title: A functional genomics screen identifies diverse transcription factors that regulate alkaloid biosynthesis in Nicotiana benthamiana.
abstract: Biosynthesis of the alkaloid nicotine in Nicotiana species is induced by insect damage and jasmonate application. To probe the transcriptional regulation of the nicotine pathway, we constructed two subtracted cDNA libraries from methyl jasmonate (MeJA)-treated Nicotiana benthamiana roots directly in a viral vector suitable for virus-induced gene silencing (VIGS). Sequencing of cDNA inserts produced a data set of 3271 expressed sequence tags (ESTs; 1898 unigenes), which were enriched in jasmonate-responsive genes, and included 69 putative transcription factors (TFs). After a VIGS screen to determine their effect on nicotine metabolism, six TFs from three different TF families altered constitutive and MeJA-induced leaf nicotine levels. VIGS of a basic helix-loop-helix (bHLH) TF, NbbHLH3, and an auxin response factor TF, NbARF1, increased nicotine content compared with control plants; silencing the bHLH family members, NbbHLH1 and NbbHLH2, an ethylene response factor TF, NbERF1, and a homeobox domain-like TF, NbHB1, reduced nicotine levels. Transgenic N. benthamiana plants overexpressing NbbHLH1 or NbbHLH2 showed increased leaf nicotine levels compared with vector controls. RNAi silencing led to both reduced nicotine and decreased levels of transcript encoding of enzymes of the nicotine pathway. Electrophoretic mobility shift assays showed that recombinant NbbHLH1 and NbbHLH2 directly bind G-box elements identified from the putrescine N-methyltransferase promoter. We conclude that NbbHLH1 and NbbHLH2 function as positive regulators in the jasmonate activation of nicotine biosynthesis.
authors.affiliation: Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9.
authors.forename: Andrea T
authors.initials: AT
authors.lastname: Todd
authors.forename: Enwu
authors.initials: E
authors.lastname: Liu
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Polvi
authors.forename: Robert T
authors.initials: RT
authors.lastname: Pammett
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Page
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D003517:Cyclopentanes / Q000378:metabolism; D018744:DNA, Plant / Q000235:genetics; D020224:Expressed Sequence Tags; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D023281:Genomics; D009538:Nicotine / Q000096:biosynthesis*; D054883:Oxylipins / Q000378:metabolism; D010802:Phylogeny; 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; D034622:RNA Interference; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D003517:Cyclopentanes; D018744:DNA, Plant; D009538:Nicotine; D054883:Oxylipins; D010937:Plant Growth Regulators; D010940:Plant Proteins; C011006:jasmonic acid