| 20202168 |
Todd AT, Liu E, Polvi SL, Pammett RT, Page JE: A functional genomics screen identifies diverse transcription factors that regulate alkaloid biosynthesis in Nicotiana benthamiana. Plant J. 2010 Feb 26.
Summary 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 dataset of 3271 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 to 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 compared to vector controls. RNAi silencing led to both reduced nicotine and decreased levels of transcripts 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. |
31(0,1,1,1) |