A functional genomics screen identifies diverse transcription factors that regulate alkaloid biosynthesis in Nicotiana benthamiana

Abstract

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 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.

APETALA2/ETHYLENE RESPONSE FACTOR and basic helix–loop–helix tobacco transcription factors cooperatively mediate jasmonate‐elicited nicotine biosynthesis

“…Twenty-four hours after transfection with hairpin (hp) gene silencing constructs, BY-2 protoplasts were elicited with MeJA and fLUC reporter activity was measured after incubation for another 24 h. The ProNsPMT2 and ProQPRT2 activities were slightly and strongly reduced, respectively, upon transfection with the ORC1 hp construct (Figure 5a), suggesting that NIC2 AP2/ERF factors including ORC1 are involved in the MeJA-mediated activation of nicotine biosynthesis in tobacco and consistent with the effects observed by RNA interference (RNAi)-mediated suppression of the NIC2 AP2/ ERF genes in tobacco plants (Shoji et al, 2010). The action of ORC1 may be invoked, at least in part, through MeJA-mediated transcriptional induction of the ORC1 gene (Goossens et al, 2003), together with that of the other NIC2 ERF and the MYC2-like bHLH factors (Rushton et al, 2008;Shoji et al, 2010;Todd et al, 2010). In addition, however, we noted that in transient expression assays, transactivation of ProNsPMT2 and ProQPRT2 by ORC1 was augmented with MeJA elicitation (Figure 5b), suggesting that the JA signal enhances the transactivation potential of the ORC1 protein.…”

“…Yet it is striking that for virtually none of the bHLH nor the NIC2 ERF activators identified so far, expression is exclusively confined to the roots, and that for many the corresponding transcripts are even encountered in all organs tested (Todd et al, 2010;Shoji et al, 2010;this study). This observation stands in sharp contrast to the almost exclusive root-specific pattern detected for many of the structural genes involved in nicotine biosynthesis.…”

“…The phylogeny of the JA-responsive AP2/ERF and bHLH factors (Rushton et al, 2008), and the outcome of various functional screens in which multiple isoforms of both TF families were shown to positively regulate nicotine biosynthesis (De Sutter et al, 2005;Todd et al, 2010;Shoji et al, 2010), suggests that tobacco plants have developed a daunting repertoire of transcriptional regulators to control their secondary metabolism. Yet it is striking that for virtually none of the bHLH nor the NIC2 ERF activators identified so far, expression is exclusively confined to the roots, and that for many the corresponding transcripts are even encountered in all organs tested (Todd et al, 2010;Shoji et al, 2010;this study).…”

“…NbbHLH1 is an interesting candidate: it is homologous to the MYC2 bHLH TF, it is induced by MeJA and it binds the G-box element of the PMT promoter in N. benthamiana, thereby relaying JA activation of nicotine biosynthesis (Todd et al, 2010). The full-length open reading frame (ORF) of the NbbHLH1 factor was transferred into the appropriate overexpression vector and tested for transient transactivation of the minimal PMT1 and the QPRT2 promoters.…”

“…These genes include those coding for ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and putrescine N-methyltransferase (PMT), all three essential for the synthesis of the pyrroline ring, as well as aspartate oxidase (AO) and quinolinate phosphoribosyltransferase (QPRT), essential for the synthesis of the pyridine ring. Recently, a functional genomics screen identified two basic helix-loop-helix (bHLH) transcription factors (TFs) that function as positive regulators in the JA activation of nicotine biosynthesis in Nicotiana benthamiana (Todd et al, 2010). The NbbHLHs are related to the Arabidopsis (Arabidopsis thaliana) bHLH transcriptional activators MYC2, MYC3 and MYC4 that are essential elements of the conserved plant JA signalling module (Lorenzo et al, 2004;Fonseca et al, 2009;Ferná ndez-Calvo et al, 2011).…”