar Phosphates metabolism
- Abstract
The Madagascar periwinkle produces monoterpenoid indole alkaloids (MIA) of high interest due to their therapeutical values. The terpenoid moiety of MIA is derived from the methyl erythritol phosphate (MEP) and seco-iridoid pathways. These pathways are regarded as the limiting branch for MIA biosynthesis in C. roseus cell and tissue cultures. In previous studies, we demonstrated a coordinated regulation at the transcriptional and spatial levels of genes from both pathways. We report here on the isolation of the 5'-flanking region (1,049 bp) of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene from the MEP pathway. To investigate promoter transcriptional activities, the HDS promoter was fused to GUS reporter gene. Agrobacterium-mediated transformation of young tobacco leaves revealed that the cloned HDS promoter displays a tissue-specific GUS staining restricted to the vascular region of the leaves and limited to a part of the vein that encompasses the phloem in agreement with the previous localization of HDS transcripts in C. roseus aerial organs. Further functional characterizations in stably or transiently transformed C. roseus cells allowed us to identify the region that can be consider as the minimal promoter and to demonstrate the induction of HDS promoter by several hormonal signals (auxin, cytokinin, methyljasmonate and ethylene) leading to MIA production. These results, and the bioinformatic analysis of the HDS 5'-region, suggest that the HDS promoter harbours a number of cis-elements binding specific transcription factors that would regulate the flux of terpenoid precursors involved in MIA biosynthesis.
- Published
- 2012
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