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Plant Molecular Biology

, Volume 73, Issue 6, pp 673–685 | Cite as

Trichome specific expression of the tobacco (Nicotiana sylvestris) cembratrien-ol synthase genes is controlled by both activating and repressing cis-regions

  • Hanane Ennajdaoui
  • Gilles Vachon
  • Cécile Giacalone
  • Isabelle Besse
  • Christophe Sallaud
  • Michel Herzog
  • Alain Tissier
Article
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Abstract

Tobacco (Nicotiana sylvestris) glandular trichomes make an attractive target for isoprenoid metabolic engineering because they produce large amounts of one type of diterpenoids, α- and β-cembratrien-diols. This article describes the establishment of tools for metabolic engineering of tobacco trichomes, namely a transgenic line with strongly reduced levels of diterpenoids in the exudate and the characterization of a trichome specific promoter. The diterpene-free tobacco line was generated by silencing the major tobacco diterpene synthases, which were found to be encoded by a family of four highly similar genes (NsCBTS-2a, NsCBTS-2b, NsCBTS-3 and NsCBTS-4), one of which is a pseudogene. The promoter regions of all four CBTS genes were sequenced and found to share over 95% identity between them. Transgenic plants expressing uidA under the control of the NsCBTS-2a promoter displayed a specific pattern of GUS expression restricted exclusively to the glandular cells of the tall secretory trichomes. A series of sequential and internal deletions of the NsCBTS-2a promoter led to the identification of two cis-acting regions. The first, located between positions -589 to -479 from the transcription initiation site, conferred a broad transcriptional activation, not only in the glandular cells, but also in cells of the trichome stalk, as well as in the leaf epidermis and the root. The second region, located between positions -279 to -119, had broad repressor activity except in trichome glandular cells and is mainly responsible for the specific expression pattern of the NsCBTS-2a gene. These results establish the basis for the identification of trans-regulators required for the expression of the CBTS genes restricted to the secretory cells of the glandular trichomes.

Keywords

Glandular trichomes Promoter Terpene biosynthesis cis-regulatory regions Tobacco Intron hairpin 
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Notes

Acknowledgments

We acknowledge the support of the Agence Nationale de la Recherche (programme Réseau Innovation Biotechnologies 2005), grant number ANR-05-PRIB02102 to AT and MH. We wish to thank the Groupement de Recherches Appliquées en Phytotechnologies of the CEA Cadarache for assistance in growing the plants.

Supplementary material

11103_2010_9648_MOESM1_ESM.ppt (240 kb)
Supplementary material 1 (PPT 240 kb)
11103_2010_9648_MOESM2_ESM.doc (125 kb)
Supplementary material 2 (DOC 125 kb)

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Hanane Ennajdaoui
    • 1
    • 4
  • Gilles Vachon
    • 2
  • Cécile Giacalone
    • 3
    • 6
  • Isabelle Besse
    • 3
  • Christophe Sallaud
    • 3
    • 7
  • Michel Herzog
    • 1
  • Alain Tissier
    • 3
    • 5
  1. 1.Laboratoire d’Ecologie AlpineUniversité Joseph Fourier and CNRS—Unité Mixte de Recherche 5553Grenoble Cedex 9France
  2. 2.Laboratoire de Physiologie Cellulaire VégétaleUniversité Joseph Fourier—Unité Mixte de Recherche 5168 CNRS/CEA/INRA/UJF—CEAGrenoble Cedex 9France
  3. 3.Librophyt, Centre de CadaracheSaint Paul-lez-DuranceFrance
  4. 4.Molecular Cell & Developmental Biology DepartmentSinsheimer LaboratoriesSanta CruzUSA
  5. 5.Institut de Recherche pour le DéveloppementUniversité Montpellier 2—Unité Mixte de Recherche Diversité et Adaptation des Plantes CultivéesMontpellier Cedex 5France
  6. 6.Laboratoire Echanges Membranaires et Signalisation, Institut de Biologie Environnementale et de BiotechnologieCommissariat à l’Energie AtomiqueSaint Paul Lez Durance CedexFrance
  7. 7.Functional and Applied Cereal Group, Biogemma, Z.I. du BrezetClermont-Ferrand Cedex 2France

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