We cloned eight transcription factors that activate lavender monoterpene synthase promoters.
In this study, we employed the Yeast One-Hybrid (Y1H) assay system to identify transcription factors that control promoters for two Lavandula × intermedia monoterpene synthase genes, linalool synthase (LiLINS) and 1,8-cineole synthase (LiCINS). The bait sequences used in the assay were either a 768-bp LiLINS, or a 1087-bp LiCINS promoter. The prey included proteins expressed in L. × intermedia floral tissue. The assay identified 96 sequences encoding proteins that interacted with one or both promoters. To explore the nature of this interaction, 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 (TFs) predicted to control these promoters. Six TFs induced expression from both promoters, two activated LiCINS promoter alone, and two did not induce expression from either promoter. The TFs identified in this study belong to various groups including those containing conserved domains typical of MYB, bZIP, NAC, GeBP and SBP-related proteins.
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This work was supported through grants and/or in-kind contributions to SSM by UBC, and the Natural Sciences and Engineering Research Council of Canada (NSERC).
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Electronic supplementary material
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Biosynthesis of linalool and 1,8-cineole in L. × intermedia. IPP: isopentenyl diphosphate, DMAPP: dimethylallyl diphosphate and GPP: geranyl diphosphate (DOCX 47 kb)
a) Schematic representation of promoter–GUS fusion constructs used in this study. LiCINS promoter (LiCINSp) and LiLINS promoter (LiLINSp) were cloned upstream of the gusA gene (encoding GUS) within the T-DNA of the pCambia1391z plant transformation vector. LB: left T-DNA border and RB: right T-DNA border. HygR: Hygromycin resistance gene. KanR: Kanamycin resistance gene (Neomycin phosphotransferase II, NPTII). b) Schematic representation of the CaMV353-TF constructs used in this study. In these constructs individual TFs were cloned downstream of a tandem CaMV 35S promoter in pGA482 plant expression vector. KanR: Kanamycin resistance gene (Neomycin phosphotransferase II, NPTII) (DOCX 25 kb)
. List of primers used for cloning of promoters and transcription factors from L. × intermedia (DOCX 14 kb)
. Spectrophotometric readings (OD595) corresponding to GUS activity in N. benthamiana plants harboring either LiLINSp:GUS or LiCINSp:GUS construct, co-expressing L. × intermedia TFs from the pGA482 vector (See Fig S2b). Results correspond to data reported in Table 1 and Fig. 2 (DOCX 15 kb)
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Sarker, L.S., Adal, A.M. & Mahmoud, S.S. Diverse transcription factors control monoterpene synthase expression in lavender (Lavandula). Planta 251, 5 (2020). https://doi.org/10.1007/s00425-019-03298-w