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

Abstract

SUMMARYTranscription 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.

DNA-binding and transcriptional activation properties of tobacco <i>NIC2</i>-locus ERF189 and related transcription factors

“…Decreased expression or dominant suppression of the NIC2-locus ERFs resulted in considerably lower transcript levels of nicotine biosynthesis genes and of nicotine levels in transgenic Nicotiana roots, whereas their overexpression gave the opposite effects (Shoji et al 2010;Todd et al 2011). Moreover, these ERF genes directly activated the transcription of PMT and QPT by recognizing GCC-box-like elements residing in their promoters (De Boer et al 2011;Shoji et al 2010;Shoji and Hashimoto 2011b). Interestingly, the tobacco NIC2-locus ERFs are closely related to ORCA3 (OCTADECANOID-RESPONSIVE Catharanthus AP2) from Catharanthus roseus, which positively controls a part of the jasmonate-inducible terpenoid indole alkaloid pathway (van der Fits and Memelink 2000), implying that the NIC2/ORCA3 ERF subfamily was recruited independently to regulate jasmonate-inducible secondary metabolism in distinct plant lineages…”

Smoking out the masters: transcriptional regulators for nicotine biosynthesis in tobacco

“…Over-expression of a certain member of NIC2-locus ERF genes increased alkaloid level dramatically in the low-nicotine nic1nic2 mutant and several-fold in wild-type tobacco, which accumulates nicotine to a high level even without the over-expression (De Boer et al 2011;Shoji et al 2010). Conversely, loss-of-function of NIC2-locus ERFs, achieved by collective down-regulation of the genes either by RNA interference or by expression of the ERFs under the control of a dominant-repressive motif, drastically decreased the alkaloid content (Shoji et al 2010).…”

“…As revealed for PMT and QPT2 genes, JA-mediated induction of these genes depends on two distinct cis-elements in their proximal promoter regions, the G box and GCC-like box (Oki and Hashimoto 2004;Xu and Timko 2004), which are recognized by MYC2 and NIC2-locus ERFs, respectively (De Boer et al 2011;Shoji et al 2010;Shoji and Hashimoto 2011b;Shoji and Hashimoto 2011c;Todd et al 2010;Zhang et al 2012) (Figure 3). These transcription factors are also induced at the transcript level by JA and cooperatively activate their target promoters when expressed transiently in tobacco cells.…”

Tobacco Nicotine Uptake Permease Regulates the Expression of a Key Transcription Factor Gene in the Nicotine Biosynthesis Pathway

“…Although these ERF homologs are likely somewhat functionally redundant, ERF189 and its closest homolog ERF199 are thought to be the major regulators of the nicotine biosynthetic pathway; overexpression of ERF189 upregulates the expression of structural genes of this pathway to a greater extent than does that of the other ERF members examined (Shoji et al, 2010), and the expression patterns of ERF189 and ERF199 overlap with those of genes in the nicotine biosynthetic pathway Shoji et al, 2010). In addition to containing ERFbinding sites, the promoters of nicotine biosynthesis genes contain functional binding sites for MYC2, which are required for their jasmonate-inducible expression (De Boer et al, 2011;Shoji and Hashimoto, 2011c). Thus, tobacco MYC2 has dual roles in nicotine biosynthesis; it positively regulates the expression of NIC2-locus ERFs and, together with these ERFs, directly binds to and activates the promoters of structural genes involved in nicotine biosynthesis (Shoji and Hashimoto, 2011c).…”

“…The basic helix-loop-helix Leu zipper-type transcription factor MYC2 is released from JAZ-mediated repression upon jasmonate perception by COI1, and activates nicotine biosynthesis genes in Nicotiana spp. (Todd et al, 2010;De Boer et al, 2011;Shoji and Hashimoto, 2011c;Zhang et al, 2012). The evolutionarily conserved MYC2 transcription factor induces the expression of the master regulator genes of nicotine biosynthesis, which encode the NICOTINE2 (NIC2)-locus ethylene response factor (ERF)-type transcription factors (Shoji and Hashimoto, 2011c).…”