The RING E3 ligase SDIR1 destabilizes EBF1/EBF2 and modulates the ethylene response to ambient temperature fluctuations in Arabidopsis

Abstract

The gaseous phytohormone ethylene mediates numerous aspects of plant growth and development as well as stress responses. The F-box proteins EIN3-binding F-box protein 1 (EBF1) and EBF2 are key components that ubiquitinate and degrade the master transcription factors ethylene insensitive 3 (EIN3) and EIN3-like 1 (EIL1) in the ethylene response pathway. Notably, EBF1 and EBF2 themselves undergo the 26S proteasome-mediated proteolysis induced by ethylene and other stress signals. However, despite their importance, little is known about the mechanisms regulating the degradation of these proteins. Here, we show that a really interesting new gene (RING)-type E3 ligase, salt- and drought-induced ring finger 1 (SDIR1), positively regulates the ethylene response and promotes the accumulation of EIN3. Further analyses indicate that SDIR1 directly interacts with EBF1/EBF2 and targets them for ubiquitination and proteasome-dependent degradation. We show that SDIR1 is required for the fine tuning of the ethylene response to ambient temperature changes by mediating temperature-induced EBF1/EBF2 degradation and EIN3 accumulation. Thus, our work demonstrates that SDIR1 functions as an important modulator of ethylene signaling in response to ambient temperature changes, thereby enabling plant adaptation under fluctuating environmental conditions.

Keywords: EBF1/EBF2; SDIR1; ambient temperature; ethylene; ubiquitination.

Fig. 1.

SDIR1 is necessary for the normal response to ethylene. (A) Dosage-response phenotypes of Col-0, sdir1-1, and sdir1-2. Etiolated seedlings were grown on MS medium supplemented with various concentrations of ACC for 3.5 d. (Scale bar, 5 mm.) (B) Phenotypes of 3.5-d-old etiolated Col-0, 35S:SDIR1ox/Col-0 (SDIR1ox) transgenic lines and ebf mutants grown on MS medium supplemented with or without the indicated concentrations of ACC. (Scale bar, 5 mm.) (C) Quantification of hypocotyl length of seedlings shown in A. (D) Quantification of hypocotyl length of the indicated genotypes shown in B. Seedlings were grown on MS medium containing various concentrations of ACC in the dark for 3.5 d. Values in C and D represent means and SD (n ≥ 15 seedlings). Statistical significances (**P < 0.01; ***P < 0.001; NS, not significantly different) were analyzed by two-way ANOVA along with Tukey’s comparison test at a significance level of 0.05.

Fig. 2.

SDIR1 positively regulates the ethylene signaling in an EIN3/EIL1-dependent manner. (A) Ethylene-induced EIN3 accumulation is disrupted in sdir1-2. The 3.5-d-old etiolated seedlings were harvested after treatment with 1 ppm ethylene for different periods of time. (B) SDIR1 promotes EIN3 protein accumulation. Seedlings were grown in the dark for 3.5 d and then treated with 20 ppm ethylene for the indicated time. The numbers below in A and B represent the ratio of EIN3 to HSP90 based on gray-value analysis normalized to the corresponding zero time points of Col-0. Anti-EIN3 antibody was used to examine the endogenous EIN3 protein levels and detection of HSP90 was used as a loading control. (C) Relative expression levels of the marker genes downstream of EIN3 in Col-0 and sdir1-2. Etiolated seedlings were treated with or without 1 ppm ethylene for 2 h. Values represent means and SD (n = 3). (D) Relative expression levels of ERF1 and BCA3 in Col-0 and SDIR1ox. Etiolated seedlings were treated with or without 20 ppm ethylene for 2 h. Values represent means and SD (n = 3). (E) Triple response phenotypes of Col-0, ein3 eil1, SDIR1ox, and SDIR1ox/ein3 eil1 transgenic plants. Seedlings were grown on MS medium supplemented with or without 10 μM ACC in the dark for 3.5 d. (Scale bar, 5 mm.) (F) Hypocotyl length of seedlings shown in F. Values represent means and SD (n ≥ 15 seedlings). (G) Relative expression level of SDIR1 in Col-0 and the indicated mutants. Values represent means and SD (n = 3). Statistical significances (*P < 0.05; **P < 0.01; ***P < 0.001; NS, not significantly different) were analyzed by one-way ANOVA in C and D, and analyzed by two-way ANOVA in F along with Tukey’s comparison test.

Fig. 3.

SDIR1 physically interacts with EBF1 and EBF2 in vivo and in vitro. (A and B) LCI assays showing the interaction between EBF1 (A) or EBF2 (B) and SDIM in Arabidopsis protoplasts. SDIM represents the mutated version of SDIR1, with a single amino acid substitution of His-234 to Tyr-234 to disrupt its E3 activity. SDIMΔTM represents an 81-amino acid deletion on the N terminus of SDIM. TM, transmembrane domain. The indicated combinations of plasmids were cotransformed into Col-0 protoplasts, and luminescence was measured after culturing under low light (2.5 μmol/m²/s) for 16 h. Cps, signal counts per second. (C) Pulldown analysis showing direct interaction between SDIR1 and EBF2 in vitro. Purified MBP and MBP-SDIR1 were immobilized with amylose resin, and then EBF2-His protein was mixed with the corresponding resin. The precipitated products were further blotted with anti-His and anti-MBP antibody, respectively. (D) Yeast two-hybrid assays showing the interactions between SDIMΔTM and EBF1/EBF2. AD and BD indicate the empty vectors pGADT7 and pGBKT7, respectively. Yeast strains were grown on selective dropout medium lacking tryptophan and leucine (SD/−WL) or lacking tryptophan, leucine, histidine and adenine (SD/−WLHA). (E) Co-IP assays showing the interaction between SDIR1 and EBF2 in Arabidopsis protoplasts. All the samples were cultured with 50 μM MG132 and with or without 100 μM ACC treatment for 16 h. Protein extracts were immunoprecipitated by anti-FLAG antibody-coated agarose beads and then the precipitated products were further blotted with anti-HA or anti-FLAG antibody. (F) Detection of endogenous EIN3 protein levels in Col-0 protoplasts treated with or without 100 μM ACC for 16 h. (G) SDIR1 interacts with EBF1/EBF2 in both the ER and the nucleus. The indicated combinations of plasmids were cotransformed into Col-0 protoplasts with or without 50 μM MG132 and 100 μM ACC, and luminescence was observed after culturing under low light for 16 h. BF indicates bright field. (Scale bar, 5 μm.)

Fig. 4.

SDIR1 directly targets EBF2 for ubiquitination and degradation. (A) Ubiquitination of EBF2 by SDIR1 in vitro. E1 (from wheat), E2 (UBCh5b), and tag-free ubiquitin (Ub) were used. Samples were blotted with anti-His antibody. (B) Immunoblot analyses of EBF2-GFP protein levels in 35S:EBF2-GFP/Col-0 and 35S:EBF2-GFP/sdir1-2. Seedlings were treated with 100 μM cycloheximide (CHX) for the indicated time. Anti-GFP and anti-HSP90 antibodies were used for detecting the corresponding proteins, respectively. (C) SDIR1 promotes the proteasomal degradation of EBF2. The 3.5-d-old etiolated seedlings were treated with or without 50 μM β-estrogen and 50 μM MG132 for 4 h. (D) SDIR1 destabilizes EBF2 both in the absence and presence of exogenous ethylene. Seedlings grown on MS medium supplemented with or without 10 μM β-estrogen in the dark for 3.5 d were treated with or without 10 ppm ethylene for 1 h. EBF2-GFP protein levels in C and D were detected by anti-GFP antibody, and detection of HSP90 was used as a loading control. F2G indicates 35S:EBF2-GFP/Col-0, iSDIR1/F2G indicates β-estrogen inducible-SDIR1/35S:EBF2-GFP. The numbers below represent the ratio of EBF2-GFP to HSP90 based on gray-value analysis normalized to the corresponding untreated groups in B, C, and D. (E) Triple response phenotypes of the indicated genotypes grown on 10 μM ACC medium supplemented with or without 20 μM β-estrogen in the dark for 3.5 d. (Scale bar, 5 mm.) (F) Hypocotyl length of seedlings shown in E. Values represent means and SD (n ≥ 15 seedlings). iE3/ee indicates β-estrogen inducible-EIN3/ein3 eil1. Statistical significance (**P < 0.01; ***P < 0.001; NS, not significantly different) was analyzed by two-way ANOVA along with Tukey’s comparison test. (G) Real-time PCR analysis of SDIR1 expression levels in the indicated genotypes. Seedlings were grown on MS medium containing 20 μM β-estrogen in the dark for 3.5 d. Values represent means and SD (n = 3).

Fig. 5.

SDIR1 modulates the ethylene signaling in response to ambient temperature change. (A) Relative transcript levels of SDIR1 in Col-0 at different temperatures. Seedlings were grown on MS medium at 22 °C in the dark for 3.5 d and then transferred to the indicated temperature for 4 h, or constantly grown at 16 °C, 22 °C, or 28 °C for 3.5 d. Values represent means and SD (n = 3). (B) Relative transcript levels of SDIR1 in etiolated Col-0 seedlings treated with ethylene at different ambient temperatures. Seedlings were constantly grown at 16 °C, 22 °C, or 28 °C for 3.5 d, and then treated with or without 1 ppm ethylene for 2 h. Values represent means and SD (n = 3). (C and D) Immunoblot analyses of GFP-EBF1 (C) and GFP-EBF2 (D) protein levels in etiolated 35S:GFP-EBF1/Col-0 (C, Upper), 35S:GFP-EBF1/sdir1-2 (C, Bottom), 35S:GFP-EBF2/Col-0 (D, Left), and 35S:GFP-EBF2/sdir1-2 (D, Right). Seedlings were grown on MS medium at 22 °C in the dark for 3.5 d and then remained at 22 °C or treated at 16 °C or 28 °C for the indicated time before sample collection. (E) Immunoblot analyses of endogenous EIN3 protein levels in etiolated Col-0, sdir1-2, and SDIR1ox. Etiolated seedlings were grown at 22 °C for 3.5 d and then remained at 22 °C or treated at 16 °C or 28 °C for 4 h unless specified. (F) ACC dosage response of Col-0 (Left) and sdir1-2 (Right) at different ambient temperatures. Seedlings were grown on MS medium containing various concentrations of ACC in the dark at 16 °C, 22 °C, or 28 °C for 3.5 d. Hypocotyl lengths were measured and normalized to the corresponding MS group in Col-0 and sdir1-2, respectively. The values represent means and SD (n ≥ 15 seedlings). (G) Immunoblot analyses of EIN3 protein levels in Col-0 at 16 °C, 22 °C, and 28 °C with or without ethylene treatment. (H) Immunoblot analyses of EIN3 protein levels in Col-0 and sdir1-2 at 22 °C and 28 °C with or without ethylene treatment. Etiolated seedlings were constantly grown at the indicated temperature for 3.5 d and then treated with or without 1 ppm ethylene for 1 h before sample collection in G and H. Statistical significances (*P < 0.05; **P < 0.01; ***P < 0.001; NS, not significantly different) were analyzed using a two-tailed Student’s t test in A and B, and by two-way ANOVA along with Tukey’s comparison test in F. GFP-EBF1 in C and GFP-EBF2 in D were detected by anti-GFP antibody. EIN3 protein levels in E, G, and H were detected with anti-EIN3 antibody. Detection of HSP90 in each immunoblot analysis was used as the loading control. The numbers below represent the ratio of GFP-EBF1 (C), GFP-EBF2 (D), or EIN3 (E, G, and H) to HSP90 based on gray-value analysis normalized to the corresponding far Left (C) or the untreated 22 °C group of Col-0 (D, E, G, and H).

Fig. 6.

A working model depicting the SDIR1-modulated ethylene signaling through degradation of EBF1/EBF2. (A) EBF1 and EBF2 negatively regulate ethylene signaling by mediating the proteasome-dependent degradation of EIN3 protein. The RING finger E3 ligase SDIR1 directly targets EBF1/EBF2 for ubiquitination and degradation, thus promoting EIN3 accumulation and the downstream ethylene response. (B) The expression level of SDIR1 is progressively induced by elevated ambient temperatures, enabling SDIR1 to mediate the temperature-induced EBF1/EBF2 degradation and EIN3 accumulation, to fine tune the ethylene response to fluctuating ambient temperatures.