An AP2 domain-containing gene, ESE1, targeted by the ethylene signaling component EIN3 is important for the salt response in Arabidopsis

Abstract

Accumulating investigations reveal that ethylene signaling is involved in the salt response in Arabidopsis (Arabidopsis thaliana), and it has been reported that overexpression of a number of ethylene response factor (ERF) genes enhances salt tolerance; however, transcriptional regulation of the ethylene signal component ETHYLENE INSENSITIVE3 (EIN3) in the salt response has not been clearly defined. Consulting microarray data and transcriptional confirmations showed that three of the ERF genes were ethylene and salt inducible, named ESE1 to ESE3. Additionally, the expression of one of the ESE genes (ESE1) was suppressed in ein2, ein3-1, eil1-3, and ein3 eil1 but enhanced in EIN3-overexpressing (EIN3ox) lines. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine, and ethylene action, AgNO₃, reduced the expression of ESE1, while ethylene overproduction eto mutants enhanced the expression of ESE1, indicating that ESE1 is an ethylene-modulated gene downstream of EIN3/EIL1. Further analyses with biochemical and molecular approaches revealed that EIN3 physically binds to the ESE1 promoter, demonstrating that ESE1 was one target of EIN3. ESE1 in turn binds to promoters of salt-related genes, such as RD29A and COR15A. Moreover, either EIN3ox or ESE1ox was sufficient to enhance transcript levels of salt-related genes and salt tolerance. In addition, ESE1ox in ein3 enhanced the salt response during seed germination and seedling development, demonstrating that ESE1 is genetically downstream of EIN3. Thus, the evidence in this report reveals that the transcriptional complex of EIN3-ESE1 is a crucial event in the salt response, thereby connecting the transcriptional regulation of EIN3 and the downstream ERF protein ESE1 in the salt response.

Figure 1.

Identification of the putative ethylene- and salt-inducible ERF genes. A, Expression of ERF genes in Arabidopsis Col-0 with or without 10 μm ACC treatment. B, Expression of ERF genes in Arabidopsis Col-0 with or without 100 mm NaCl treatment. Transcript levels of the ERF genes are indicated relative to the level of the control in Col-0 taken as 1, referring to the transcripts of TUB4 in the same sample. Error bars (sd) are based on three independent experiments.

Figure 2.

The expression of ESE1 is modulated by ethylene signaling components. A, Expression of ESE1 in Arabidopsis Col-0, ein2, ein3-1, eil1-3, ein3 eil1-3, and EIN3ox lines with or without 100 mm NaCl treatment. P values (each genotype versus Col-0 under salt induction) were determined by two-tailed Student’s t test assuming equal variance (* P < 0.05). B, Expression of ESE1 in Col-0, eto1, eto2, and eto3, and treatment with 10 μm ACC and inhibitors (5 μm each) of ethylene perception and biosynthesis in Col-0, respectively. Transcripts of the ESE1 gene are indicated relative to the level of the control in Col-0 (taken as 1), referring to the transcripts of TUB4 in the same sample. Error bars (sd) are based on three independent experiments.

Figure 3.

The ESE1 promoter is identified by EIN3 in yeast and in plants. A, Interaction of the full length of EIN3 with different fragments of the ESE1 promoter. pD1, pD2, pD3, and pD4 indicate the reporters carrying different fragments of the ESE1 promoter. B, Interaction of the ESE1 promoter fragment D4 with different lengths of EIN3. pEIN3-F, pEIN3-N, and pEIN3-C indicate the effectors carrying full-length protein and N-terminal and C-terminal portions of EIN3. Left panels in A and B show the reporter constructs carrying the ESE1 promoter fragments and the effector constructs carrying different domains of EIN3, respectively, in the yeast one-hybrid assay. Numbers indicate the positions of the nucleotides at the 5′ or 3′ end of each fragment relative to the translation start site in A or the amino acids in B. The right panels in A and B show β-galactosidase activity. +, Cotransformation of pGBKT7-53 and pGADT7-Rec T as a positive control; –, cotransformation of pGBKT7-lam and pGADT7-Rec T as a negative control. C, Identification of EIN3 on the ESE1 promoter in vivo. The predicted ATGTA box for the putative binding sites for EIN3 protein in the promoters of ESE1 and EBF2 is indicated by the black box, and the numbers indicate the positions of the fragments relative to the putative transcriptional start site. The right panel shows the ChIP enrichment detected by qPCR amplification using immunoprecipitated DNA of α-myc antibody as a template. ACC was used to treat seedlings. Two EIN3ox lines displayed similar results; the qPCR data are from EIN3ox-4. Error bars (sd) are based on three independent experiments. The interaction of the EBF2 promoter with EIN3 (Konishi and Yanagisawa, 2008) was used as a positive control. An unrelated DNA sequence from the TUB4 gene was used as an internal control.

Figure 4.

The ESE1 promoter is recognized by EIN3 in a transient expression assay. The left panel shows a schematic diagram of the effector and reporter constructs used in an Agrobacterium-mediated transient cotransformation expression assay. The coding domain of EIN3 is fused downstream of cauliflower mosaic virus 35S in pCAMBIA1303. The promoter fragments of ESE1 and EBF2 are fused upstream of the GUS gene in pCAMBIA1381Z. The gray box indicates binding sites for the EIN3 protein. The right panel shows the GUS activity resulting from transient cotransformation with reporter and effector constructs. Error bars (sd) are based on three independent experiments. MU, 4-Methylumbelliferyl β-d-glucuronide.

Figure 5.

The ESE1 promoter physically interacts with the EIN3 protein in vitro. A, Interaction of EIN3 with the ESE1 promoter. The top panel shows the positions of the probes used in EMSA. Numbers indicate the positions of the nucleotides at the 5′ and 3′ ends of each probe relative to the translation start site. The bottom panel displays the detection of probes after reaction with the N-terminal or C-terminal area of EIN3 and the GST protein (as a negative control). B, Interaction confirmation using mutated probe D4P2-3M after reaction with the N-terminal area of EIN3 or the GST protein (as a negative control). The sequences of D4P2-3 and D4-P2-3M are described in “Materials and Methods.” C, Competition assay of unlabeled probe D4P2-3 after reaction with the N-terminal area of EIN3 in the presence of labeled probe D4P2-3.

Figure 6.

ESE1 regulates the salt response during germination and seedling growth. A, Position of the T-DNA insertion in the ESE1 promoter region. UTR, Untranslated region. B, Expression level of ESE1 in Col-0 and the ese1 mutant. Transcripts of ESE1 are indicated relative to these of Col-0 taken as 1, referring to the transcripts of TUB4 in the same sample. C, Seed germination under 100 mm NaCl treatment for 96 h. About 100 seeds were used for each treatment. Results are averages of three replicates. D, Phenotype observation under 100 mm NaCl treatment for 7 d. About 20 to 25 seedlings in triplicate were used for each line. E, Fresh weight of each shoot. The data were obtained from an average of 10 shoots in triplicate. Error bars indicate sd. P values (each genotype versus Col-0 after salt induction) were determined by two-tailed Student’s t test assuming equal variance (* P < 0.05, ** P < 0.01).

Figure 7.

EIN3 and ESE1 coactivate the expression of downstream salt-related genes. Transcript levels of these genes are indicated relative to the level of wild-type Col-0 taken as 1, referring to the transcripts of TUB4 in the same sample. Error bars (sd) are based on three independent experiments.

Figure 8.

ESE1 identifies the promoters of salt-related genes in vivo and in vitro. A, Detection of the interaction of ESE1 with DRE. Competition was conducted with unlabeled DRE. GST protein is a negative control. B, Detection of the interaction of ESE1 with the GCC box. Competition was conducted with unlabeled GCC box. C, ChIP enrichment tested by qPCR using immunoprecipitated DNA of α-myc antibody as a template shows the binding of ESE1 to the regulatory region of downstream genes. Error bars (sd) are based on three independent experiments. An unrelated DNA sequence from the TUB4 gene was used as an internal control.

Figure 9.

Model of the EIN3-ESE1 transcriptional complex in the regulation of expression of salt-related genes. TF, Transcription factor.