Unveiling two new trichome-specific promoters of interest for metabolic engineering in Nicotiana tabacum

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 level of NtRbS-T1 and NtMALD1 , two newly identified genes expressed in glandular trichomes, with those of NtCYP71D16 , NtCBTS2α , NtCPS2 , NtLTP1 , which were reported in the literature to be specifically expressed in glandular trichomes. The latter were previously investigated separately, preventing any accurate comparison of their expression level. We show that NtRbcS-T1 and NtMALD1 are specifically expressed in glandular trichomes just like NtCYP71D16 , NtCBTS2α , NtCPS2 , while NtLTP1 was also expressed in other leaf tissues as well as in the stem. Transcriptional fusions of all six promoters to the GUS-VENUS reporter gene were introduced in N. tabacum by Agrobacterium -mediated transformation. Most transgenic lines displayed GUS activity in tall glandular trichomes. In some transgenic lines, except for pNtLTP1:GUS-VENUS , this expression was specific. In other transgenic lines, GUS expression was extended to other tissues, probably resulting from a position effect during transgene integration. We discuss alternatives to overcome this lack of tissue specificity in some transgenic lines, should some of these promoters be used in the context of metabolic engineering in N. tabacum .