Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis

Abstract

Plant responses to ethylene are mediated by regulation of EBF1/2-dependent degradation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Here, we report that the level of EIL1 protein is upregulated by ethylene through an EBF1/2-dependent pathway. Genetic analysis revealed that EIL1 and EIN3 cooperatively but differentially regulate a wide array of ethylene responses, with EIL1 mainly inhibiting leaf expansion and stem elongation in adult plants and EIN3 largely regulating a multitude of ethylene responses in seedlings. When EBF1 and EBF2 are disrupted, EIL1 and EIN3 constitutively accumulate in the nucleus and remain unresponsive to exogenous ethylene application. Further study revealed that the levels of EBF1 and EBF2 proteins are downregulated by ethylene and upregulated by silver ion and MG132, suggesting that ethylene stabilizes EIN3/EIL1 by promoting EBF1 and EBF2 proteasomal degradation. Also, we found that EIN2 is indispensable for mediating ethylene-induced EIN3/EIL1 accumulation and EBF1/2 degradation, whereas MKK9 is not required for ethylene signal transduction, contrary to a previous report. Together, our studies demonstrate that ethylene similarly regulates EIN3 and EIL1, the two master transcription factors coordinating myriad ethylene responses, and clarify that EIN2 but not MKK9 is required for ethylene-induced EIN3/EIL1 stabilization. Our results also reveal that EBF1 and EBF2 act as essential ethylene signal transducers that by themselves are subject to proteasomal degradation.

Figure 1.

EIL1 Is a Nuclear Protein That Is Stabilized by Ethylene and the Proteasome Inhibitor MG132. (A) Ethylene treatment stabilizes EIL1 protein in wild-type Col-0 but not in ein2 mutant. Etiolated seedlings grown on MS medium for 4 d were treated with ethylene (C₂H₄, 20 ppm) gas for 0, 1, 4, and 12 h before tissues were harvested for immunoblot assays using anti-EIL1 antibody. A nonspecific band was used as a loading control. (B) MG132 treatment stabilizes EIL1 protein. Suspension cell cultures derived from wild-type Col-0 were treated with mock (0.5% DMSO), ACC (100 μM), or MG132 (50 μM) for 4 h before tissues were harvested for immunoblot assays. (C) Overexpression of EIL1 (EIL1ox) or EIL1-GFP (EIL1-GFPox) caused ethylene hypersensitivity. (Top) Three-day-old etiolated seedlings grown on MS medium supplemented without or with 10 μM ACC. (Bottom) Quantification of hypocotyl lengths in the top panel. Each bar represents the average length (±sd) of at least 20 seedlings. Experiments were repeated three times with similar results. (D) Ethylene and MG132 treatments stabilize EIL1-GFP protein. Six-day-old light-grown EIL1-GFPox seedlings (Col seedlings used for control) were treated with mock (0.5% DMSO), ethylene (20 ppm), and MG132 (50 μM) alone or in combination for 4 h before tissues were harvested for immunoblot assays. (E) ACC and MG132 treatments promote EIL1-GFP protein accumulation in the nucleus. Four-day-old etiolated seedlings were treated with 100 μM ACC (MS medium used for control) or 50 μM MG132 (0.5% DMSO in MS medium used for control) for 4 h before tissues were used for detecting GFP inflorescence (the top four panels show ×40 GFP images; the bottom two show differential interference contrast and GFP merged). (F) EIL1 protein overaccumulates in the ein3 ebf1 ebf2 triple mutant. Proteins were extracted from 2-week-old light-grown seedlings and were subjected to immunoblots with anti-EIL1 antibody. [See online article for color version of this figure.]

Figure 2.

EIL1 Mutation Partially Suppresses Phenotypes of the ebf1/ebf2 Single and Double Mutants. (A) The triple response phenotype of 3-d-old etiolated seedlings grown on MS medium alone or supplemented with 10 μM ACC. (B) Graphical quantification of hypocotyl lengths in (A). (C) Fifty-day-old soil-grown plants, showing that the semidwarf phenotype of the ebf1 mutant is rescued by the eil1, but not the ein3, mutation. (D) Graphical quantification of plant height in (C). Each bar represents the average length (±sd) of at least 10 plants. eil1 ebf1 shows a statistically significant difference (Student’s t test) compared with ebf1 (P < 0.001), whereas ein3 ebf1 does not (P > 0.1). (E) Genomic DNA analyses of eil1 ebf1 ebf2 triple mutant by PCR. eil1-1 contains a transposon insertion, and the amplification band is ~200 bp larger than that of the wild type. Both ebf1-1 and ebf2-1 contain T-DNA insertion, and no band is amplified under the amplification conditions. The primers used are labeled on the right. The detailed primer sequence information is provided in Supplemental Table 1 online. (F) Graphical quantification of hypocotyl lengths of 3-d-old etiolated seedlings grown on MS medium supplemented without or with 10 μM ACC. (G) Ten-day-old light-grown seedlings. The eil1 ebf1 ebf2 triple mutant was growth arrested at the seedling stage. Each bar in (B) and (F) represents the average length (±sd) of at least 20 plants. Experiments were repeated three times with similar results. [See online article for color version of this figure.]

Figure 3.

A Functional EIL1-GFP Protein in the ein3 ebf1 ebf2 Triple Mutant Is Not Responsive to Exogenous Ethylene. (A) Forty-five-day-old transgenic plants overexpressing EIL1-GFP in ein3 ebf1 ebf2 (OE2/tm and OE3/tm). The ein3 ebf1 ebf2 triple mutant (tm) is shown for comparison. (B) Flowers of the ein3 ebf1 ebf2 triple mutant (tm) and OE/tm transgenic plants, showing the stunted sepal/petal and protruding gynoecium in OE/tm. (C) Constitutive accumulation of the EIL1-GFP fusion protein in the nuclei of the OE2/tm petals (from left to right, ×40 DIC, ×40 GFP, and DIC and GFP merged). (D) RT-PCR analysis of EIL1 and ethylene marker gene expression. RNA extracted from 45-d-old soil-grown plants was used. (E) Graphical quantification of hypocotyl lengths of OE/tm etiolated seedlings grown on MS medium without or with 10 μM ACC for 3 d. Each bar represents the average length (±sd) of at least 20 seedlings. (F) Constitutive accumulation of EIL1-GFP in the nuclei of the OE3/tm hypocotyl tissue without or with ACC treatment.

Figure 4.

A Functional EIN3-FLAG Protein in the ein3 eil1 ebf1 ebf2 Quadruple Mutant Is Not Responsive to Exogenous Ethylene. (A) The triple response phenotype of 3-d-old ein3 eil1 ebf1 ebf2 (qm, left seedling in each image) and estradiol-inducible EIN3-FLAG in the qm background (iE/qm, right seedling in each image) etiolated seedlings grown on MS medium supplemented with increased concentrations of estradiol. (B) Response to ACC (10 μM) of 3-d-old Col, qm, and iE/qm etiolated seedlings grown on MS medium supplemented with variable concentrations of estradiol. (C) Graphical quantification of hypocotyl lengths in response to various concentrations of estradiol with or without ACC (10 μM). Each bar represents the average length (±sd) of at least 20 seedlings. (D) ACC treatment does not enhance the accumulation of EIN3-3FLAG fusion protein. Proteins were extracted from 6-d-old light-grown seedlings treated with indicated concentrations of estradiol for 8 h, treated with or without 100 μM ACC for another 4 h, and subjected to immunoblots using anti-FLAG antibody. (E) RT-PCR analysis of ERF1 expression in iE/qm transgenic plants. Total RNA was prepared from 6-d-old light-grown transgenic plants treated with indicated concentrations of estradiol for 8 h and then treated with or without 100 μM ACC for another 4 h. [See online article for color version of this figure.]

Figure 5.

EBF1/EBF2 Genetically Act Downstream of CTR1 and EIN2. (A) The triple response phenotype of 3-d-old etiolated seedlings grown on MS medium with or without 10 μM ACC. (B) Graphical quantification of hypocotyl lengths of various mutants in (A). Each bar represents the average lengths (±sd) of at least 20 seedlings. (C) Sixty-day-old representative plants grown on soil, showing that neither the ein2 nor the ctr1 mutation was able to suppress the phenotype of ein3 ebf1 ebf2, which was fully suppressed by the eil1 mutation. [See online article for color version of this figure.]

Figure 6.

The Levels of EBF1/EBF2 Proteins Are Upregulated by Ag⁺ and MG132. (A) Reduced ethylene sensitivity of 35S-EBF1-TAP, 35S-EBF2-TAP, 35S-EBF1-GFP, and 35S-EBF2-GFP etiolated seedlings grown on MS medium supplemented with 10 μM ACC for 3 d. (B) Immunoblot assay of EBF1-TAP protein (detected by anti-MYC antibody) in 6-d-old light-grown 35S-EBF1-TAP seedlings. (C) Immunoblot assay of EBF2-TAP protein (detected by anti-MYC antibody) in 6-d-old light-grown seedlings. (D) Treatments with Ag⁺ and MG132 but not ACC promoted the accumulation of EBF1-GFP and EBF2-GFP in the nuclei of the transgenic seedlings. Seedlings grown on MS medium were treated with ethylene (20 ppm) for the indicated time, or ACC (100 μM), AgNO₃ (20 μM for GFP detection and 100 μM for immunoblot assays), and MG132 (50 μM) for 4 h before being subjected to immunoblot assays or GFP detection. [See online article for color version of this figure.]

Figure 7.

EIN2 Is Required for Ethylene-Induced EIN3 Stabilization and EBF1/EBF2 Degradation. (A) The triple-response phenotype of 3-d-old etiolated seedlings grown on MS medium supplemented with or without 10 μM ACC. (B) Immunoblot assay of EIN3 protein in EIN3ox and EIN3ox ein2 seedlings treated with mock (0.5% DMSO), ACC (100 μM), CHX (100 μM) or MG132 (50 μM) for 4 h. The relative EIN3 protein levels were calculated after normalization with loading controls and listed. Experiments were repeated two times with similar results. (C) Analysis of ethylene-regulated gene expression (ERF1 and EBF2) by quantitative real-time RT-PCR for 6-d-old light-grown seedlings treated with or without ethylene (20 ppm) for 4 h. Data presented are mean values of three biological repeats with standard deviation. (D) Immunoblot assay of EBF1-TAP protein (detected by anti-MYC antibody) in 6-d-old light-grown 35S:EBF1-TAP ein2 seedlings. (E) Immunoblot assay of EBF2-GFP protein (detected by anti-GFP antibody) in 6-d-old light-grown 35S:EBF2-GFP ein2 seedlings. (F) The GFP fluorescence was detected in the roots of 3-d-old 35S:EBF2-GFP ein2 etiolated seedlings treated with 100 μM ACC, 20 μM AgNO₃, or 50 μM MG132 for 4 h. [See online article for color version of this figure.]

Figure 8.

Ethylene-Induced EBF1 and EBF2 Degradation Is Independent of EIN3/EIL1. Ag⁺ and MG132 treatments promote the nuclear accumulation of EBF1-GFP and EBF2-GFP in the ein3 eil1 mutant background. The GFP fluorescence was detected in the roots of 3-d-old etiolated seedlings treated with mock (0.5% DMSO), 20 μM AgNO₃, or 50 μM MG132 for 4 h.

Figure 9.

MKK9 Is Not Involved in the Ethylene Signaling Pathway. (A) The triple response phenotype of 3-d-old Col-0 and mkk9-5 etiolated seedlings grown on MS medium supplemented with indicated concentrations of ACC, showing similar ethylene sensitivity. (B) Graphical quantification of hypocotyl lengths in (A). (C) The triple response phenotype of indicated genotypes grown on MS medium supplemented with or without 10 μM ACC for 3 d, showing a similar phenotype between mkk9 ctr1 and ctr1. (D) Graphical quantification of hypocotyl lengths in (C). (E) The rosette phenotype of 35-d-old plants, showing no difference between mkk9 ctr1 and ctr1. (F) Quantitative analysis of hypocotyl lengths. Five-day-old light-grown seedlings germinated on water-agar medium supplemented without or with 10 μM ACC. (G) Immunoblot assay of EIN3 protein in 4-d-old etiolated seedlings of the indicated genotypes treated with or without ethylene gas (20 ppm) for 4 h. (H) Analysis of ethylene-regulated gene ERF1 expression by quantitative real-time RT-PCR in 6-d-old light-grown seedlings treated with or without ethylene (20 ppm) for 4 h. Error bars indicate sd (n = 3). Each bar in (B), (D), and (F) represents the average length (±sd) of at least 20 seedlings. Experiments were repeated three times with similar results. [See online article for color version of this figure.]

Figure 10.

A Proposed Model Illustrating the Ethylene Signaling Pathway That Requires EIN2 and EBF1/EBF2 to Regulate the Functions of EIN3 and EIL1. The ethylene signal is perceived by a linear pathway involving the receptors, CTR1, EIN2, and EBF1/EBF2, to ultimately activate EIN3 and EIL1 transcription factors. Ethylene acts to promote the proteasomal breakdown of EBF1/EBF2, which form the SCF complex to target EIN3/EIL1 for proteolysis. Both EIN2 and EBF1/EBF2 are indispensible for ethylene-induced EIN3/EIL1 accumulation. EIN3 and EIL1 act cooperatively but differentially in the ethylene response pathway. The MKK4/MKK5- and MKK9-activated MPK3/MPK6 kinases are believed to enhance ethylene production by upregulating ACS abundance, although MKK9-MPK3/MPK6 were reported to directly phosphorylate EIN3 and modulate its stability (Yoo et al., 2008). Several ethylene signaling components (ETR2, EIN2, EBF1/EBF2, and EIN3/EIL1) are subject to ubiquitin/proteasome-mediated degradation, with the responsible E3 ligases for the receptors and EBF1/EBF2 remaining unidentified. Arrows and bars represent positive and negative regulations, respectively. The solid lines indicate direct regulation, whereas the dotted lines indicate either indirect regulation or regulation in an unknown manner.