Methyl jasmonate induced expression of the tobacco putrescine N-methyltransferase genes requires both G-box and GCC-motif elements

Abstract

Putrescine N-methyltransferase (PMT) catalyzes the first committed step of nicotine biosynthesis, converting putrescine into N-methylputrescine. A variety of chemical, environmental, and developmental cues have been implicated in its regulation. Here we have examined the differential expression of beta-glucuronidase (GUS) transgenes under the control of the transcriptional regulatory sequences of four distinct members of the NtPMT gene family from tobacco (Nicotiana tabacum L.). BY-2 cell cultures expressing various NtPMT promoter-GUS constructs were examined for their response to treatment with various combinations of methyl jasmonate (MeJA), auxin (AUX), and ethylene (ETH). All four NtPMT gene promoters examined were inducible by MeJA, although the extent of the induction varied dramatically, with the NtPMT1a promoter being the most responsive. High AUX levels in the cell growth media repressed NtPMT::GUS transgene expression and inhibited their MeJA-induced transcription. Treatment of BY-2 cells with ETH alone did not result in a significant alteration in NtPMT::GUS expression. However, similar to AUX, ETH treatment led to the suppression of MeJA-induced transcription. Detailed deletion analysis of the NtPMT1a gene promoter showed that as little as 111 bp upstream of the transcriptional start site were sufficient to confer MeJA-responsiveness. Deletion of a conserved G-box element (GCACGTTG) at -103 to -96 bp completely abolished MeJA-responsiveness. Further mutagenesis studies revealed that in addition to a functional G-box, MeJA-responsiveness of the NtPMT1a promoter also required a TA-rich region and a GCC-motif (TGCGCCC) located at -80 to -69 bp and -62 to -56 bp relative to the start site, respectively. A synthetic G-box tetramer (4 X syn G-box) fused to a -83 bp fragment from the NtPMT1a promoter (containing the TA-rich region, GCC-box, and TATA-box) displayed a 30-fold induction by MeJA treatment, whereas when the 4 X syn G-box was fused to a minimal (-46 bp) promoter fragment derived from the CaMV 35S gene, no induction by MeJA treatment was detected. Our results indicate that multiple intersecting signal transduction pathways and different transcriptional regulatory factors are involved in mediating JA-responsiveness of NtPMT expression in tobacco.

Clustered Transcription Factor Genes Regulate Nicotine Biosynthesis in Tobacco

“…The 236-bp promoter region of PMT2 faithfully reported the spatio-temporal expression patterns of PMT and contained two functionally important cis-elements, a G-box element and a GCC-box element (Shoji et al, 2000b;Oki and Hashimoto, 2004;Xu and Timko, 2004). We first tested a DNA probe that covered the entire promoter region upstream of the predicted TATA box (P0; 2236 to -105, numbered from the first ATG) and three overlapping 30-bp probes within P0 (P1, 2182 to 2153; P2, 2162 to 2133; and P3, 2142 to 2113) ( Figure 8A).…”

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

“…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.…”

Molecular regulation of nicotine biosynthesis

“…PMT and other genes involved in nicotine formation show basal low expression in the root of undamaged tobacco plants, but wounding and jasmonate treatment to the leaf increase the gene expression levels 3-4 folds (Shoji et al 2000;Sinclair et al 2000). Functional analysis of the tobacco PMT promoter revealed that jasmonate-induced expression requires G-box and GCC-motif elements in the proximal region of the PMT promoter, which are often found in jasmonate-responsive promoters (Oki and Hashimoto 2004;Xu and Timko 2004). Ethylene supplied simultaneously with jasmonic acid effectively abrogated jasmonate activation of the PMT and A622 promoters.…”