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

Laboratoire d'Ecologie Alpine, Université Joseph Fourier and CNRS-Unité Mixte de Recherche 5553, 2233, rue de piscine, BP 53, 38041 Grenoble Cedex 9, France.
Plant Molecular Biology (Impact Factor: 4.26). 08/2010; 73(6):673-85. DOI: 10.1007/s11103-010-9648-x
Source: PubMed


Tobacco (Nicotiana sylvestris) glandular trichomes make an attractive target for isoprenoid metabolic engineering because they produce large amounts of one type of diterpenoids, alpha- and beta-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.

Download full-text


Available from: Gilles Vachon, Nov 20, 2014
  • Source
    • "In both apple and transgenic tobacco, the MdAAT2 promoter was highly active in stigmas and also in the glandular trichomes of transgenic tobacco leaves and sepals (Fig. 5a, b; Supplementary Fig. S3). Glandular trichomes are specialized organs located on the surface of the aerial parts of many higher plant species, and these structures have been shown to produce an abundance of secondary metabolites in certain species (Ennajdaoui et al. 2010; Wagner et al. 2004). Moreover, esters could be detected in apple leaves, and some ester compounds were only detected during phytophagous mite attack (Llusia and Penuelas 2001). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Volatile esters are major factors affecting the aroma of apple fruits, and alcohol acyltransferases (AATs) are key enzymes involved in the last steps of ester biosynthesis. The expression of apple AAT (MdAAT2) is known to be induced by salicylic acid (SA) or ethylene in apple fruits, although the mechanism of its transcriptional regulation remains elusive. In this study, we reveal that two apple transcription factors (TFs), MdMYB1 and MdMYB6, are involved in MdAAT2 promoter response to SA and ethylene in transgenic tobacco. According to electrophoretic mobility shift assays, MdMYB1 or MdMYB6 can directly bind in vitro to MYB binding sites in the MdAAT2 promoter. In vivo, overexpression of the two MYB TFs can greatly enhance MdAAT2 promoter activity, as demonstrated by dual luciferase reporter assays in transgenic tobacco. In contrast to the promoter of MdMYB1 or MdMYB6, the MdAAT2 promoter cannot be induced by SA or ethephon (ETH) in transgenic tobacco, even in stigmas in which the MdAAT2 promoter can be highly induced under normal conditions. However, the induced MYB TFs can dramatically enhance MdAAT2 promoter activity under SA or ETH treatment. We conclude that MdMYB1 and MdMYB6 function in MdAAT2 responses to SA and ethylene in transgenic tobacco, suggesting that a similar regulation mechanism may exist in apple.
    Plant Molecular Biology 06/2014; 85(6). DOI:10.1007/s11103-014-0207-8 · 4.26 Impact Factor
  • Source
    • "TpGAS was found to be expressed much higher in the trichomes compared in the other tissues (Majdi et al., 2011). Trichome specific expression of a diterpene synthase in transgenic tobacco was recently reported (Ennajdaoui et al., 2010). To obtain higher production of free parthenolide in heterologous plants host, it would be a good option to try tissue specific expression in trichomes to prevent conjugation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew that are required for the biosynthesis of parthenolide, using a combination of 454 sequencing of a feverfew glandular trichome cDNA library, co-expression analysis and metabolomics. When parthenolide biosynthesis was reconstituted by transient co-expression of all pathway genes in Nicotiana benthamiana, up to 1.4 μg g−1 parthenolide was produced, mostly present as cysteine and glutathione conjugates. These relatively polar conjugates were highly active against colon cancer cells, with only slightly lower activity than free parthenolide. In addition to these biosynthetic genes, another gene encoding a costunolide and parthenolide 3β-hydroxylase was identified opening up further options to improve the water solubility of parthenolide and therefore its potential as a drug.
    Metabolic Engineering 05/2014; 23. DOI:10.1016/j.ymben.2014.03.005 · 6.77 Impact Factor
  • Source
    • "To test N. benthamiana as a transient expression system for the production of plant diterpenes, we chose the cembratrien-ol (CBT-ol) synthase encoded by the trichome-specific NsCBTS2a gene from Nicotiana sylvestris[33]. CBTS enzymes from tobacco are class I terpene synthases which directly cyclize GGPP to a mixture of α- and β-CBT-ol. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis. Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2. With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.
    Plant Methods 12/2013; 9(1):46. DOI:10.1186/1746-4811-9-46 · 3.10 Impact Factor
Show more