Abstract
Nicotine biosynthesis in tobacco (Nicotiana tabacum L.) is highly regulated by jasmonic acid (JA). Two nuclear loci, A and B (renamed NIC1 and NIC2) have been identified that mediate JA-inducible nicotine formation and total alkaloid accumulation. NIC2 was recently shown to be a cluster of seven genes encoding Apetala2/Ethylene-Response Factor (AP2/ERF)-domain transcription factors (TFs) in Group IX of the tobacco AP2/ERF family. Here we report the characterization of several NtERF TF genes that are not within the NIC2 locus, but required for methyl JA (MeJA)—induced nicotine biosynthesis. Expression of NtERF1, NtERF32, and NtERF121 is rapidly induced (<30 min) by MeJA treatment. All three of these TFs specifically bind the GCC box-like element of the GAG motif required for MeJA-induced transcription of NtPMT1a, a gene encoding putrescine N-methyltransferase, the first committed step in the synthesis of the nicotine pyrrolidine ring. Ectopic overexpression of NtERF32 increases expression of 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. We conclude that NtERF32 and related ERF genes are important non-NIC2 locus associated transcriptional regulators of nicotine and total alkaloid formation.
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Acknowledgments
We thank Jennifer Murphy, Matthew Kastner, Logan Breaud, Ryan Thompson and Ashley Taylor for technical support. We also thank Alain Goossens and Sofie Tilleman for providing the tobacco BY-2 cell culture line, and Chengalrayan Kudithipudi and Alec Hayes for helpful comments. The authors are responsible for the content of the work and distribution of materials integral to the findings presented in this article. This work was supported in part by a grant from Altria Client Services, Inc.
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Sears, M.T., Zhang, H., Rushton, P.J. et al. NtERF32: a non-NIC2 locus AP2/ERF transcription factor required in jasmonate-inducible nicotine biosynthesis in tobacco. Plant Mol Biol 84, 49–66 (2014). https://doi.org/10.1007/s11103-013-0116-2
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DOI: https://doi.org/10.1007/s11103-013-0116-2