Role of ERF transcription factors in jasmonate-inducible regulation of nicotine biosynthesis in tobacco

Plants often respond to biotic and abiotic stresses with large-scale transcriptional reprogramming of basic metabolism. Wounding and herbivore attack results in the synthesis and accumulation of jasmonates (such as jasmonic acid (JA) methyl jasmonate (MeJA)) followed by activation of a signal transduction pathway leading to the transcriptional activation or repression of JA-dependent genes. In wild Nicotiana species and cultivated tobacco ( Nicotiana tabacum L.) elevated levels of JA result in activation of the biosynthesis of nicotine and related pyridine alkaloids. The biosynthesis of nicotine begins with the polyamine putrescine that is converted to N-methylputrescine by an enzyme known as putrescine N-methyltransferase (NtPMT), key regulatory enzyme in nicotine synthesis. Transcriptional regulation of the NtPMT expression is one of the most important points in nicotine biosynthesis. Here, I have characterized cis-regulatory elements in the NtPMT1a promoter that are necessary and/or sufficient for JA-induced NtPMT1a transcription. I have demonstrate that novel tripartite motif, termed the GAG motif, consisting of a G box-like element, an AT-rich region, and a GCC box-like element is required for maximum NtPMT1a transcription after JA treatment. I have shown that the G box-like element and GCC-like box are necessary and sufficient to direct MeJA-responsive NtPMT1a expression leading to nicotine formation. I have further demonstrated that these elements are the binding sites for two major types of transcription factors (TF), MYC and ERF, respectively. I have shown that NtERF32, a member of Group IX within the tobacco ERF gene family, is rapidly induced by MeJA and specifically binds to the GCC-like box within the GAG motif. Ectopic overexpression of NtERF2 increases expression of several genes in nicotine biosynthetic pathway including NtPMT1a in vivo and elevates total alkaloid contents, whereas RNAi-mediated knockdown of NtERF32 reduces the mRNA levels of multiple genes in the nicotine biosynthetic pathway including NtPMT1a and quinolinate phosphoribosyltransferase (NtQPT2 ), and lowers nicotine and total alkaloid levels. Based on these studies I conclude the GAG motif represents a molecular switch that is responsible for activating transcription of the NtPMT genes and that NtERF32 is an important part of the early molecular switch that activates expression of the nicotine biosynthetic genes.