Gene expression and biochemistry of isoprenoid biosynthesis in the glandular secretory trichomes of Artemisia annua

Abstract

The Chinese herb Artemisia annua possesses small 10-cell biseriate glandular trichomes on the surface of its aerial tissues. These trichomes were isolated from floral tissue using a Bead Beater based method. Expression patterns of expressed sequence tags from a trichome library whose Basic Local Alignment Search Tool (BLAST) results suggested a possible function in terpenoid metabolism were investigated by RT-PCR. Known terpenoid biosynthetic enzymes, such as amorpha-4,11-diene synthase showed a high degree of trichome-specific expression. In order to investigate cell specific gene expression within the trichome, the promoter for the gene encoding amorpha-4,11-diene synthase was isolated but the lack of an efficient transformation protocol in A. annua hindered reporter gene localization experiments. Traditional and whole-mount in situ hybridization techniques were used to further the study of cell specific gene expression within the glandular trichome. An RNA probe constructed from the sequence of amorpha-4,11-diene synthase localized expression to the 2nd and 3rd subapical cell pairs of the glandular trichomes. This suggests that at least part of the artemisinin biosynthetic pathway resides within the lower cell pairs. To better understand the genes involved in terpenoid biosynthesis in A. annua, the full length sequence of a short chain alcohol dehydrogenase highly represented in an expressed sequence tag library and shown to have trichome-specific expression by RT-PCR, was cloned. Heterologous expression in Escherichia coli demonstrated that the enzyme was capable of oxidizing a wide range of monoterpenols to their corresponding ketone forms. All of this data helps us to better understand the organization of expression and biochemistry of terpenoids in A. annua glandular trichomes.