Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis

Abstract

Jasmonates (JA) are important regulators of plant defense responses that activate expression of many wound-induced genes including the tomato proteinase inhibitor II (pin2) and leucine aminopeptidase (LAP) genes. Elements required for JA induction of the LAP gene are all present in the -317 to -78 proximal promoter region. Using yeast one-hybrid screening, we have identified the bHLH-leu zipper JAMYC2 and JAMYC10 proteins, specifically recognizing a T/G-box AACGTG motif in this promoter fragment. Mutation of the G-box element decreases JA-responsive LAP promoter expression. Expression of JAMYC2 and JAMYC10 is induced by JA, with a kinetics that precedes that of the LAP or pin2 transcripts. JAMYC overexpression enhanced JA-induced expression of these defense genes in potato, but did not result in constitutive transcript accumulation. Using footprinting assays, an additional protected element was identified, located directly adjacent to the T/G-box motif. Mutation of this element abolishes JA response, showing that recognition of this duplicated element is also required for gene expression. Knockout mutants in the AtMYC2 homolog gene of Arabidopsis are insensitive to JA and exhibit a decreased activation of the JA-responsive genes AtVSP and JR1. Activation of the PDF1.2 and b-CHI, ethylene/JA-responsive genes, is, however, increased in these mutants. These results show that the JAMYC/AtMYC2 transcription factors function as members of a MYC-based regulatory system conserved in dicotyledonous plants with a key role in JA-induced defense gene activation.

Figure 1.

Amino acid sequences and patterns of expression of the JAMYC bHLH-Zip transcription factors. (A) JAMYC2 and JAMYC10 deduced amino acid sequences. The bHLH-Leu Zip domain is shown as a gray box, and the acidic region is underlined. The start point of the partial clones isolated by one hybrid screening is indicated as an arrowhead. (B) Northern analysis of tomato leaves induced by wounding or application of MeJA. (C) Pattern of tissue-specific expression at 30 min of MeJA treatment. Each lane was loaded with 30 μg of total RNA. Blots were hybridized with probes corresponding to the 3′-noncoding regions of JAMYC2 or JAMYC10, or to the LAP cDNA clone as indicated. Equal loading was verified by EtBr staining of the gel.

Figure 2.

Characterization of the JAMYC DNA recognition motif. (A) Hydroxyl radical interference assays identify a T/G-box element in the -125LAP promoter fragment as the recognition site for the His-C2 protein. The -125LAP probe was asymmetrically labeled with ³²P-dCTP and treated with hydroxyl radical before incubation with the purified protein. Free probe (N) and retarded bands (S1 and S2) were purified by PAGE and separated on a sequencing gel. A G + A Maxam reaction (GA) on the same fragment was loaded as marker. Protected residues are shown as a black box on the nucleotide sequence on the left. Identical results were obtained for both His-C2 and His-C10 proteins. (B) Binding activity of His-C2 protein in gel retardation assays. End-labeled LAP^T/G-BOX double-stranded oligonucleotides containing an intact T/G-box motif were used as probe. The oligonucleotide pairs LAP^T/G-BOX, with an intact motif, or LAP^t/g-box, in which the T/G-box motif was mutagenized, were used as competitors for binding. Oligonucleotides were added in 50- to 500-fold excess for competition. (C) JAMYC2 recognizes a G-box motif required for JA induction of the pin2 promoter. Binding of the His-C2 protein to the LAP^T/G-BOX probe was competed by incubation with a 50- to 500-fold excess of the unlabeled PIN2^G-BOX (intact G-box) or PIN2^g-box (mutated G-box) oligonucleotide pairs. (D) Site-directed mutagenesis of the LAP promoter T/G-box motif. Transgenic potato plants carrying either the -317LAP or the -317LAP^t/g-box constructs were grown on soil for 4 wk, and GUS activity was measured in noninduced leaves or in leaves treated for 24 h with 50 μM MeJA. Dots represent the values of GUS activity measured in each individual transformant. (E) DNA binding and trans-activation activity of the JAMYC2 and JAMYC10 transcription factors in BY2 cells. The reporter constructs -317LAP:GUS and -317LAP^t/g-box:GUS, including intact or mutagenized copies of the T/G-box motif, were cotransfected with the effector plasmids 2x35S:JAMYC2 (JAMYC2) and 2X35S:JAMYC10 (JAMYC10), expressing the JAMYC proteins under control of the 2x35S promoter. The same vector without insert (2x35S) was used as a control. A 35S:Luciferase plasmid was also used as internal control to normalize cotransfection efficiency. Histograms represent the mean GUS/LUC values for each set of replicates.

Figure 3.

Potato JAMYC2 and JAMYC10 overexpressers show an enhanced induction of the LAP and pin2 genes upon MeJA treatment. (A) LAP and pin2 gene expression in wild-type controls (control) and the 35S:JAMYC2 and 35S:JAMYC10 transgenic potato plants after 6 h of treatment with increasing concentrations of MeJA. Each lane was loaded with 20 μg of total RNA. Blots were hybridized with the LAP, pin2, and JAMYC2 or JAMYC10 probes as indicated. Equal loading was verified by EtBr staining of the gel. (B) Histograms represent the levels of expression of LAP and pin2 as quantified by densitometric scanning of the blot.

Figure 4.

DMS in vivo footprinting of the proximal LAP promoter region identified two G residues in a GAGTA repeated motif that are protected from methylation and shown to be required for JA response. (A) DMS in vivo footprinting of the -270 to -3 LAP promoter region. Control tomato leaves (control) and leaves of plants treated with 50 μM MeJA (8, 16, and 24 h) were directly incubated with DMS. DNA was extracted from these leaves, cleaved with piperidine, and subjected to LmPCR. Amplification of in vitro methylated DNA (naked) is also included as control. Protected G residues are indicated by filled circles and residues with enhanced reactivity (hypersensitivity) by open circles. The position of both protected and hyperreactive residues is indicated on the left. (B) Deletion analysis and site-directed mutagenesis of the proximal LAP promoter region. Transgenic potato plants carrying the -317LAP, -125LAP, -78LAP, and -317LAP^gagta constructs fused to the uidA gene were grown on soil for 4 wk, and GUS activity was measured in noninduced leaves or in leaves treated for 24 h with 50 μM MeJA. The histogram shows the average values of GUS activity detected in nontreated and MeJA-treated plants. (C) Nucleotide sequence of the proximal LAP promoter. Hypermethylated G residues, GAGTA repeats, and the T/G-box motif are shown in the -125LAP promoter context. Mutations introduced to yield construct -317LAP^gagta are indicated.

Figure 5.

The Arabidopsis AtMYC2 gene shares strong homology with JAMYC2 and JAMYC10 and is rapidly induced by JA. (A) Phylogenetic tree generated by CLUSTALW alignment of the tomato JAMYC2 and JAMYC10 proteins and related bHLH proteins from Arabidopsis. (B) RNA blot analysis of the induction of AtMYC2 by MeJA. Total RNA was isolated from 3-week-old Col-0 plants grown in soil after different times of MeJA application, and 30 μg was loaded per line. Equal loading was verified by rRNA visualization after EtBr staining.

Figure 6.

Arabidopsis lines carrying an insertion within the AtMYC2 gene are insensitive to JA and exhibit altered JA-regulated gene expression. (A) Northern analysis of the atmyc2-1 (SALK_040500) and atmyc2-2 (SALK_083483) mutants. Fifteen micrograms of total RNA from Col-0 and atmyc2 mutant seedlings noninduced or treated for 8 h with 50 μM MeJA was loaded per lane and hybridized with the AtMYC2 probe. (B) MeJA inhibition of root growth in the atmyc2 mutants and Col-0 seedlings grown for 10 d on MS plates, or MS plates with 10, 50, and 100 μM MeJA. (C) MeJA induction of the VSP, JR1, PDF1.2, and b-CHI transcripts in Col-0 and the atmyc2 mutants. Fifteen micrograms of total RNA from noninduced seedlings (NI) or seedlings treated for 8 h with 50 μM MeJA (JA) was loaded per lane and hybridized with the indicated probes. (D) Complementation of the atmyc2-2 JA-insensitive root growth phenotype by overexpression of the JAMYC2 and JAMYC10 proteins (JAMYC/atmyc2-2 lines). Seeds were grown for 10 d on MS medium containing 50 μM MeJA. (E) Molecular complementation of the atmyc2-2 phenotype by expression of the Arabidopsis AtMYC2 gene or the tomato JAMYC factors. Fifteen micrograms of total RNA from seedlings treated for 8 h with 50 μM MeJA was loaded per lane and hybridized with the VSP and PDF1.2 probes.

Figure 7.

Model for the proposed function of AtMYC2 and ERF1 in cross-talk regulation of the JA and JA/ET pathways in Arabidopsis. Herbivore and pathogenic attack promote a transient increase of both JA and ethylene. JA is involved in systemic activation of wound-responsive genes, and ethylene prevents local expression of these genes. A positive interaction between JA and ethylene is involved in activation of the JA/ET-pathway regulated genes in response to necrotrophic pathogen attack. Whereas ERF1 activates JA/ET-regulated gene expression (Lorenzo et al. 2003), AtMYC2 is involved in activation of JA-regulated genes and also in repression of JA/ET-induced gene expression. Arrows and bars indicate positive and negative interactions, respectively. A possible function of ERF1 in repression of the JA-regulated pathway is indicated by a question mark. (OGAs) Oligogalacturonides; (JA) jasmonic acid; (ET) ethylene.