Identification of a bipartite jasmonate-responsive promoter element in the Catharanthus roseus ORCA3 transcription factor gene that interacts specifically with AT-Hook DNA-binding proteins

Abstract

Jasmonates are plant signaling molecules that play key roles in defense against certain pathogens and insects, among others, by controlling the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the APETALA2-domain transcription factor ORCA3 is involved in the jasmonate-responsive activation of terpenoid indole alkaloid biosynthetic genes. ORCA3 gene expression is itself induced by jasmonate. By loss- and gain-of-function experiments, we located a 74-bp region within the ORCA3 promoter, which contains an autonomous jasmonate-responsive element (JRE). The ORCA3 JRE is composed of two important sequences: a quantitative sequence responsible for a high level of expression and a qualitative sequence that appears to act as an on/off switch in response to methyl jasmonate. We isolated 12 different DNA-binding proteins having one of four different types of DNA-binding domains, using the ORCA3 JRE as bait in a yeast (Saccharomyces cerevisiae) one-hybrid transcription factor screening. The binding of one class of proteins bearing a single AT-hook DNA-binding motif was affected by mutations in the quantitative sequence within the JRE. Two of the AT-hook proteins tested had a weak activating effect on JRE-mediated reporter gene expression, suggesting that AT-hook family members may be involved in determining the level of expression of ORCA3 in response to jasmonate.

Figure 1.

The ORCA3 promoter contains a MeJA-responsive element. A, Schematic representation of the T-DNA transferred to C. roseus suspension cells in the ORCA3 promoter studies. The CAT and hygromycin phosphotransferase genes are under the control of the CaMV 35S promoter, while GUS is under the control of an ORCA3 promoter derivative. Genes in the T-DNA have terminators from the nopaline synthase gene or the pea (Pisum sativum) ribulose-1,5-bisphosphate carboxylase-small subunit-encoding E9 or 3C genes. LB and RB, Left and right T-DNA borders. B, mRNA levels of the GUS and CAT transgenes and the endogenous ORCA3 gene in representative transgenic BIX cell lines containing the ORCA3 promoter constructs Δ826-GUS or 4D-GUS-47 treated with 100 μm CHX, 10 μm MeJA, or both compounds combined for different number of hours. Blots shown in the Δ826 and 4D segments were hybridized with a GUS probe. Both cell lines showed identical patterns of CaMV 35S-driven CAT mRNA and endogenous ORCA3 mRNA accumulation. The examples shown here are from the 4D line. Film exposure times were 15 h for GUS and CAT and 48 h for ORCA3.

Figure 2.

Localization of the JRE by 5′ deletion analysis of the ORCA3 promoter. A, Schematic representation of 5′ deletion derivatives of the ORCA3 promoter fused to GUS. Construct 6Tcyt-Δ88 contains six copies of the constitutive cyt-1 element of the T-DNA T-CYT gene fused to the ORCA3 Δ88 promoter. The position of the JRE deduced from this analysis is indicated. B, Northern blots showing GUS, ORCA3, and CAT mRNA levels in two independent transgenic BIX cell lines for each deletion construct. Cells were treated with 10 μm MeJA (M) or DMSO (C) at a final concentration of 0.1% for 5 h. Numbering of nts in the ORCA3 promoter is relative to the ATG start codon of translation.

Figure 3.

Localization of the JRE. A, Schematic representation of internal deletion constructs within the Δ264 context. B, Position of fragments A to D derived from the Δ264 promoter. Fragments were tetramerized and fused to the CaMV 35S minimal promoter and the GUS reporter gene. C, Northern blots showing GUS, ORCA3, and CAT mRNA levels in two independent transgenic BIX cell lines for each construct. Cells were treated for 5 h with 10 μm MeJA (M) or DMSO (C) at a final concentration of 0.1%. Numbering of nts in the ORCA3 promoter is relative to the ATG start codon of translation.

Figure 4.

Block scanning mutagenesis identified two sequences in the D region of the ORCA3 promoter that are important for the MeJA response. A, The wild-type sequence of the D region is shown. Numbering of mutations is given above the sequence. In each mutant, boxed nts were changed into their complementary nt. B, Northern blots showing GUS, ORCA3, and CAT mRNA levels in two independent transgenic BIX cell lines for each construct. Cells were incubated for 5 h with 10 μm MeJA (M) or DMSO (C) at a final concentration of 0.1%.

Figure 5.

In vitro binding of proteins encoded by representative one-hybrid clones of each class to wild-type or mutated derivatives of fragment D. Fragments indicated at the top of each segment were used as probes in in vitro binding assays using the protein indicated at the bottom of each segment. D represents wild-type D fragment, and numbers 1 to 7 represent mutated fragments M1 to M7 as shown in Figure 4A. Mutations affecting the quantitative sequence and the qualitative sequence are overlined with broken and solid lines, respectively. Dots indicate free probes, whereas asterisks indicate specific DNA-protein complexes. All members of each class gave qualitatively similar binding patterns to the example shown here (data not shown).

Figure 6.

Transactivation analysis of AT-hook proteins. Effect of AT-hook proteins expressed from the CaMV 35S promoter on 4D-GUS-47 reporter gene expression in bombarded cells of C. roseus cell line MP183L. Means of triplicate bombardments corrected for protein amounts with ses are shown.

Figure 7.

AT-Hook gene expression analysis. Northern blots showing mRNA levels corresponding to AT-hook genes, ORCA3, and the Rps9 control gene. MP183L cells were treated with 10 μm MeJA for different number of hours.

Figure 8.

JA-responsive expression conferred by the bipartite JRE. A, Comparison of JREs. Alignment of JREs in the promoters of the genes encoding Catharanthus ORCA3 (CrORCA3), tomato (LeLAP), Arabidopsis (AtVSP1), potato (StPI-II), soybean (GmVspB), and Arabidopsis 12-oxo-phytodienoic acid-10,11-reductase (AtOPR1). Similarities with the ORCA3 JRE are highlighted. B to D, Models for JA-responsive expression conferred by the bipartite JRE. B, Without JA, a repressor protein is bound to the qualitative DNA sequence, silencing expression. In the presence of JA, the repressor is inactivated, allowing expression following binding of AT-hook protein to the quantitative sequence (C), or the qualitative sequence binds an activator protein (D), which stimulates expression together with the AT-hook protein. In the latter case, the AT-hook protein can be DNA bound irrespective of the presence of JA. Activator in D and repressor in B can be differently modified forms of the same protein.