Salt-induced stabilization of EIN3/EIL1 confers salinity tolerance by deterring ROS accumulation in Arabidopsis

Abstract

Ethylene has been regarded as a stress hormone to regulate myriad stress responses. Salinity stress is one of the most serious abiotic stresses limiting plant growth and development. But how ethylene signaling is involved in plant response to salt stress is poorly understood. Here we showed that Arabidopsis plants pretreated with ethylene exhibited enhanced tolerance to salt stress. Gain- and loss-of-function studies demonstrated that EIN3 (ETHYLENE INSENSITIVE 3) and EIL1 (EIN3-LIKE 1), two ethylene-activated transcription factors, are necessary and sufficient for the enhanced salt tolerance. High salinity induced the accumulation of EIN3/EIL1 proteins by promoting the proteasomal degradation of two EIN3/EIL1-targeting F-box proteins, EBF1 and EBF2, in an EIN2-independent manner. Whole-genome transcriptome analysis identified a list of SIED (Salt-Induced and EIN3/EIL1-Dependent) genes that participate in salt stress responses, including several genes encoding reactive oxygen species (ROS) scavengers. We performed a genetic screen for ein3 eil1-like salt-hypersensitive mutants and identified 5 EIN3 direct target genes including a previously unknown gene, SIED1 (At5g22270), which encodes a 93-amino acid polypeptide involved in ROS dismissal. We also found that activation of EIN3 increased peroxidase (POD) activity through the direct transcriptional regulation of PODs expression. Accordingly, ethylene pretreatment or EIN3 activation was able to preclude excess ROS accumulation and increased tolerance to salt stress. Taken together, our study provides new insights into the molecular action of ethylene signaling to enhance plant salt tolerance, and elucidates the transcriptional network of EIN3 in salt stress response.

Figure 1. ACC/Ethylene pretreatment or enhanced ethylene signaling increases salt tolerance.

(A) Plants were grown on MS medium with or without 10 µM ACC for 5 d and then transferred onto MS medium supplemented with 200 mM NaCl for 3 d. Plants were also transferred onto MS medium as controls. (B) Survival rate of plants shown in (A). Seedling death was scored as complete bleaching of cotyledons and leaves. Values are mean ± SD from 25 seedlings per replicate (n = 3 replicates). (Student's t test, *P<0.05 and **P<0.01). (C) Relative electrolyte leakage of plants shown in (A). Values are mean ± SD from 50 seedlings per replicate (n = 3 replicates). (Student's t test, *P<0.05 and **P<0.01). (D) Survival rate of plants pretreated with air (Air) or 20 ppm ethylene (ET) for 5 d and then transferred onto MS medium supplemented with 200 mM NaCl. Survival rates were calculated on the second, third and fourth day. Values are mean ± SD from 20 seedlings per replicate (n = 4 replicates). (Student's t test, *P<0.05 and **P<0.01). (E) Survival rate of plants pretreated with air or 20 ppm ethylene for indicated time and then transferred onto medium supplemented with 200 mM NaCl. Survival rates were calculated on the third day after transfer. Values are mean ± SD from 20 seedlings per replicate (n = 4 replicates). A5E0: 5 d of air treatment. A2E1A2: 2 d of air followed by 1 d of ethylene then 2 d of air treatment. A1E2A2: 1 d of air followed by 2 d of ethylene then 2 d of air treatment. A0E5: 5 d of ethylene treatment. ET: ethylene. (Student's t test, *P<0.05 and **P<0.01).

Figure 2. Salt treatment promotes protein accumulation and transcriptional activity of EIN3 in both EIN2-dependent and EIN2-independent manners.

(A) Salt treatment promotes EIN3 protein accumulation in wild type. 5-d-old seedlings were treated with 200 mM NaCl for 3 h and 6 h. Protein was extracted and subjected to immunoblots using anti-EIN3 antibody. A nonspecific band was used as a loading control. (B) Salt treatment promotes EIN3 protein accumulation in ein2-5 mutant. Experiments were repeated three times with similar results. (C) Histochemical analysis of 5xEBS:GUS transgenic plants. (D) 5xEBS:GUS activity in Col-0 or ein2-5 background was measured. Two biological replicates and three technical replicates were performed (Student's t test, *P<0.05 and **P<0.01). (E–G) Real-time RT-PCR analysis of gene expression of ERF1 (E), ERF2 (F) and PDF1.2 (G).

Figure 3. Salt treatment promotes EBF1/EBF2 protein degradation in an EIN2-independent manner.

(A) Immunoblot assays of EBF1/2-MYC protein in Col-0. Transgenic seedlings overexpressing EBF1/2-MYC in Col-0 grown on MS medium supplemented with or without 10 µM ACC for 5 d were subjected to 200 mM NaCl for 3 h and 6 h. A nonspecific band was used as a loading control. (B) Immunoblot assay of EBF2-GFP protein in ein2-5 background. Transgenic seedlings overexpressing EBF2-GFP in ein2-5 background grown on medium supplemented with or without 10 µM ACC for 5 d were subjected to 200 mM NaCl for 3 h and 6 h. Experiments were repeated three times with similar results. (C) Salt induced EBF2 protein degradation was inhibited by MG132. 5-d-old plants were treated with 200 mM NaCl or/and 50 µM MG132 for 6 h. Experiments were repeated three times with similar results. (D) GFP fluorescence of 35S:EBF2-GFP in the roots of ein2-5 mutant. The seedlings grown on MS medium for 5 d were treated with 200 mM NaCl and/or 50 µM MG132 for 6 h.

Figure 4. Transcriptome profiling analyses identify salt-regulated EIN3/EIL1-dependent genes.

(A) and (B) Venn diagrams showing the overlaps among transcripts induced or repressed (q<0.05 and 5-fold change as a cutoff) by salt in Col-0, ein3eil1 and EIN3ox plants. (C) and (D) Hierarchical clusters displaying the salt-induced expression of those SIED (Salt-Induced EIN3/EIL1-Dependent) and SRED (Salt-Repressed EIN3/EIL1-Dependent) genes in Col-0, ein3eil1 and EIN3ox plants. A total of 114 SIED genes (C) and 14 SRED genes (D) were included in the cluster (Table S1 and S2). (E) and (F) Venn diagrams showing the overlaps between transcripts induced or repressed (2-fold cutoff) by salt in Col-0 and transcripts induced or repressed (2-fold cutoff) by overexpression of EIN3 (comparing transcriptome of EIN3ox versus Col-0 under unstressed condition).

Figure 5. Functional characterization of SIED genes identifies a novel regulator of salt tolerance.

(A) Plants were grown on MS medium for 5 d and then transferred onto MS medium supplemented with 200 mM NaCl for 4 d. Experiments were repeated three times with similar results. (B) Survival rate of plants shown in (A). Values are mean ± SD from at least 50 seedlings per replicate (n = 4 replicates). (C) qRT-PCR analysis of SIED1 expression. (D) Histochemical analysis of SIED1 expression in Col-0, ein3eil1 and EIN3ox plants. (E) pSIED1:GUS activity in Col-0, ein3eil1 or EIN3ox background (Student's t test, **P<0.01 and ***P<0.001). (F) Overexpression of SIED1 in wild-type enhanced salt tolerance. Seedlings were grown on MS medium for 5 d and then transferred onto MS medium supplemented with 200 mM NaCl for 4 d. Experiments were repeated three times with similar results. (G) Survival rate of plants shown in (F). Values are mean ± SD from at least 50 seedlings per replicate (n = 3 replicates). (H) and (I) Overexpression of SIED1 in ein3eil1 or sied1 backgrounds enhanced salt tolerance. Fresh weight (H) and survival rate (I) were measured (Student's t test, **P<0.01 and ***P<0.001).

Figure 6. EIN3 increases activity of peroxidases through transcriptional regulation of POD genes directly.

(A) qRT-PCR analysis of PODs expression. (B) Measurement of POD activity. The treated seedlings in (A) were also used (Student's t test, *P<0.05 and **P<0.01). (C) Schematic diagrams of putative EIN3 Binding Site (EBS) (arrows) in the promoters of two POD genes. The 1 kb upstream sequences are shown, and the translational start sites (ATG) are shown at position +1. (D) ChIP-qPCR assays of the promoter regions of POD genes from DNA of Col-0 seedlings with anti-EIN3 antibody. A Tubulin 8 fragment was amplified as control. Three biological replicates and two technical replicates were performed with similar results. (E) EMSA showing the interaction between the EBS containing region of POD genes and EIN3 protein. GST-tagged EIN3 N-terminus fusion protein was incubated with biotin-labeled DNA fragment. Competition for the biotin-labeled promoter region was done by adding an excess of unlabeled wild-type probe (competitor) or mutated probe (mutant competitor). Two biological replicates and two technical replicates were performed with similar results.

Figure 7. ROS accumulation in salt-treated Col-0, ein3eil1, EIN3ox and ebf2 plants.

(A) Fluorescence microscopy images of ROS (indicated by H₂DCFDA fluorescence). Seedlings grown on MS medium supplemented with or without 10 µM ACC for 5 d were subjected to salt treatment. BF: bright field. Experiments were repeated three times with similar results. (B) DAB staining of seedlings under normal conditions or salt treatment. Seedlings grown on MS medium supplemented with or without 10 µM ACC for 5 d were treated with 200 mM NaCl for 6 h, and used for DAB staining. (C) H₂O₂ content in the seedlings in (B) (Student's t test, *P<0.05 and **P<0.01). (D) qRT-PCR analysis of DEFL expression (a ROS marker gene) (Student's t test, *P<0.05 and **P<0.01).