Inside glandular trichomes : pleiotropic drug resistance transporters in Artemisia annua and specialized metabolism in Nicotiana tabacum

Abstract

The secondary metabolism is highly active in plant glandular trichomes, which have therefore been extensively studied to elucidate the related biosynthetic pathways. In contrast, the transport of secondary metabolites remains poorly understood. In this work, we developed tools to study enzymes involved in the synthesis and secretion of secondary metabolites in trichomes of Artemisia annua and Nicotiana tabacum. A. annua glandular trichomes produce the sesquiterpene artemisinin, a strong antimalarial drug. However how this molecule is secreted out of the trichomes is unknown. As several Pleiotropic Drug Resistance (PDR) transporters are involved in diterpenes transport, we made the hypothesis that this sesquiterpene might also be transported by a PDR transporter. We identified 15 putative A. annua PDR genes in the DNA databases. Two of them, AaPDR1 and AaPDR2, were found to be highly expressed in trichomes. Their cDNAs were cloned and their expression in heterologous and homologous systems was investigated in order to study their transport activity. However, the role of AaPDR2 might be to transport other sesquiterpene(s) than artemisinin since it is expressed in T-shaped trichomes which, unlike glandular trichomes, do not synthesize artemisinin. N. tabacum bears two types of glandular trichomes, short glandular trichomes (SGTs) and tall glandular trichomes (TGTs). TGTs have been largely studied and are known to produce diterpenes and sugar esters, which are the main compounds of the trichome exudate. In contrast, SGTs have been poorly characterized. Using centrifugation on Percoll density gradients, we set up a method to obtain samples enriched in tall and short trichomes with a purity of 93% and 86%, respectively. The proteomes of these samples were compared by mass spectrometry using a quantitative methodology (isobaric tags for relative and absolute quantitation), which led to the identification of 461 protein groups. TGTs specifically contain enzymes involved in diterpenes and lipids biosynthesis. Proteins that are more abundant in SGTs belong to the translation machinery (ribosomal proteins), primary metabolism (phosphoenolpyruvate carboxykinase, alanine or aspartate amino transferases) or secondary metabolism (polyphenol oxidase). These data open the way to more focused approaches aimed at deciphering the physiological roles of SGTs.(AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 201