Towards the identification of secondary metabolite transporters in glandular trichomes of Mentha x piperita and Nicotiana tabacum

Mentha x piperita produces and accumulates large amounts of monoterpenes, C10 molecules, in glandular trichomes, specialized organs which develop at the leaf and stem surface. While the biosynthetic pathway of these monoterpenes has received much attention, little is known about their transmembrane transport. As several Pleiotropic Drug Resistance (PDR) ABC transporters were shown to be involved in the transport of diterpenes, C20 molecules, we hypothesized that the transport of monoterpenes could also be carried out by such transporters. RT-PCR amplification using degenerated primers and M. x piperita trichome RNA was performed, leading to the identification of two PDR sequences, one of which, MpPDR1, was entirely cloned. Specific antibodies were raised and used to confirm the localization of MpPDR1 expression in M. x piperita glandular trichomes. Further work revealed that the protein is localized in the plasma membrane, supporting a role in the secretion of metabolites outside the cell. In order to investigate its physiological role, silencing of MpPDR1 expression was undertaken. While transgenic M. x piperita plants expressing the β-glucuronidase reporter gene were obtained, no silenced plants could be regenerated, indicating possible interference with regeneration. In a parallel approach to identify transporters putatively involved in secondary metabolites transport, a proteomic analysis of isolated trichomes was carried out. The feasibility was demonstrated with Nicotiana tabacum trichomes, revealing various enzymes related to the synthesis of secondary metabolites, as well as several transporters that might be responsible for their transport. Purification of M. x piperita glandular trichomes was then performed by centrifugation on a Percoll gradient, opening the way to a proteomic analysis.