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Cloning and functional characterization of genes involved in the biosynthesis and secretion of essential oil constituents of Lavandula Demissie, Zerihun Abebe


Several members of the genus Lavandula produce valuable essential oils (EOs) that are primarily constituted of regular and irregular monoterpenes, the C₁₀ class of terpenes. We isolated over 22,000 ESTs from leaves, flowers and glandular trichomes of L. angustifolia and L. x intermedia species to facilitate the discovery of genes regulating the biosynthesis and secretion of EOs. In this study, we identified and studied genes involved in regular and irregular monoterpene biosythesis and secretion. One of these genes, the L. x intermedia lavandulyl pyrophosphate synthase (LiLPPS), catalyzes the head–to–middle condensation of two DMAPP units to produce lavandulyl pyrophosphate in vitro. The apparent Km and kcat of LiLPPS were 208 µM and 0.1 s -¹, respectively. LiLPPS is a novel cis prenyl transferase family member and its identification elucidated the biosynthetic origin of irregular monoterpenes in Lavandula EOs. L. angustifolia β-phellandrene synthase (LaβPHLS) and L. x intermedia 1,8-cineole synthase (LiCINS) transformed geranyl pyrophosphate primarily to β-phellandrene and 1,8-cineole, respectively. The apparent Km and kcat of LaβPHLS were 6.6 µM and 1.8 x 10-² s-¹ while those of LiCINS were 5.8 µM and 8.8 x 10-³ s-¹, respectively. LaβPHLS transcripts were highly abundant in young leaves where β-phellandrene is actively produced. LiCINS mRNA levels paralleled the 1,8-cineole content in flowers of three lavender species, and developmental stages of L. x intermedia inflorescence indicating that the production of 1,8 cineole is likely controlled through transcriptional regulation of LiCINS. The genomic CINS of Lavandula species had identical exon-intron architecture and coding sequences, but intron analysis suggests that LiCINS was most likely inherited from L. latifolia. We also identified the L. angustifolia ABCB1 (LaABCB1) that mediates the efflux of vinblastine when expressed in Xenopus laevis oocytes. Treating oocytes with an oxidative uncoupler, ATPase inhibitor, classical ABC blocker and selected Lavandula de novo monoterpenes inhibited efflux mediated by LaABCB1. LaABCB1 transcripts were constitutively expressed in the EO producing tissues of L. angustifolia, L. latifolia and L. intermedia plants. Both the expression pattern and the in vitro activity of LaABCB1 indicate that it plays a role in monoterpene trafficking in the lavender oil glands.

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