Clustered Transcription Factor Genes Regulate Nicotine Biosynthesis in Tobacco  

Abstract

Tobacco (Nicotiana tabacum) synthesizes nicotine and related pyridine alkaloids in the root, and their synthesis increases upon herbivory on the leaf via a jasmonate-mediated signaling cascade. Regulatory NIC loci that positively regulate nicotine biosynthesis have been genetically identified, and their mutant alleles have been used to breed low-nicotine tobacco varieties. Here, we report that the NIC2 locus, originally called locus B, comprises clustered transcription factor genes of an ethylene response factor (ERF) subfamily; in the nic2 mutant, at least seven ERF genes are deleted altogether. Overexpression, suppression, and dominant repression experiments using transgenic tobacco roots showed both functional redundancy and divergence among the NIC2-locus ERF genes. These transcription factors recognized a GCC-box element in the promoter of a nicotine pathway gene and specifically activated all known structural genes in the pathway. The NIC2-locus ERF genes are expressed in the root and upregulated by jasmonate with kinetics that are distinct among the members. Thus, gene duplication events generated a cluster of highly homologous transcription factor genes with transcriptional and functional diversity. The NIC2-locus ERFs are close homologs of ORCA3, a jasmonate-responsive transcriptional activator of indole alkaloid biosynthesis in Catharanthus roseus, indicating that the NIC2/ORCA3 ERF subfamily was recruited independently to regulate jasmonate-inducible secondary metabolism in distinct plant lineages.

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

“…These findings are also in agreement with those of Shoji et al (2010), who showed that gain-and loss-of-function of another clade 2-1 NIC2-locus ERF, i.e. ERF189, increased and decreased significantly the accumulation of nicotine in transgenic tobacco hairy roots, respectively.…”

“…These NIC2-locus ERF TFs are close homologues of the C. roseus ORCA3 and showed both functional redundancy and divergence in their capacity to regulate nicotine biosynthesis. Similar to the involvement of ORCA3 in TIA biosynthesis, NIC2-locus ERFs activate most of the known structural genes in the tobacco nicotine biosynthesis pathway and bind a GCC motif in the PMT promoter (Shoji et al, 2010). In agreement with these findings, we previously showed that two JA-inducible AP2/ERF TFs from tobacco, ORC1 and JAP1, positively regulate the PMT promoter in a protoplast-based transient expression assay (Goossens et al, 2003;De Sutter et al, 2005).…”

“…In agreement with these findings, we previously showed that two JA-inducible AP2/ERF TFs from tobacco, ORC1 and JAP1, positively regulate the PMT promoter in a protoplast-based transient expression assay (Goossens et al, 2003;De Sutter et al, 2005). ORC1 and JAP1 both belong, similar to all the NIC2 AP2/ERFs, to group IX of ERF genes, and are also known as ERF221 (one of the NIC2 AP2/ERFs in clade 2-1) and ERF10 (clade 1), respectively (Rushton et al, 2008;Shoji et al, 2010).…”

“…In fact, it turned out that the NIC2 locus, one of the long-sought regulatory NIC loci that positively regulate nicotine biosynthesis in tobacco, comprised at least seven clustered AP2/ ERF encoding genes (Shoji et al, 2010). These NIC2-locus ERF TFs are close homologues of the C. roseus ORCA3 and showed both functional redundancy and divergence in their capacity to regulate nicotine biosynthesis.…”

Elicitation of tobacco alkaloid biosynthesis by disrupted spores and filtrate of germinating spores of the arbuscular mycorrhizal fungiGlomus etunicatum

“…genes in each step (Shoji et al 2010). In our experiments, we chose to analyze the expression of the genes coding for PMT, A622, and CYP82E4 because of the availability of a known gene sequence in the public databases and also due to their key location in different branches of the pyridine alkaloids biosynthesis pathway in tobacco.…”