Structural and regulatory genes controlling the biosynthesis of essential oil constituents in Lavender

This thesis describes research conducted to enhance our understanding of essential oil (EO) metabolism in lavender (Lavandula). Specific experiments were carried out in three areas. First, we developed a comprehensive transcriptomic database to facilitate the discovery of novel structural and regulatory essential oil biosynthetic genes in lavender. The database includes 101,618 contigs (N50 = 831 bp), over 75% of which were successfully annotated. Annotated sequences include full length transcripts for all previously reported genes involved in the MVA and MEP pathways of isoprenoid metabolism, all prenyltransferases involved in the isoprenoid biosynthesis, and all terpene synthase genes cloned from various lavender species. They also contain novel EO-related genes, as exemplified by the S-linalool synthase (Li(S)-LINS) gene we cloned using this database. Further, the database contains 1633 TFs, representing 2.1% of the lavenders' transcriptome, some of which have been shown to regulate secondary metabolism in plants. The most abundant TF families were bHLH (209), WRKY (190), and MYB (189) followed by AP2 (79), bZIP (77), and zinc finger (19), NAC (13), and MYC (9). Second, we isolated the 5' upstream genomic DNA (promoter) sequences for linalool synthase (LiLINS) and 1,8-cineole synthase (LiCINS) genes from L. x intermedia and used them to conduct a Yeast One-Hybrid Assay in order to identify TFs that regulate the expression of LiLINS and LiCINS genes in this plant. The assay identified 96 proteins that interacted with one or both promoters. To elucidate the nature of this interaction further, the LiLINS and LiCINS promoter fragments were each fused to the E. coli gusA (GUS) reporter gene. The constructs were separately transformed into tobacco (Nicotiana benthamiana) leaves, co-expressing individually a subset of ten representative transcription factors. Six TFs induced expression from both promoters, two activated LiCINS promoter alone, and two did not induce expression from either promoter. Finally, we isolated and functionally characterized genes for two acetyltransferases, LiLAT-3 & LiLAT-4, which convert some of the monoterpene EO constituents into their respective esters in lavender. Our results, which have enhanced our understanding of EO biosynthesis in plants, will help improve EO yield and composition in lavender and other plants through plant breeding and biotechnology.Arts and Sciences, Irving K. Barber School of (Okanagan)Biology, Department of (Okanagan)Graduat