Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP), an ethylene-inducible, GCC box DNA-binding protein interacts with an ocs element binding protein

Abstract

Ocs elements are a group of promoter sequences required for the expression of both pathogen genes in infected plants and plant defense genes. Genes for ocs element binding factors (OBFs), belonging to a specific class of basic-region leucine zipper (bZIP) transcription factors, have been isolated in a number of plants. Using protein-protein interaction screening with OBF4 we have isolated AtEBP, an Arabidopsis protein that contains a novel DNA-binding domain, the AP2/EREBP domain. One class of proteins that contain this domain are the tobacco ethylene-responsive element binding proteins (EREBPs). The EREBPs bind the GCC box that confers ethylene responsiveness to a number of pathogenesis related (PR) gene promoters. AtEBP expression is inducible by exogenous ethylene in wild-type plants and AtEBP transcripts are increased in the ctr1-1 mutant, where ethylene-regulated pathways are constitutively active. Electrophoretic mobility-shift assay and DNase I footprint analysis revealed that AtEBP can specifically bind to the GCC box. Interestingly, the highest level of AtEBP expression was detected in callus tissue, where ocs elements are very active. Synergistic effects of the GCC box with ocs elements or the related G-box sequence have been previously observed, for example, in the ethylene-induced expression of a PR gene promoter. Our results suggest that cross-coupling between EREBP and bZIP transcription factors occurs and may therefore be important in regulating gene expression during the plant defense response.

Figure 6

Analysis of the DNA-binding properties of AtEBP. (A) Analysis of AtEBP binding to the GCC wild-type (wt) sequence from the tobacco PRB-1b promoter (−201 to −191) and a GCC mutant (m) sequence. Lanes 1 and 5 contained only the free probes. Lanes 2 and 6 contained 5 ng, lanes 3 and 7 contained 25 ng, and lanes 4 and 8 contained 50 ng of AtEBP. Lanes 9 and 10 contained 100 ng of GST–AtEBP. (B) The oligonucleotide sequences for the GCC wild-type and mutant probes. The mutated residues in the GCC mutant probe are outlined. The 5′ end of each probe contained a BamHI site.

Figure 1

Sequence of the AtEBP cDNA clone and the predicted amino acid sequence. The predicted amino acid sequence for the longest ORF is shown directly below the nucleotide sequence. A putative nuclear localization signal is underlined. The AtEBP GenBank database accession number is Y09942Y09942.

Figure 2

AtEBP and OBF4 interact in an in vitro assay. One hundred nanograms of labeled OBF4 protein (OBF4*) was incubated with equal amounts of either glutathione-agarose beads or beads where the GST or GST–AtEBP proteins were already bound. The amount of OBF4* protein (40 kDa) that was retained after washing the glutathione-agarose beads was purified, analyzed by SDS/PAGE, and then detected by autoradiography. Lanes 1 and 2 reflect nonspecific binding of OBF4*; lane 3 reflects the specific binding of OBF4* to GST–AtEBP bound to the glutathione-agarose beads. Aliquots of the OBF4* input are shown in lane 4 (2%) and lane 5 (10%).

Figure 3

AtEBP has homology to plant DNA-binding proteins. Comparison of a region of the predicted amino acid sequence of the Arabidopsis AtEBP protein to the DNA-binding domain of the tobacco EREBPs (D38123, D38126, D38124, D38125), and similar sequences present in the Arabidopsis TINY (X94698) and APETALA2 (U12546) proteins using the Genetics Computer Group gap program. Identical amino acids are marked by black boxes. A region with a high potential for forming an amphipathic helix is indicated by a double arrow.

Figure 4

Analysis of the AtEBP gene family in Arabidopsis. Southern bolt analysis of Arabidopsis genomic DNA using the AtEBP cDNA clone as a probe. Each lane contained 10 μg of genomic DNA that was digested with the indicated enzyme. At left, a 1-kb DNA ladder (Bethesda Research Laboratories) was used for the molecular length markers.

Figure 5

Analysis of AtEBP RNA expression patterns RNA gel blot analysis with the AtEBP cDNA clone as a probe was performed using 5 μg (A and B) or 15 μg (C and D) of total RNA from 8-day-old Arabidopsis seedlings or callus tissue. Equal amounts of RNA were loaded as determined by ethidium bromide staining. (A) Seedlings compared with callus tissue. (B) Seedlings following treatment with the indicated amounts (mM) of cadmium (Cd), copper (Cu) or hydrogen peroxide (H₂O₂). (C) Seedlings following treatment with phosphate buffer pH 7.0 (lane 1), or phosphate buffer pH 7.0 containing 1 mM ethephon (lane 2) or 1 mM kinetin (lane 3). (D) Wild-type (lane 1) compared with the mutant seedlings: ctr1-1 (lane 2) and etr1-3 (lane3).

Figure 7

DNase I footprint analysis of AtEBP binding to the GCC box in the PRB-1b promoter. A PRB-1b promoter fragment corresponding to nucleotides −205 to −155 was labeled on both strands (bottom, top) and incubated with no protein (lanes 2, 5, 7, and 10), 50 ng AtEBP (1x; lanes 3 and 8), or 100 ng AtEBP (2x; lanes 4 and 9). The reactions were analyzed on a 8% sequencing gel together with G+A sequencing ladders generated from the same PRB-1b promoter fragments. The GCC box on the PRB-1b promoter is indicated by arrows.