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

Abstract

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.

The Tomato Terpene Synthase Gene Family

“…4A). TPS36 shows the highest identity to the diterpene-producing cembratrienol synthases from Nicotiana sylvestris (Ennajdaoui et al, 2010). It encodes an enzyme that catalyzes the formation of an unidentified sesquiterpene from z,z-FPP (Fig.…”

“…While expression of TPS genes in trichomes of various species was reported (Bertea et al, 2006;Schilmiller et al, 2009;Ennajdaoui et al, 2010;Falara et al, 2010), intraspecies variation of TPS gene expression in trichomes of different organs and differences between closely related species have not yet been examined in detail. Comparison between the expression patterns of the tomato TPS genes in trichomes isolated from leaf versus stem revealed both qualitative and quantitative differences consistent with the distinct volatile profiles of the two organs.…”

Trichome Specific Expression: Promoters and Their Applications

“…However, more efficient silencing was obtained with intronhairpin constructs targeting the exon 2 of the CBTS genes, under the control of the CBTS2a gene itself. In this case, the best transgenic lines had almost no CBT-diols detectable (Ennajdaoui et al, 2010). There is also the possibility to exploit natural variation in N. tabacum.…”

“…In tobacco, the pathway to the cembratrien-diols was elucidated and shown to involve two steps. The first is encoded by a multigene family of diterpene synthases, the cembratrien-ol synthases (CBTS), which altogether account for the mix of the two stereoisomers of CBT-ol ( and ) (Wang and Wagner, 2003;Ennajdaoui et al, 2010). The second step is carried out by a cytochrome P450 mono-oxygenase which hydroxylates the CBT-ols at a specific position Wang and Wagner, 2003).…”

“…When the inhibitory region is deleted, expression can be detected in the whole leaf epidermis as well as in patches in roots. On the contrary, when the activating region is deleted, no expression at all can be detected (Ennajdaoui et al, 2010). This indicates that the cell specific expression is the result of the unique combination of a broad activating region with an inhibitory region which restricts expression to the desired cells.…”