Tobacco MYC2 Regulates Jasmonate-Inducible Nicotine Biosynthesis Genes Directly and By Way of the NIC2-Locus ERF Genes

Abstract

In Arabidopsis, the MYC2-family basic helix-loop-helix transcription factors mediate transcriptional regulation of jasmonate-responsive genes, and their transcriptional activities are suppressed by physical interactions with jasmonate-ZIM domain (JAZ) proteins. Jasmonate-inducible nicotine formation in Nicotiana plants has been shown to be suppressed by tobacco JAZ proteins, and be regulated by both MYC2-related and NIC2-locus ethylene response factor (ERF) transcription factors. We here show that tobacco MYC2 (NtMYC2) recognizes the G-box sequences, 5'-CAC(G/A)T(G/T)-3', found in the proximal promoter regions of several nicotine biosynthesis genes, including Putrescine N-Methyltransferase 2 (PMT2) and Quinolinate Phosphoribosyltransferase 2 (QPT2). Transient transactivation assays using cultured tobacco cells showed that NtMYC2 and NIC2-locus ERF189 additively activated the PMT2 and QPT2 promoters depending on their cognate binding sites. RNA interference (RNAi) silencing of NtMYC2 in tobacco hairy roots strongly decreased transcript levels of jasmonate-responsive structural genes, including those involved in nicotine biosynthesis, as well as the NIC2-locus ERF genes. Conversely, ERF189 was not required for the expression of NtMYC2. NtMYC2, but not ERF189, interacted with tobacoo JAZs in a yeast two-hybrid assay. These results indicate that NtMYC2 controls nicotine biosynthesis genes in two combinatorial ways, by directly binding the G-box in the target promoters and by up-regulating the NIC2-locus ERF genes.

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

“…The transcript levels of ERF189 were similarly reduced to less than 40% of control plants in the RNAi roots, whereas those of MYC2 were not significantly affected. Because MYC2 is regulated not only transcriptionally but also posttranslationally, by its physical interaction with JAZ repressors (Chini et al, 2007), we analyzed the expression of the MYC2-regulated PROTEINASE INHIBITOR II (PI-II; Balandin et al, 1995;Shoji and Hashimoto, 2011c) to establish whether the transcriptional activator activity of MYC2 was affected in the silenced lines. PI-II expression was similar in the NUP1-RNAi and control lines, indicating that NUP1 regulates ERF189 but not MYC2.…”

“…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).…”

“…(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). At least seven ERF genes encoding a subclade of the tobacco group IXa subfamily are clustered at the NIC2 locus, also known as the B locus (Legg and Collins, 1971), and all of these genes are deleted in the lownicotine tobacco nic2 mutant (Shoji et al, 2010).…”

“…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).…”

“…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).…”