A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis

Abstract

The PDF1.2 gene of Arabidopsis encoding a plant defensin is commonly used as a marker for characterization of the jasmonate-dependent defense responses. Here, using PDF1.2 promoter-deletion lines linked to the beta-glucoronidase-reporter gene, we examined putative promoter elements associated with jasmonate-responsive expression of this gene. Using stably transformed plants, we first characterized the extended promoter region that positively regulates basal expression from the PDF1.2 promoter. Second, using promoter deletion constructs including one from which the GCC-box region was deleted, we observed a substantially lower response to jasmonate than lines carrying this motif. In addition, point mutations introduced into the core GCC-box sequence substantially reduced jasmonate responsiveness, whereas addition of a 20-nucleotide-long promoter element carrying the core GCC-box and flanking nucleotides provided jasmonate responsiveness to a 35S minimal promoter. Taken together, these results indicated that the GCC-box plays a key role in conferring jasmonate responsiveness to the PDF1.2 promoter. However, deletion or specific mutations introduced into the core GCC-box did not completely abolish the jasmonate responsiveness of the promoter, suggesting that the other promoter elements lying downstream from the GCC-box region may also contribute to jasmonate responsiveness. In other experiments, we identified a jasmonate- and pathogen-responsive ethylene response factor transcription factor, AtERF2, which when overexpressed in transgenic Arabidopsis plants activated transcription from the PDF1.2, Thi2.1, and PR4 (basic chitinase) genes, all of which contain a GCC-box sequence in their promoters. Our results suggest that in addition to their roles in regulating ethylene-mediated gene expression, ethylene response factors also appear to play important roles in regulating jasmonate-responsive gene expression, possibly via interaction with the GCC-box.

Figure 1.

Basal and MeJA-responsive expression of GUS activity from the PDF1.2 promoter deletion lines. A, Average basal GUS activity (fluorescence units per milligram of protein) in each of the PDF1.2 promoter deletion constructs. Each bar represent data from five independent transgenic lines derived from six replicates in two independent experiments. Shaded bars, Plants grown in sterile conditions; black bars, plants grown in soil. B, The PDF1.2 promoter reporter constructs that were placed in the binary vector pBI101.3 used to generate transgenic plant lines. C, Average induction values from each promoter deletion construct in MeJA-treated plants. Induction data were generated from five independent transgenic lines for sterile-grown seedlings, whereas two representative lines were used for soil-grown plants. GUS activity assays were performed on 20 T₂ plants for each transgenic line. Bars indicate se.

Figure 2.

Delineation of enhancer element using quantitative transient expression analysis of the PDF1.2-promoter deletion constructs in Arabidopsis leaves. Relative expression data for each construct represent the mean of GUS/luciferase ratios for at least six separate leaf samples. Bars indicate se. The data shown here are compiled from several independent bombardment experiments that produced similar results.

Figure 3.

A through C, Further confirmation that the GCC-box confers jasmonate responsiveness to the PDF1.2 promoter using quantitative analysis of stably transformed plants. C, Results are presented as average -fold induction values from two to five transgenic lines harboring each PDF1.2 promoter deletion construct. A, The basal expression of GUS from each construct is also given. The mGCC construct is the same as construct P3 but otherwise contains three substitutions in the first (G–T), fourth (G–T), and sixth (C–A) nucleotides (shown in bold) to convert the core GCCGCC box sequence to TCCTCA. B, Construct pIB contains a 20-nucleotide-long sequence including the GCC-box and the surrounding nucleotides from the -270 to -250 of the PDF1.2 promoter linked to a truncated 35S cauliflower mosaic virus (CaMV) promoter. se bars are also shown for each construct.

Figure 4.

A, Expression profiling of selected ERF genes by northern blot in a time-course analysis. Left panel, Expression in response to MeJA; right panel, expression in response to ethylene for each gene. Top and bottom panels represent RNA from control (C) and treated (T) plants, respectively. B, Induction ratios of the Arabidopsis ERF1, AtERF2, and PDF1.2 genes obtained by real-time quantitative reverse transcriptase (RT)-PCR after MeJA treatment.

Figure 5.

Arabidopsis ERF genes respond to fungal inoculation. A, Macroarray analysis showing -fold induction of four Arabidopsis ERFs after challenge by incompatible fungal pathogen A. brassicicola in inoculated (local) leaves and uninoculated (systemic) leaves of the same plant. Samples were collected 5 h after inoculation. Bars show -fold induction of each gene compared with the equivalent tissue from inoculated plants. Expression was determined using ImageQuant software, and values were adjusted against β-tubulin, as a loading control. B, Induction ratios (local) of the Arabidopsis AtERF2 and PDF1.2 genes obtained by real-time quantitative RT-PCR after inoculation with A. brassicicola.

Figure 6.

Overexpression of AtERF2 activates defense gene expression in Arabidopsis. Shown here are -fold expression values of the AtERF2, PDF1.2, Thi2.1, and PR4 genes in two independent stably transformed transgenic T₂ lines containing the 35S-AtERF2 transgene over those observed in untransformed control plants. Error bars are also shown for each line.