The Arabidopsis ETHYLENE RESPONSE FACTOR1 regulates abiotic stress-responsive gene expression by binding to different cis-acting elements in response to different stress signals

Abstract

ETHYLENE RESPONSE FACTOR1 (ERF1) is an upstream component in both jasmonate (JA) and ethylene (ET) signaling and is involved in pathogen resistance. Accumulating evidence suggests that ERF1 might be related to the salt stress response through ethylene signaling. However, the specific role of ERF1 in abiotic stress and the molecular mechanism underlying the signaling cross talk still need to be elucidated. Here, we report that ERF1 was highly induced by high salinity and drought stress in Arabidopsis (Arabidopsis thaliana). The salt stress induction required both JA and ET signaling but was inhibited by abscisic acid. ERF1-overexpressing lines (35S:ERF1) were more tolerant to drought and salt stress. They also displayed constitutively smaller stomatal aperture and less transpirational water loss. Surprisingly, 35S:ERF1 also showed enhanced heat tolerance and up-regulation of heat tolerance genes compared with the wild type. Several suites of genes activated by JA, drought, salt, and heat were found in microarray analysis of 35S:ERF1. Chromatin immunoprecipitation assays found that ERF1 up-regulates specific suites of genes in response to different abiotic stresses by stress-specific binding to GCC or DRE/CRT. In response to biotic stress, ERF1 bound to GCC boxes but not DRE elements; conversely, under abiotic stress, we observed specific binding of ERF1 to DRE elements. Furthermore, ERF1 bound preferentially to only one among several GCC box or DRE/CRT elements in the promoter region of its target genes. ERF1 plays a positive role in salt, drought, and heat stress tolerance by stress-specific gene regulation, which integrates JA, ET, and abscisic acid signals.

Figure 1.

Expression profile of ERF1. A, qRT-PCR analyses of ERF1 induction by abiotic stresses. Total RNA was extracted from plants harvested at the indicated times after each treatment. Two-week-old seedlings were dried on Whatman 3MM paper (Drought), treated with 150 mm NaCl (NaCl), treated with 400 mm mannitol (Mannitol), or incubated at 37°C (Heat). The amplification of ACTIN2 was used as an internal control to normalize all data. The level of the transcript before stress treatments was set to 1.0. Three independent experiments were performed with similar results. Error bars indicate se (ANOVA; *P < 0.05). B, Fluorescence microscopy images of Arabidopsis protoplast. Constructs of 35S:GFP or 35S:ERF1-GFP were translocated into Arabidopsis protoplast by polyethylene glycol transfection. The expression of the introduced genes was detected after 16 h. Nuclei are shown with 4′,6-diamino-phenylindole (DAPI) staining. Bars = 20 μm. C, GUS staining of ERF1 promoter:GUS transgenic plants. Three-week-old homozygous plants (G3 and G6) were either mock treated or treated with 50 μm JA, 150 mm NaCl, 30 min of drought stress, or 1 h of heat shock stress (45°C). Histochemical GUS staining was performed overnight on 10 seedlings for each experiment.

Figure 2.

ABA inhibition effect on ERF1 expression. In qRT-PCR and GUS staining analyses, 2-week-old wild-type or ERF1 promoter:GUS transgenic plants were either mock treated or treated with 50 μm JA, 50 μm ET, both JA + ET, or together with 50 μm ABA. A, qRT-PCR analyses of ERF1 and AREB1 under ABA treatment. Total RNA was extracted from 2-week-old plants harvested at the indicated times after 50 μm ABA treatment. Three independent experiments were performed with similar results. B, qRT-PCR analyses of ERF1 under different combinations of hormone treatments. Three independent experiments were performed with similar results. Error bars indicate se. C, Hormone applications on GUS staining of ERF1 promoter:GUS transgenic plants. Samples were collected after 1 h of each treatment.

Figure 3.

Drought- and salt-tolerant phenotypes of 35S:ERF1 transgenic Arabidopsis. A, Expression levels of ERF1 mRNA in 35S:ERF1 (OE5 and OE6) and ERF1 RNAi (RNAi7 and RNAi15) transgenic plants. B, Drought tolerance of wild-type (WT), 35S:ERF1, and ERF1 RNAi transgenic plants after withholding water for 12 to 16 d and rehydration for 4 d (Recover). C, Three-week-old plants were irrigated with different concentrations of NaCl solution (100 mm for 4 d, 200 mm for another 4 d, and 300 mm for the rest of the time). These experiments were repeated three times with similar results. D, Survival rates of wild-type, 35S:ERF1, and ERF1 RNAi transgenic plants under drought and salt stress. Error bars indicate se (Student’s t test; *P < 0.001). E, Seed germination rates of 35S:ERF1 and ERF1 RNAi transgenic plants under salt stress treatment. ERF1 overexpressed and RNAi seeds were germinated under different concentrations of NaCl. The germination rates were calculated after 3 d (top panel) and 5 d (bottom panel). Results are averages of three replicates. Error bars indicate se (Student’s t test; *P < 0.05, **P < 0.01). F, Root elongation assays. Three-day-old seedlings were transferred to an MS agar plate with 150 mm NaCl and incubated vertically for 7 d before root lengths were measured. Results are averages of three replicates. Error bars indicate se (Student’s t test; *P < 0.05).

Figure 4.

Water loss in detached leaves and the influence of ERF1 overexpression on ABA-mediated stomatal closure. A, Water loss from detached leaves as a function of time in Col-0 and 35S:ERF1 plants (OE3 and OE6). This experiment was repeated three times with similar results. Values are means of the percentage of leaf water loss ± se (n = 15). Error bars indicate se (ANOVA; *P < 0.05). B, Micrographs representing the dynamics of ABA-mediated stomatal closure in Col-0 and 35S:ERF1 plants. C, Stomatal apertures were measured on epidermal peels of wild-type (WT), 35S:ERF1 (OE5 and OE6), and ERF1 RNAi (RNAi7 and RNAi15) transgenic plants. Stomata were preopened under light for 2.5 h and then incubated in the indicated concentrations of ABA for 2.5 h under light. This experiment was repeated three times with the same trend. Values are means ± se (n > 60). Error bars indicate se (ANOVA; *P < 0.05). D, Infrared thermal images of 3-week-old 35S:ERF1 (OE3, OE5, and OE6) and wild-type (Col) plants.

Figure 5.

Pro and ABA contents in ERF1 transgenic plants. Total Pro or ABA was prepared from 3-week-old Arabidopsis grown on MS agar plates. Pro contents were also measured after treating with 0.4 m mannitol for 24 h. Data are presented as means and se of three replications. A, Pro contents in ERF1 transgenic plants. Error bars indicate se (ANOVA; *P < 0.01). DW, Dry weight; WT, wild type. B, ABA contents of ERF1 transgenic plants. Error bars indicate se (Student’s t test; *P < 0.01). FW, Fresh weight.

Figure 6.

Effects of ABA, ET, and JA on ERF1 gene expression. The relative mRNA amounts of ERF1 were analyzed by qRT-PCR (the expression level of Col-0 was set to 1). Total RNA was prepared from 3-week-old Arabidopsis grown on MS agar plates treated with 0.4 m mannitol for 24 h or 150 mm NaCl for 1 h. Data represent means and se of three replications. Error bars indicate se (ANOVA; *P < 0.01). A, Effects of high salinity on ERF1 gene expression in aba2, abi1, and abi2 knockout mutants. B, Effects of high salinity and drought stress on ERF1 gene expression in etr1-1 and ein2-5 mutants. WT, Wild type. C, Effects of high salinity and ABA on ERF1 gene expression in ctr1 mutants. D, Effects of high salinity and JA on ERF1 gene expression in jar1-1 mutants.

Figure 7.

Venn diagram and validation of selected microarray data. A, Venn diagram representing the distribution of drought-, high salinity-, and heat shock (HS)-responsive ERF1 up-regulated genes. The numbers in parentheses indicate total numbers of ERF1 up-regulated genes that showed expression ratios > 2 in the microarray analysis of drought, high salinity, and heat shock stress responses. B, Expression analysis of ERF1 up-regulated genes in the 35S:ERF1 plants. Total RNA was prepared from 3-week-old Arabidopsis plants from one line of Col-0, two independent 35S:ERF1 lines, and two independent ERF1 RNAi lines, R7 and R15. The relative mRNA amount of ERF1 up-regulated genes was analyzed by qRT-PCR (the expression level of Col-0 was set to 1). Data represent means and se of three replications. Error bars indicate se (Student’s t test; *P < 0.01). WT, Wild type.

Figure 8.

Heat shock stress tolerance of the 35S:ERF1 and wild-type (WT) plants. One-week-old seedlings of wild-type or 35S:ERF1 (OE5 and OE6) plants were incubated at 45°C for 1 h. After heat stress treatment (HS), plants were grown under normal conditions for 1 week. Percentages of surviving plants are indicated. More than 30 plants were used per test, and each test was repeated three times. Error bars indicate se (Student’s t test; *P < 0.005).

Figure 9.

ERF1 binding affinity to the DRE element and the GCC box in selected ERF1 stress-responsive downstream gene promoters. A, In the downstream gene promoters, the sequence regions used for ChIP assays are marked. Black marks, GCC box; gray marks, DRE elements. B, ChIP assays. Fragments showing significant enrichment are underlined. For ChIP assays, 3-week-old 35S:ERF1-GFP transgenic plants under normal conditions (Normal), treated with 1 h of 50 μm JA (JA), 30 min of dehydration (Drought), 1 h of 150 mm NaCl (Salt), or 1 h of heat shock stress (45°C; Heat) were used. Three measurements were averaged for individual assays. The values in Col-0 plants were set to 1 after normalization against ACT2 for qRT-PCR analysis. Error bars indicate se (Student’s t test; *P < 0.05, **P < 0.01).

Figure 10.

Proposed model of ERF1 function in the regulation of biotic stress- and abiotic stress-responsive gene expression. ERF1 positively regulates both biotic and abiotic stress responses. ERF1 induction required both ET and JA signaling under abiotic stress and was negatively regulated by ABA. It is not clear if the negative effect of ABA on ERF1 expression resulted from a direct effect or indirectly through affecting JA-ET signaling (dashed line). Under different stress conditions, such as pathogen infection, dehydration, high salinity, and heat shock, ERF1 activates specific sets of stress response genes by targeting to specific cis-elements (GCC boxes during biotic stress and DRE elements during abiotic stress). The factors controlling the stress-specific promoter targeting of ERF1 remain unknown.