The RING E3 Ligase SDIR1 Destabilizes EBF1/EBF2 and Modulates the Ethylene Response to Ambient Temperature Fluctuations in Arabidopsis

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

Dongdong Haoa,b,c,1, Lian Jina,b,1, Xing Wena,b, Feifei Yud, Qi Xied, and Hongwei Guoa,b,2

aInstitute of Plant and Food Science, Department of Biology, Southern University of Science and Technology, 518055 Shenzhen, China; bKey Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes, Southern University of Science and Technology, 518055 Shenzhen, China; cState Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, 100871 Beijing, China; and dState Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, 100101 Beijing, China

Edited by Joseph J. Kieber, University of North Carolina at Chapel Hill, Chapel Hill, NC, and accepted by Editorial Board Member Joseph R. Ecker January11, 2021 (received for review December 4, 2020) The gaseous phytohormone ethylene mediates numerous aspects ER-integrated receptor proteins (5, 7, 9). Upon ethylene binding of plant growth and development as well as stress responses. The to its receptors, the Raf-like protein kinase constitutive triple F-box proteins EIN3-binding F-box protein 1 (EBF1) and EBF2 are response 1 (CTR1)-mediated phosphorylation of ethylene in- key components that ubiquitinate and degrade the master tran- sensitive 2 (EIN2) is inhibited, leading to the proteolytic cleav- scription factors ethylene insensitive 3 (EIN3) and EIN3-like 1 (EIL1) age and translocation of EIN2 from the ER membrane to the in the ethylene response pathway. Notably, EBF1 and EBF2 them- nucleus (8, 10–15). The downstream transcription factors EIN3 selves undergo the 26S proteasome-mediated proteolysis induced and EIN3-like 1 (EIL1) are then stabilized to regulate the ex- by ethylene and other stress signals. However, despite their im- pression of the vast majority of ethylene-responsive genes (16–18). portance, little is known about the mechanisms regulating the The stability of the EIN3/EIL1 proteins is tightly regulated by degradation of these proteins. Here, we show that a really inter- EIN3-binding F-box protein 1 (EBF1) and EBF2 through the 26S esting new gene (RING)-type E3 ligase, salt- and drought-induced proteasome-mediated protein degradation pathway (19–21). The

ring finger 1 (SDIR1), positively regulates the ethylene response protein levels of EBF1/EBF2 are down-regulated by ethylene but PLANT BIOLOGY and promotes the accumulation of EIN3. Further analyses indicate up-regulated by Ag+ and the 26S proteasome inhibitor MG132, that SDIR1 directly interacts with EBF1/EBF2 and targets them for suggesting that ethylene promotes the accumulation of EIN3/EIL1 ubiquitination and proteasome-dependent degradation. We show at least partly by inducing EBF1/EBF2 proteasomal degradation that SDIR1 is required for the fine tuning of the ethylene response (22). In addition, ethylene also inhibits the translation of EBF1/ to ambient temperature changes by mediating temperature- EBF2 mRNA to further lower the abundance of the EBF1/EBF2 induced EBF1/EBF2 degradation and EIN3 accumulation. Thus, our proteins (23, 24). work demonstrates that SDIR1 functions as an important modulator EBF1 and EBF2 are vital for ethylene signaling, with distinct of ethylene signaling in response to ambient temperature changes, but overlapping roles in regulating EIN3 stability (25). The ebf1 thereby enabling plant adaptation under fluctuating environmental ebf2 double mutants exhibit striking growth defects, including conditions. Significance SDIR1 | EBF1/EBF2 | ubiquitination | ethylene | ambient temperature As a gaseous plant hormone, ethylene plays a vital role in plant he survival of sessile organisms such as plants is threatened growth and development as well as the adaptive responses to Tby various environmental perturbations. However, plants environmental stimuli. EBF1 and EBF2, which ubiquitinate and have developed elaborate mechanisms enabling the rapid percep- degrade the master transcription factors of ethylene signaling tion of the changing conditions along with highly orchestrated (EIN3/EIL1), have emerged as signaling hubs to regulate vari- molecular responses, resulting in extraordinary morphological ous physiological processes. Here, we characterized a RING plasticity. One of these mechanisms is the utilization of plant hor- finger E3 ligase SDIR1 that can directly destabilize EBF1/EBF2 to mones, which are small organic molecules produced endogenously promote the ethylene response. Notably, SDIR1 mediates the in trace quantities (1). Ethylene is the first identified gaseous plant temperature-induced protein degradation of EBF1/EBF2 to fine hormone, exerting a role in a wide variety of plant growth and tune the ethylene signaling at different ambient temperatures. development processes, as well as the adaptive responses to biotic This work elucidates another layer of ethylene signal trans- and abiotic stresses (2). Extensive studies of ethylene have been duction and advances our understanding of the complex conducted due to its great commercial value in agriculture and mechanisms underlying the environmental control of ethylene horticulture, particularly its exceptional importance in controlling signaling. fruit ripening and leaf and flower senescence (2–4). Author contributions: D.H., L.J., X.W., and H.G. designed research; D.H. and L.J. per- Over the past three decades, a number of critical components formed research; F.Y. and Q.X. contributed new reagents/analytic tools; D.H., L.J., X.W., of the ethylene response pathway have been identified using a and H.G. analyzed data; and D.H., L.J., X.W., and H.G. wrote the paper. mutational analysis of the so-called “triple response,” charac- The authors declare no competing interest. terized by dark-grown seedlings that form short, swollen hypo- This article is a PNAS Direct Submission. J.J.K. is a guest editor invited by the cotyls and roots and exaggerated apical hooks when exposed to Editorial Board. ethylene or its precursor 1-amino-cyclopropane-1-carboxylic acid Published under the PNAS license. (ACC) (5–9). Using genetic and molecular approaches, a largely 1 D.H. and L.J. contributed equally to this work. linear ethylene signaling pathway from hormone perception at 2To whom correspondence may be addressed. Email: [email protected]. the endoplasmic reticulum (ER) membrane to transcriptional This article contains supporting information online at https://www.pnas.org/lookup/suppl/ regulation in the nucleus has been established (2, 3). In the doi:10.1073/pnas.2024592118/-/DCSupplemental. model plant Arabidopsis thaliana, ethylene is perceived by five Published February 1, 2021.

PNAS 2021 Vol. 118 No. 6 e2024592118 https://doi.org/10.1073/pnas.2024592118 | 1of10 Downloaded by guest on September 26, 2021 severe dwarfism and seedling lethality, suggesting that EBF1 and response of sdir1 mutants. Results showed that compared with EBF2 are essential for plant growth and development (19–21). the wild type (Col-0), the two mutant alleles sdir1-1 and sdir1-2 In addition to ethylene, EBF1 and EBF2 are widely involved in exhibited a significantly decreased sensitivity to the concentrations the response to many other stimuli. For example, cold stress of ACC from 0 to 0.5 μM(Fig.1A and C). With the concentration induces EIN3 protein accumulation by destabilizing the EBF1 of ACC increased, the differences between the mutants and Col- protein (26). High salinity also promotes EBF1/EBF2 degrada- 0 were attenuated (SI Appendix, Fig. S1 A and B), indicating that tion to stabilize EIN3 (27). It was reported that EBF1/EBF2 are SDIR1 is peculiarly necessary for the response to low concentra- directly targeted for ubiquitination by constitutive photomorpho- tions of ethylene. We further examined the phenotypes of SDIR1ox genesis 1 (COP1), which plays important roles in sensing light transgenic plants and found that the hypocotyls of SDIR1ox were fluences during seedling emergence from the soil, but not in the constitutively shorter than Col-0 in the absence of exogenous ACC. ethylene-induced stabilization of EIN3 (28). Upon reaching the Upon ACC treatment, SDIR1ox showed stronger triple response soil surface, the red light receptor phytochrome B (phyB) termi- phenotypes, similar to the ethylene-hypersensitive mutants ebf1-1 nates the ethylene response by enhancing the interaction between and ebf2-1 (20), as evidenced by significantly shorter hypocotyls EBF1/EBF2 and EIN3 to ensure deetiolation (29). Moreover, and roots than Col-0 (Fig. 1 B and D). In addition, we also exam- EBF1/EBF2 directly destabilizes phytochrome-interacting factor 3 ined the responses of these genotypes to ethylene treatment. Con- (PIF3) via proteasome-dependent protein degradation to facilitate sistently, sdir1-2 was less sensitive than Col-0 in response to lower photomorphogenesis and improve freezing tolerance (30, 31). concentrations of ethylene (SI Appendix,Fig.S1C and D). Mean- Overall, EBF1 and EBF2 are emerging as signaling hubs to mod- while, similar to ebf1-1 and ebf2-1,theSDIR1ox transgenic plants ulate various physiological processes in plants; therefore, uncover- showed hyperresponsiveness to ethylene treatment (SI Appendix, ing the molecular mechanisms of EBF1/EBF2 regulation would be Fig. S1 E and F). Together, these data suggest that SDIR1 is a of particular relevance in the context of plant fitness. positive regulator of the ethylene response. The ubiquitin-proteasome degradation system (UPS) plays roles in nearly every aspect of plant biology, such as modulating SDIR1 Works Upstream of EIN3/EIL1 to Positively Regulate the Ethylene phytohormone signaling pathways (32, 33). The UPS activity in- Signaling. Upon ethylene treatment, the master transcription fac- volves a three-step enzymatic cascade including the E1, E2, and tor EIN3 accumulates rapidly to induce the downstream ethylene E3 enzymes, of which E3 ligases are responsible for substrate response (16, 20). To further determine the function of SDIR1 in recognition. In this study, we revealed that a really interesting new ethylene signaling, we analyzed EIN3 protein accumulation and gene (RING)-type E3 ligase, salt- and drought-induced ring finger the induction of downstream genes in sdir1-2 and SDIR1ox 1 (SDIR1), positively regulates ethylene signaling in Arabidopsis. (SDIR1ox #8 was used to represent SDIR1ox in the following SDIR1, the expression of which is induced by salt and drought, is experiments). Given the aberrant phenotypes of the sdir1 mutants localized on the ER membrane with its C terminus facing the were more apparent at low concentrations of ethylene, we first cytoplasm (34, 35). This enzyme is known to promote the response determined the EIN3 protein levels upon 1 ppm ethylene treat- to abscisic acid (ABA) and high salinity, while it enhances plant ment. Our results showed that the ethylene-induced EIN3 accu- resistance to drought stress (34). SDIR1-interacting protein 1 mulation was compromised in sdir1-2 compared with Col-0, (SDIRIP1), the first identified substrate of SDIR1, acts down- supporting a prominent role of SDIR1 in this condition (Fig. 2A). stream of SDIR1 and upstream of the transcription factor ABA When treated with a relatively high concentration of ethylene (20 insensitive 5 (ABI5) to negatively regulate the responses to ABA ppm), there was no significant difference in EIN3 abundance and salt stress (35). SDIR1 and its orthologs in other plant species between Col-0 and sdir1-2, except that the levels of EIN3 protein are widely involved in stress responses, especially the resistance to in sdir1-2 were slightly lower than Col-0 with treatment for 2 h (SI drought stress in crop plants (36–41). Recently, TaSDIR1-4A, a Appendix,Fig.S2A). This result was consistent with the mild SDIR1 ortholog in wheat (Triticum aestivum), was found to be a phenotypes of sdir1 mutants at high concentrations of ethylene. negative regulator of grain size, and might be regulated by ethyl- To further study the effect of SDIR1 on EIN3 accumulation, ene response factor 3 (TaERF3) (42). These results indicate that we also examined the EIN3 protein levels in SDIR1ox. We no- SDIR1 is an important stress and growth regulator in both Ara- ticed that the EIN3 protein in SDIR1ox was more abundant than bidopsis and crop plants. Col-0 even in the absence of ethylene (Fig. 2B), which may ex- Here, we show that sdir1 mutants display reduced sensitivity to plain the constitutively shorter hypocotyl of SDIR1ox. After ethylene, while SDIR1-overexpressing transgenic lines (SDIR1ox) ethylene treatment, the EIN3 protein accumulated to signifi- have an enhanced ethylene response. Mechanistic studies reveal cantly higher levels in SDIR1ox compared with Col-0 (Fig. 2B). that SDIR1 directly interacts with and ubiquitinates EBF1/EBF2, In addition to ethylene, similar results were obtained with ACC mediating the proteasome-dependent degradation of EBF1/ applications (SI Appendix, Fig. S2B). The differences in EIN3 EBF2. The expression of SDIR1 is induced by elevated ambient accumulation among these genotypes were abolished upon fur- temperatures. Accordingly, ethylene signaling is altered alongside ther treatment with the 26S proteasome inhibitor MG132 (SI increases in the ambient temperature, which is partially dependent Appendix, Fig. S2B). Moreover, the levels of EIN3 mRNA were on SDIR1-mediated EBF1/EBF2 degradation and EIN3 accu- not altered by SDIR1 or ethylene regardless of the treatment mulation. Overall, our work demonstrates that SDIR1 functions as concentration and time (SI Appendix, Fig. S2C). These results an important modulator to fine tune the ethylene response during indicate that SDIR1 promotes EIN3 accumulation at the post- ambient temperature fluctuations. translational level. Consistently, the expression levels of EIN3 target genes, including ethylene response factor 1 (ERF1), hookless Results 1 (HLS1), beta carbonic anhydrase 3 (BCA3), and MYB domain SDIR1 Is Necessary for the Normal Response to Ethylene. In order to protein 14 (MYB14) (16), were lower in sdir1-2, while some of identify new regulators in ethylene signaling, we screened a them (ERF1 and BCA3) were higher in SDIR1ox than Col-0 after collection of Arabidopsis stress-related mutants in search of those treatment with ethylene (Fig. 2 C and D). These results suggest with abnormal triple responses. The seedlings were grown on the that SDIR1 promotes the ethylene response, likely by affecting Murashige and Skoog (MS) medium supplemented with or EIN3 accumulation. without 0.5 μM ACC, a relatively low dose that was used to To assess this possibility, the coding sequence of SDIR1 driven screen weak ethylene-insensitive (wei) mutants (43). Among them, by the 35S promoter was overexpressed in the ein3 eil1 back- the RING-type E3 ligase sdir1 mutants attracted our interest. To ground. As expected, the SDIR1ox/ein3 eil1 transgenic lines further confirm the phenotype, we examined the ethylene dosage showed the same phenotype as ein3 eil1 both in the absence and

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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.

presence of ACC (Fig. 2 E and F), even though the SDIR1 ex- SDIR1 is responsible for the interaction (Fig. 3 A and B and SI pression levels in the SDIR1ox/ein3 eil1 transgenic lines were Appendix,Fig.S3B). In coimmunoprecipitation (co-IP) assays, comparable to SDIR1ox (Fig. 2G). These data indicate that the myc-SDIR1 was coprecipitated with GFP-EBF1 using GFP function of SDIR1 is dependent on EIN3/EIL1. Besides, ethylene antibody-conjugated beads (SI Appendix,Fig.S3C, Top). Recip- production is not evidently affected by SDIR1, further supporting rocally, GFP-EBF1 was coprecipitated with HA-SDIR1 using HA the role of SDIR1 in ethylene signaling rather than its biosynthesis antibody-conjugated beads (SI Appendix,Fig.S3C, Bottom), pro- (SI Appendix,Fig.S2D). We therefore conclude that SDIR1 viding further evidence for the SDIR1-EBF1 interaction in planta. functions upstream of EIN3/EIL1 to positively regulate ethylene In addition, an in vitro pulldown assay demonstrated a direct in- signaling. teraction between the MBP-tagged SDIR1 protein and His-tagged EBF2 protein (Fig. 3C). SDIR1 Physically Interacts with EBF1 and EBF2. To further explore Moreover, we performed yeast two-hybrid (Y2H) assays to the mechanism of SDIR1 in ethylene signaling, we investigated the verify the interaction. The results showed that SDIMΔTM but interaction between SDIR1 and the ethylene signaling components not SDIR1ΔTM strongly interacted with EBF1/EBF2 (Fig. 3D), using luciferase (LUC) complementation imaging (LCI) assays in which suggests that the E3 ligase activity of SDIR1 may hinder or Arabidopsis protoplasts. SDIR1 is a RING finger protein and was abolish this interaction in yeast cells. In accordance, SDIRIP1 reported to be a functional E3 ligase (34). To avoid substrate deg- was previously identified in a Y2H screen using SDIMΔTM but radation, we used a mutated version of SDIR1, named SDIM, with not SDIR1ΔTM as the bait (35). To further dissect the regions a single amino acid substitution of His-234 to Tyr-234 to block its E3 that mediate the interaction, we constructed a series of deletion ligase activity without affecting the interaction with its substrate (35). mutants lacking regions of SDIR1/SDIM or EBF1/EBF2 (SI We found that SDIM specifically interacts with EBF1/EBF2 but not Appendix, Fig. S3D). The Y2H results showed that the entire C other signaling components, including the receptor ethylene re- terminus of SDIR1 was necessary for its interaction with EBF1/ sponse 1 (ETR1), CTR1, and EIN2, in the protoplasts (Fig. 3 A and EBF2, while the leucine-rich repeat domains of EBF1/EBF2 were B and SI Appendix,Fig.S3A). LCI assays conducted in tobacco both necessary and sufficient for the interaction with SDIMΔTM (Nicotiana benthamiana) leaves further confirmed the SDIM-EBF2 (SI Appendix,Fig.S3E). Taken together, these results demonstrate interaction (SI Appendix,Fig.S3B). In addition, we observed a that SDIR1 directly interacts with EBF1 and EBF2 in vivo and relatively strong interaction between EBF1/EBF2 and SDIMΔTM in vitro. (containing an 81-amino acid deletion on the N-terminal trans- To further examine the effect of ethylene on the interaction membrane domain), indicating that the cytoplasmic C terminus of between SDIR1 and EBF1/EBF2, we conducted the co-IP assays

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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.

in Arabidopsis protoplasts with or without ACC treatment. HA- the nucleus, to in both the cytosol and the nucleus (Fig. 3G and EBF2 was coprecipitated with FLAG-SDIR1, suggesting the SI Appendix, Fig. S3G). SDIR1 didn’t interact with the C ter- in vivo association between SDIR1 and EBF2. However, this minus of EIN2 even in the presence of MG132 (SI Appendix, Fig. interaction was not significantly affected by ACC treatment S3H), suggesting the specificity of the SDIR1-EBF1/EBF2 in- (Fig. 3E). The levels of EIN3 protein in protoplast increased in teraction in this system. Besides, ethylene seemingly had no ef- the presence of ACC, indicating the treatment was effective in fect on the localization of SDIR1 (SI Appendix, Fig. S3I), as well our experiment (Fig. 3F). Next, we explored the subcellular lo- as the subcellular localization of the SDIR1-EBF2 interaction calization of the SDIR1-EBF1/EBF2 interaction. It was reported (Fig. 3G). Overall, these results reveal that SDIR1 specifically in- that the localization of SDIR1 is not affected by the His-to-Tyr teracts with EBF1 and EBF2 in an ethylene-independent manner. mutation in SDIM (35), so we first examined the colocalization of SDIM and EBF2. We found that mCherry-EBF2 localized to SDIR1 Directly Targets EBF1/EBF2 for Ubiquitination and Degradation. both the nucleus and cytoplasm in the tobacco leaves, and par- As SDIR1 is an active RING finger E3 ligase (34, 35) and in- tially colocalized with GFP-SDIM in the cytosol (SI Appendix, teracts with EBF1/EBF2 (Fig. 3 and SI Appendix, Fig. S3), we Fig. S3F). To determine the subcellular localization of the in- speculated that EBF1 and EBF2 are the substrates of SDIR1. To teraction, we performed a bimolecular fluorescence comple- verify this hypothesis, we carried out an in vitro ubiquitination mentation (BiFC) assay in protoplast. In the absence of MG132, assay using EBF2-His and MBP-tagged E3 ligases. In the pres- we couldn’t detect evident interaction signal between SDIR1 and ence of the universal E1, E2, and tag-free ubiquitin, EBF2 was EBF1/EBF2, which may be caused by the SDIR1-mediated polyubiquitinated by MBP-SDIR1 but not the MBP protein alone degradation of EBF1/EBF2 (Fig. 3 G, Left). Upon MG132 (Fig. 4A). Moreover, when the transmembrane domain was de- treatment, we observed that SDIR1 interacted with EBF1/EBF2 leted, SDIR1ΔTM still possessed the ability to ubiquitinate EBF2 in both the cytosol (partially colocalized with the ER marker) in vitro (Fig. 4A). These results indicate that the C terminus of and the nucleus (Fig. 3 G, Middle and Right). Nevertheless, the SDIR1 directly interacts with and ubiquitinates EBF2. localizations of the interaction were not always the same in dif- Since ubiquitination leads to the 26S proteasome-dependent ferent cells, which varied from mainly in the cytosol or mainly in degradation of the target proteins, we next examined the effect

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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)orEBF2(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 cul- tured 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.)

of SDIR1 on EBF1/EBF2 protein stability. Upon the translation EBF2 protein was significantly decreased when the expression of inhibitor cycloheximide treatment, the EBF1 and EBF2 proteins SDIR1 was induced by β-estrogen in the iSDIR1/35S:EBF2-GFP degraded gradually, with half-lives of less than 60 min in Col-0, transgenic seedlings, which could be restored by the MG132 but these were greatly prolonged in sdir1-2 (SI Appendix, Fig. application (Fig. 4C and SI Appendix, Fig. S4C). These results S4A and Fig. 4B), indicating that the degradation of EBF1/EBF2 suggest that SDIR1 promotes the proteasome-dependent deg- is inhibited by the sdir1 mutation. In contrast, overexpression of radation of EBF2 in Arabidopsis. Consistently, transient expres- SDIR1 significantly lowered the abundance of EBF1 protein (SI sion experiments in tobacco leaves also showed that SDIR1 Appendix, Fig. S4B). Besides, we constructed a β-estrogen-in- attenuated EBF2 protein accumulation and MG132 abolished ducible expression vector of SDIR1 (iSDIR1) and transformed it the effect of SDIR1 (SI Appendix, Fig. S4D). We found that into the 35S:EBF2-GFP/Col-0 background. The accumulation of SDIR1 down-regulated the levels of EBF2 protein in the absence

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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 μMMG132for4h.(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,andD.(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).

or presence of exogenous ethylene (Fig. 4D and SI Appendix, Fig. findings provide further support for the effect of SDIR1 on S4E), consistent with the finding that ethylene is not required for EBF1/EBF2 stability. In addition, we also tested the necessity of the SDIR-EBFs interaction (Fig. 3 E and G). Based on these SDIR1 E3 activity for its ability to regulate the ethylene signal- results, we conclude that SDIR1 is the E3 ligase that directly ing. As expected, the SDIM protein harboring the mutation of targets EBF1/EBF2 for ubiquitination and proteasome-dependent His-234 to Tyr-234 could not ubiquitinate EBF2 in vitro (SI degradation. Appendix, Fig. S5A), though the mutation didn’t affect its in- The 35S:EBF2-GFP/Col-0 transgenic lines display ethylene teraction with EBF1/EBF2 (Fig. 3 and SI Appendix, Fig. S3). insensitivity because of excessive accumulation of EBF2, which Notably, SDIMox transgenic plants exhibited a wild-type re- impedes EIN3 accumulation even in the presence of ethylene sponse to ethylene but not a hyperresponsive phenotype as (20, 22). Consistent with the above biochemical results, the in- SDIR1ox did, which may be due to the inability of SDIM to duction of SDIR1 expression using β-estrogen partially restored degrade EBF1/EBF2 protein (SI Appendix, Fig. S5 B–D). These the ethylene response of 35S:EBF2-GFP/Col-0 (Fig. 4 E and F). results indicate that the E3 ligase activity is necessary for the Furthermore, the extents of the phenotypic rescue among the function of SDIR1 in ethylene signaling, further supporting the three independent iSDIR1/EBF2-GFP transgenic lines were finding that SDIR1 modulates the ethylene response by directly correlated to their SDIR1 expression levels (Fig. 4 E–G). These controlling the protein stability of EBF1/EBF2.

6of10 | PNAS Hao et al. https://doi.org/10.1073/pnas.2024592118 The RING E3 ligase SDIR1 destabilizes EBF1/EBF2 and modulates the ethylene response to ambient temperature fluctuations in Arabidopsis Downloaded by guest on September 26, 2021 SDIR1 Mediates the Alteration of Ethylene Signaling in Response to the fold change of EIN3 abundance was larger at 16 °C (Fig. 5 G, Ambient Temperature Change. We next sought to explore the bi- Left). In contrast, EIN3 protein levels were higher at 28 °C than ological significance underlying the SDIR1-mediated degrada- 22 °C both in the absence and presence of ethylene, whereas the tion of EBF1/EBF2. Previous studies revealed that SDIR1 is ethylene-induced fold change of EIN3 abundance was lower at involved in the response to ABA, salt, and drought stresses (34, 28 °C (Fig. 5 G, Right). These results provide an explanation for 35). Coincidently, high salinity promotes the degradation of why ethylene sensitivity is higher at 16 °C and lower at 28 °C and EBF1/EBF2 protein to stabilize EIN3 protein, which confers the also indicate that temperature regulates the ethylene response resistance to salt stress (27). Therefore, we first examined the partly by controlling the abundance of EIN3 protein. Meanwhile, effect of SDIR1 on the salt-induced EIN3 accumulation. Con- the ethylene-induced EIN3 accumulation was suppressed in sistently, the levels of EIN3 protein were induced by salt; how- sdir1-2 both at 22 °C and 28 °C (Fig. 5H). Taken together, these ever, the EIN3 protein abundance in sdir1-2 was comparable to results lead us to conclude that SDIR1 mediates the alteration of Col-0 after salt treatment (SI Appendix, Fig. S6), indicating that ethylene signaling in response to ambient temperature change SDIR1 is not involved in the salinity-induced destabilization of through the degradation of the EBF1 and EBF2 proteins. EBF1/EBF2 protein. In addition to salt and drought, we found In addition to the protein abundance, the transcriptional ac- that the transcript levels of SDIR1 were also significantly induced tivity of EIN3 is also subjected to regulation in response to by elevated ambient temperatures, either using a transient or myriad signals (44–48). Our results above showed that the levels long-term temperature treatment (Fig. 5A). Moreover, ambient of EIN3 protein were up-regulated while the ethylene responses temperature seemed to modulate the effect of ethylene on were weakened by elevated ambient temperatures, implying that SDIR1 gene expression. The transcript levels of SDIR1 were not EIN3 protein may be less active at higher temperatures. To test affected by ethylene at 16 °C, but obviously decreased after this possibility, we examined the transcript levels of a selection of ethylene treatment at 28 °C (Fig. 5B). These results suggest that EIN3 target genes at different ambient temperatures (16). The SDIR1 may play a role in the communication between the eth- expression of these genes was in general induced by ethylene in ylene response and ambient temperature. The stability of the an EIN3/EIL1-dependent manner, but to variable extents at EBF1/EBF2 protein is directly controlled by SDIR1; thus, we different temperatures (SI Appendix, Fig. S8). Although the ex- next examined whether ambient temperature could regulate the pression levels of most EIN3 target genes were lower at 16 °C abundance of EBF1/EBF2. The levels of EBF1/EBF2 protein than at 22 °C, there were no significant differences in the basal decreased as the temperature increased from 16 °C to 28 °C, level as well as ethylene induction of several of them between which was partly mediated by SDIR1 as the sdir1 mutation 28 °C and 22 °C (SI Appendix, Fig. S8). Given the levels of EIN3 greatly disrupted the temperature-induced degradation of EBF1/ protein are obviously higher at 28 °C than 22 °C, it is reasonable to PLANT BIOLOGY EBF2 protein without affecting the corresponding mRNA levels speculate that the transcriptional activity of EIN3 may be pro- of EBF1/EBF2 (Fig. 5 C and D and SI Appendix,Fig.S7A and B). gressively repressed with the increase of ambient temperature. Consistently, elevated ambient temperatures enhanced the accumulation of EIN3 protein, which was attenuated by the sdir1 Discussion mutation and magnified by the overexpression of SDIR1 (Fig. 5E). Given that the control of EIN3/EIL1 stability mediated by EBF1/ Meanwhile, EIN3 mRNA levels remained unchanged or only EBF2 is vital for the ethylene response (20–22), the significance slightly increased with a transient or long-term temperature of the regulation on EBF1/EBF2 abundance and/or activity is treatment, respectively, and there were no significant differ- apparent. Emerging evidence has also demonstrated that EBF1 ences in EIN3 transcript levels among Col-0, sdir1-2,andSDIR1ox and EBF2 act as signaling hubs to regulate many aspects of plant at different temperatures (SI Appendix,Fig.S7C and D).Inad- growth and stress responses (26–31). Despite their importance, dition, we also confirmed the effect of temperature using the the regulatory mechanisms of the EBF1 and EBF2 functions 35S:EIN3-GFP/ein3 eil1 transgenic plants and found that the remain poorly understood. In this study, we identified a RING-type abundance of EIN3 protein was obviously induced by temperature E3ligase,SDIR1,thatdirectly interacts with EBF1/EBF2 and elevation either with a transient or long-term treatment (SI Ap- mediates their ubiquitination-dependent degradation, thus pro- pendix, Fig. S7E). moting EIN3 accumulation and the downstream ethylene response Next, we analyzed the ethylene response at different temper- (Fig. 6A). Moreover, the transcript levels of SDIR1 are gradually atures. We found that ethylene responsiveness in Col-0 gradually up-regulated as the ambient temperature increases, enabling decreased as the temperature was increased from 16 °C to 28 °C SDIR1 to mediate the temperature-induced degradation of EBF1/ (Fig. 5 F, Left), suggesting that the regulation of EBF1/EBF2 and EBF2 and promote EIN3 accumulation to modulate the ethylene EIN3 abundance by temperature may influence the ethylene response at different ambient temperatures (Fig. 6B). Overall, we response under different temperature conditions. In accordance, propose that SDIR1 may function as an important modulator co- this temperature effect was disturbed in sdir1-2, in which rela- ordinating the response to ethylene and temperature signals by di- tively small differences in ethylene responsiveness were observed rectly destabilizing EBF1/EBF2 (Fig. 6). Our work provides another as the temperature increased (Fig. 5 F, Right). To exclude the layer for ethylene signal transduction and insights into the com- possible differences in the conversion of ACC to ethylene at munication between plants and their surrounding environments. different temperatures, we also did the dose–response directly The UPS plays important roles in hormone signaling, includ- with ethylene treatment. Consistently, the ethylene sensitivity ing in the perception of auxin, jasmonic acid, and gibberellin, as was down-regulated by elevated ambient temperature, whose well as the signal transduction of ABA and strigolactone (33). effect was impaired by the sdir1 mutation (SI Appendix, Fig. Multiple steps of ethylene biosynthesis and signal transduction S7F). These results suggest that SDIR1 plays an important role are also tightly controlled by the UPS, including those involving in the alteration of the ethylene response by ambient tempera- ACC synthase 4 (ACS4)/ACS5/ACS9 (49–51), ACS7 (52), ETR2 ture, likely by mediating the degradation of the EBF1/EBF2 (53), EIN2 (54), EBF1/EBF2 (22, 28), and EIN3/EIL1 (19–22). protein during temperature fluctuations. To further explore the Despite the importance of EBF1/EBF2 in controlling the ethylene molecular mechanism underlying the temperature-modulated response, the mechanism underlying the proteasome-dependent ethylene response, we examined the EIN3 protein levels at dif- degradation of EBF1/EBF2 is poorly understood. The E3 ligase ferent temperatures following an ethylene treatment. Compared COP1 was reported to target EBF1/EBF2 for degradation; how- with the levels at 22 °C, the basal levels of EIN3 were lower at ever, it specifically functions in sensing light fluences during the 16 °C. After the ethylene treatment, EIN3 accumulation was protrusion of seedlings from the soil and is not involved in ethylene- induced; although it was still less abundant at 16 °C than 22 °C, induced EIN3 stabilization (28). Here, our results demonstrate that

Hao et al. PNAS | 7of10 The RING E3 ligase SDIR1 destabilizes EBF1/EBF2 and modulates the ethylene response to https://doi.org/10.1073/pnas.2024592118 ambient temperature fluctuations in Arabidopsis Downloaded by guest on September 26, 2021 ABC

D E

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).

SDIR1 is another E3 ligase directly controlling EBF1/EBF2 abun- ethylene produced during ripening are typically less than 1 ppm dance, which represents a hierarchical action of the E3 ligases in (57). Even under some stress conditions, such as cold, high sa- ethylene signaling. Unlike COP1, SDIR1 is necessary for the nor- linity, potassium deprivation, iron and phosphorus deficiency, mal response to ethylene, as sdir1 mutants exhibit reduced ethylene ethylene production in Arabidopsis only increases by 1.5- to sensitivity and EIN3 accumulation (Figs. 1 and 2 and SI Appendix, 2-fold (58–62), which seems still less than the high concentration Fig. S1), which explicitly suggests a regulatory role for SDIR1 in (10 ppm) used in our experiments. Hence, the response to rel- ethylene signaling. atively low concentrations of ethylene might also have profound We noticed that SDIR1 plays a more prominent role in re- impacts on plants with physiological significance. In addition to sponse to low concentrations of ethylene (Fig. 1A and SI Ap- the proteasomal degradation of the EBF1/EBF2 proteins, the pendix, Fig. S1 A–D). In fact, the ethylene levels produced by inhibition of EBF1/EBF2 mRNA translation mediated by EIN2 plants in the vegetative growth phase are normally low (55). It also plays a vital role in the ethylene response, which is partic- was reported that dark-grown Col-0 seedlings produce only 0.001 ularly evident following treatments with high concentrations of to 0.002 ppm ethylene per seedling in 3 d from germination (56). ethylene (23, 24). How the two regulatory modules coordinate to Besides, ethylene can stimulate the ripening of fruit at concen- regulate ethylene signaling remains an open question. None- trations as low as 0.01 ppm (56), and the physiological levels of theless, it is conceivable that the two parallel mechanisms may

8of10 | PNAS Hao et al. https://doi.org/10.1073/pnas.2024592118 The RING E3 ligase SDIR1 destabilizes EBF1/EBF2 and modulates the ethylene response to ambient temperature fluctuations in Arabidopsis Downloaded by guest on September 26, 2021 A B S6). Similarly, COP1 destabilizes EBF1/EBF2 but is not involved in the typical ethylene-induced accumulation of EIN3. These obser- vations suggest that E3 ligases degrade their substrates likely under given conditions to specifically and coordinately regulate plant adaptations to environmental cues. Therefore, whether SDIR1 also participates in the cross-talk between ethylene and other environmental signals would be an interesting topic for future studies. Given that SDIR1 has a great importance in improving the stress tolerance of crop plants, our elucidation of its roles in the responses to ambient temperature and ethylene will present new opportunities for the utilization of SDIR1 in agriculture. In addition to SDIR1, ethylene response may also be affected by many other factors during ambient temperature fluctuations. Since many aspects of ethylene effect are realized through the Fig. 6. A working model depicting the SDIR1-modulated ethylene signaling action of auxin, the temperature-modulated auxin response may through degradation of EBF1/EBF2. (A) EBF1 and EBF2 negatively regulate contribute to the alteration of ethylene response at different ethylene signaling by mediating the proteasome-dependent degradation of ambient temperatures (43, 66, 67). Besides, it was reported that EIN3 protein. The RING finger E3 ligase SDIR1 directly targets EBF1/EBF2 for the activation of phyB results in the degradation of EIN3 by ubiquitination and degradation, thus promoting EIN3 accumulation and the enhancing the association of EBF1/EBF2 and EIN3 (29); hence, downstream ethylene response. (B) The expression level of SDIR1 is progres- it is likely that the high ambient temperature deactivation of sively induced by elevated ambient temperatures, enabling SDIR1 to mediate phyB might also participate in the regulation of EIN3 function. the temperature-induced EBF1/EBF2 degradation and EIN3 accumulation, to fine tune the ethylene response to fluctuating ambient temperatures. In addition, dramatic chromatin remodeling like histone modi- fications is observed during ambient temperature change (64), and EIN3 activity was reported to be regulated by histone modi- operate in different states of ethylene signaling, together keeping fications as well (48, 68). Therefore, it is conceivable that the EBF1/EBF2 protein levels under tight control, which is essential transcriptional activity of EIN3 at the chromatin level may be for the proper activation of the ethylene response upon the affected by temperature change. In line with this notion, our ex- perception of various environmental stimuli. pression analysis with a selection of EIN3 target genes also sug- Plants are continuously exposed to changes in environmental gested a relatively reduced activity of EIN3 at high ambient PLANT BIOLOGY conditions throughout their lifecycle, with light and ambient temperature. Nonetheless, further investigation is needed to fully temperature being two of the most prominent cues affecting understand the mechanisms and importance of the temperature- their growth and development. As a growth and stress hormone, controlled ethylene response. ethylene is vital for plant adaptations to environmental changes (2–4, 63). COP1, a key component in light signaling, directly Materials and Methods targets EBF1/EBF2 for ubiquitination and degradation, provid- Detailed descriptions of plant materials, growth conditions, and treatments ing a direct link between the light and ethylene signaling path- are described in SI Appendix, SI Materials and Methods. Generation of trans- ways (28). Here, our work demonstrates that ethylene signaling is genic plants and mutants, hypocotyl and root length measurements, RNA also regulated by ambient temperature (Fig. 5). We found that extraction and real-time PCR, total protein extraction and immunoblotting, an elevated ambient temperature promotes EBF1/EBF2 degra- ethylene production measurement, transient expression in N. benthamiana, dation and EIN3 accumulation to modulate the ethylene response, LCI assay, BiFC assay, co-IP assay, pulldown assay, yeast two-hybrid assay, which is partially dependent on SDIR1. In the natural world, in vitro ubiquitination assay, and microscopy were carried out according to temperature fluctuates mildly under the soil, but varies greatly at protocols described in SI Appendix, SI Materials and Methods. the surface of the soil (4, 64), indicating a possible temperature Data Availability. All study data are included in the article and/or SI Appendix. challenge for seedlings emergence from the soil. Previous studies have revealed essential functions for ethylene in seedling germina- ACKNOWLEDGMENTS. We thank Huawei Zhang (Chinese Academy of tion (28, 29, 65); therefore, it is plausible that the SDIR1-mediated Sciences) for kindly providing purified SDIR1ΔTM protein and suggestions. modulation of ethylene signaling may enable a germinating seedling We also thank all members of the H.G. laboratory for helpful discussions and to adapt to temperature fluctuations immediately after its emer- suggestions. This work was funded by the National Natural Science Founda- tion of China (Grant 31800231 to L.J. and Grant 31970308 to X.W.), Shenz- gence. Interestingly, despite the established functions of SDIR1 and hen Science and Technology Program (KQTD20190929173906742 to H.G.), ethylene signaling in response to salt stress, SDIR1 is not involved and Key Laboratory of Molecular Design for Plant Cell Factory of Guang- in the salt-induced stabilization of EIN3 protein (SI Appendix,Fig. dong Higher Education Institutes (SUSTech) (2019KSYS006 to H.G.).

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