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Molecular cloning and functional characterization of Catharanthus roseus hydroxymethylbutenyl 4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway

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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.

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Acknowledgments

This research was financially supported by the Ministère de l’Enseignement Supérieur et de la Recherche (MENRT) by a grant from the University of Tours. O. Ginis was financed by a fellowship from the Région Centre. We thank Emeline Marais for help in maintaining cell cultures. We also thank Dr Andrew J. Simkin for careful revision of the manuscript.

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  1. Université François Rabelais de Tours, EA 2106, Biomolécules et Biotechnologies Végétales, 31 avenue Monge, 37200, Tours, France

    Olivia Ginis, Vincent Courdavault, Céline Melin, Arnaud Lanoue, Nathalie Giglioli-Guivarc’h, Benoit St-Pierre, Martine Courtois & Audrey Oudin

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  1. Olivia Ginis
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Ginis, O., Courdavault, V., Melin, C. et al. Molecular cloning and functional characterization of Catharanthus roseus hydroxymethylbutenyl 4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway. Mol Biol Rep 39, 5433–5447 (2012). https://doi.org/10.1007/s11033-011-1343-8

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