Mathieu Pottier1,2, Raphaëlle Laterre1, Astrid Van Wessem1, Aldana M Ramirez1, Xavier Herman1, Marc Boutry1, Charles Hachez3. 1. Louvain Institute of Biomolecular Science and Technology, Université catholique de Louvain, 1348, Louvain-la-Neuve, Belgium. 2. InBioS-PhytoSYSTEMS, Laboratory of Plant Physiology, University of Liège, 4000, Liège, Belgium. 3. Louvain Institute of Biomolecular Science and Technology, Université catholique de Louvain, 1348, Louvain-la-Neuve, Belgium. charles.hachez@uclouvain.be.
Abstract
MAIN CONCLUSION: pRbcS-T1 and pMALD1, two new trichome-specific promoters of Nicotiana tabacum, were identified and their strength and specificity were compared to those of previously described promoters in this species. Nicotiana tabacum has emerged as a suitable host for metabolic engineering of terpenoids and derivatives in tall glandular trichomes, which actively synthesize and secrete specialized metabolites. However, implementation of an entire biosynthetic pathway in glandular trichomes requires the identification of trichome-specific promoters to appropriately drive the expression of the transgenes needed to set up the desired pathway. In this context, RT-qPCR analysis was carried out on wild-type N. tabacum plants to compare the expression pattern and gene expression level of NtRbcS-T1 and NtMALD1, two newly identified genes expressed in glandular trichomes, with those of NtCYP71D16, NtCBTS2α, NtCPS2, and NtLTP1, which were reported in the literature to be specifically expressed in glandular trichomes. We show that NtRbcS-T1 and NtMALD1 are specifically expressed in glandular trichomes like NtCYP71D16, NtCBTS2α, and NtCPS2, while NtLTP1 is also expressed in other leaf tissues as well as in the stem. Transcriptional fusions of each of the six promoters to the GUS-VENUS reporter gene were introduced in N. tabacum by Agrobacterium-mediated transformation. Almost all transgenic lines displayed GUS activity in tall glandular trichomes, indicating that the appropriate cis regulatory elements were included in the selected promoter regions. However, unlike for the other promoters, no trichome-specific line was obtained for pNtLTP1:GUS-VENUS, in agreement with the RT-qPCR data. These data thus provide two new transcription promoters that could be used in metabolic engineering of glandular trichomes.
MAIN CONCLUSION: pRbcS-T1 and pMALD1, two new trichome-specific promoters of Nicotiana tabacum, were identified and their strength and specificity were compared to those of previously described promoters in this species. Nicotiana tabacum has emerged as a suitable host for metabolic engineering of terpenoids and derivatives in tall glandular trichomes, which actively synthesize and secrete specialized metabolites. However, implementation of an entire biosynthetic pathway in glandular trichomes requires the identification of trichome-specific promoters to appropriately drive the expression of the transgenes needed to set up the desired pathway. In this context, RT-qPCR analysis was carried out on wild-type N. tabacum plants to compare the expression pattern and gene expression level of NtRbcS-T1 and NtMALD1, two newly identified genes expressed in glandular trichomes, with those of NtCYP71D16, NtCBTS2α, NtCPS2, and NtLTP1, which were reported in the literature to be specifically expressed in glandular trichomes. We show that NtRbcS-T1 and NtMALD1 are specifically expressed in glandular trichomes like NtCYP71D16, NtCBTS2α, and NtCPS2, while NtLTP1 is also expressed in other leaf tissues as well as in the stem. Transcriptional fusions of each of the six promoters to the GUS-VENUS reporter gene were introduced in N. tabacum by Agrobacterium-mediated transformation. Almost all transgenic lines displayed GUS activity in tall glandular trichomes, indicating that the appropriate cis regulatory elements were included in the selected promoter regions. However, unlike for the other promoters, no trichome-specific line was obtained for pNtLTP1:GUS-VENUS, in agreement with the RT-qPCR data. These data thus provide two new transcription promoters that could be used in metabolic engineering of glandular trichomes.
Entities:
Keywords:
Cembratrien-ol synthase; Copal-8-ol diphosphate synthase; Cytochrome P450 oxygenase; Lipid transfer protein; Major allergen mal D 1.0501; Rubisco small subunit