title: APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis.
abstract: Transcription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix-loop-helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.
authors.affiliation: School of Biological Sciences, Monash University, Melbourne, Vic. 3800, Australia.
authors.forename: Kathleen
authors.initials: K
authors.lastname: De Boer
authors.forename: Sofie
authors.initials: S
authors.lastname: Tilleman
authors.forename: Laurens
authors.initials: L
authors.lastname: Pauwels
authors.forename: Robin
authors.initials: R
authors.lastname: Vanden Bossche
authors.forename: Valerie
authors.initials: V
authors.lastname: De Sutter
authors.forename: Rudy
authors.initials: R
authors.lastname: Vanderhaeghen
authors.forename: Pierre
authors.initials: P
authors.lastname: Hilson
authors.forename: John D
authors.initials: JD
authors.lastname: Hamill
authors.forename: Alain
authors.initials: A
authors.lastname: Goossens
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D030008:Catharanthus / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D009538:Nicotine / Q000096:biosynthesis*; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D054883:Oxylipins / Q000378:metabolism*; D010766:Phosphorylation; D010937:Plant Growth Regulators / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D003517:Cyclopentanes; D009538:Nicotine; D051738:Origin Recognition Complex; D054883:Oxylipins; D010937:Plant Growth Regulators; D010940:Plant Proteins; C011006:jasmonic acid