Dear Editor,

Ethylene gas is a classic phytohormone regulating many aspects of plant development and defense. Molecular and genetic studies have revealed a highly conserved signaling pathway starting from the ER membrane-associated receptors to transcription factors in the nucleus1. EIN2 (ETHYLENE INSENSITIVE2) is an essential positive regulator of ethylene signaling in Arabidopsis thaliana, as loss-of-function ein2 mutants are completely insensitive to ethylene2. EIN2 encodes a 1 294 amino acid protein that comprises a membrane-spanning amino terminus and a functionally unknown carboxyl terminus3. EIN2 was reported to localize at the ER membrane when transiently expressed in tobacco leaf cells4. EIN3 (ETHYLENE INSENSITIVE3) and its homolog EIL1 (EIN3-LIKE1) are two nuclear-localized transcription factors genetically acting downstream of EIN25,6. However, it remains a long mystery on how the ethylene signal is transmitted from ER-located EIN2 into the nucleus to modulate EIN3/EIL1-directed transcription. Here we report that the carboxyl end of EIN2 (CEND) is a trafficking signal translocating from ER membrane to the nucleus. Ethylene signal promotes the cleavage of CEND from ER-located EIN2, and facilitates its nuclear localization to stabilize EIN3 protein.

It was reported that constitutive expression of EIN2 carboxyl terminal end (CEND, amino acids 459-1 294) leads to partial activation of ethylene responses in light-grown plants3, implying that CEND might function in signal transmission. The CEND fragment contains a putative nuclear localization signal (NLS, amino acids 1 262-1 269), which is highly conserved among numerous EIN2 orthologs of angiosperms (Figure 1A)7. To determine whether CEND is nuclear-localized, we expressed CEND-GFP in tobacco leaf cells and found that GFP fluorescence was detected in both nucleus and cytoplasm (Figure 1B). By contrast, deletion of the NLS retained the CEND-GFP fusion protein exclusively in the cytoplasm (Figure 1B), suggesting that the NLS sequence is responsible for CEND's nuclear localization. Constitutive expression of CEND has been shown to induce ethylene response phenotypes, including elongated hypocotyl of light-grown seedlings, compacted rosette, reduced fertility, abnormal flower with protruded gynoecium, and activation of downstream gene expression3. Similarly, constitutive expression of CEND-GFP was also able to induce these ethylene responses (Figure 1C, Supplementary information, Figure S1A and S1B). In contrast, removal of the NLS eliminated the ability of CEND to activate ethylene signaling (Figure 1C, Supplementary information, Figure S1A and S1B), indicating that the nuclear localization of EIN2 CEND is required for its action.

Figure 1
figure 1

EIN2 is subjected to ethylene-induced processing and nuclear translocation that is necessary and sufficient for the activation of ethylene-regulated responses. (A) Schematic representation of EIN2 and alignment of a predicted nuclear localization sequence (NLS). (B) Transient expression of CEND-GFP and CEND-GFP with a deletion of the putative NLS (CEND-ΔNLS-GFP) in tobacco leaves. DAPI was used as the nuclei marker. (C) Phenotypes of 4-week-old adult plants of the indicated genotypes. (D) Comparison of phenotypes of 4-week-old adult plants with or without 60 μM DEX application. (E) The hypocotyl lengths of 5-day-old light-grown seedlings on 1/2 MS plate containing DEX of the indicated concentrations. Mean ± SD, n > 10. *** indicates P < 0.001 in two-tailed Student's t-test. (F) Immunoblot assay indicates the levels of endogenous EIN3 protein in 5-day-old light-grown seedlings with or without 100 μM DEX treatment for 4 h. (G, H) Subcellular localization of EIN2 (G) and EIN2 N-terminus (H) in Arabidopsis suspension cells. EIN2-GFP and EIN21-479-GFP were transiently expressed in Arabidopsis suspension cells. Cnx1-mRFP and HDEL-mRFP were co-transformed as ER markers, respectively. DIC, differential interference contrast. Scale bars, 20 μm. (I) GFP fluorescence in the roots of 3-day-old etiolated seedlings of 35S:EIN2-GFP/ein2-5 transgenic plants with or without ACC treatment. Arrows indicate the nuclei. Scale bars, 20 μm. (J) Ethylene signal promotes the cleavage of EIN2 C-terminus. Arabidopsis suspension cells stably expressing EIN2-GFP were immunoprecipitated with the GFP-Trap_A beads and immunoblotted with anti-GFP antibody. Five C-terminal fragments (C1-C5) are indicated by asterisk (*). (K) Subcellular fraction and immunoblot analysis of EIN2 fragments. Proteins were fractionated from Arabidopsis suspension cells stably expressing EIN2-GFP. Nuc, nucleus; Cyto, cytoplasm. Histone H3 and cFBPase were used as controls of nuclear and cytosolic fractions, respectively.

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To address whether the nuclear localization of CEND is sufficient to activate ethylene signaling, we employed a glucocorticoid receptor (GR) system, which has been widely used to induce nuclear translocation of transcriptional regulators upon dexamethasone (DEX) treatment in animals and plants8,9. We found that transgenic plants constitutively expressing CEND-GR in the ein2-5 mutant were indistinguishable from ein2-5 plants without DEX application (Figure 1D and 1E, Supplementary information, Figure S1C–S1E). Upon DEX treatments, CEND-GR/ein2-5 plants exhibited elongated hypocotyls, abnormal flowers with protruded gynoecia, reduced fertility, and activation of ethylene-responsive gene expression, all reminiscent of CEND-expressing plants (Figure 1D and 1E, Supplementary information, Figure S1C–S1E). Consistent with these constitutive ethylene response phenotypes, we found that promoting the nuclear transport of CEND-GR by DEX application notably increased EIN3 protein level (Figure 1F). Therefore, we conclude that the nuclear localization of CEND is both required and sufficient for the activation of ethylene signaling.

A previous study demonstrated that EIN2 transiently expressed in tobacco leaf cells was localized at the ER membrane4. Our above results indicate that CEND is partly located in the nucleus, which is essential for its function. To reconcile this seemingly discrepancy, we re-examined the subcellular localization of EIN2. We verified the functionality of the EIN2-GFP fusion protein based on its ability to complement the ein2-5 mutant (Supplementary information, Figure S2A). Consistent with previous report, EIN2-GFP was co-localized with the ER marker protein when transiently expressed in tobacco leaf cells (Supplementary information, Figure S2B). However, when treated with ACC, EIN2-GFP was also observed in the nucleus (Supplementary information, Figure S2C and S2D). We further expressed EIN2-GFP in protoplasts of Arabidopsis PSB-D suspension cells and detected the GFP fluorescence in both ER and nucleus (Figure 1G). By comparison, a fusion protein of GFP and EIN2 N-terminal membrane-spanning domain (amino acids 1-479) was exclusively detected in ER membrane (Figure 1H), implying that the N-terminal end is responsible for EIN2's location on ER. We also detected weak fluorescence in the nucleus of transgenic Arabidopsis plants expressing EIN2-GFP upon ACC treatment, but not in untreated plants (Figure 1I). Taken together, these results indicate that ethylene treatment facilitates the translocation of EIN2-GFP from ER membrane into the nucleus.

Given the above results, we hypothesized that CEND could be a trafficking molecule transported into the nucleus when ethylene signal is on. To test this hypothesis, we generated an Arabidopsis suspension cell line stably expressing EIN2-GFP. Immunoblot assays with anti-GFP antibody showed that, in addition to full-length EIN2-GFP, at least five smaller fragments (designated as C1-C5) were detected in the transformed cell line (Figure 1J). Interestingly, ACC treatment hardly altered the pattern or intensity of these fragments, whereas treatment with silver ion or aminoethoxyvinylglycine (AVG), inhibitor of ethylene perception or biosynthesis, respectively, led to the disappearance of those fragments (Figure 1J). This result indicates that EIN2 C-terminal end could be cleaved and ethylene signal induces the cleavage(s). The lack of an ACC effect implied that Arabidopsis suspension cells are already saturated with high ethylene production, likely induced by mechanical stresses during cell preparation and propagation. It also provides an explanation for the observed nuclear localization of EIN2-GFP in Arabidopsis protoplasts even without ACC treatment (Figure 1G).

To further confirm that EIN2 C-terminal end could be cleaved and translocated into the nucleus, we isolated nuclear fraction (Nuc) and soluble cytosolic fraction (Cyto) from the EIN2-GFP-expressing Arabidopsis suspension cells. Immunoblot assay with anti-GFP antibody showed that full-length EIN2-GFP as well as C1 and C3 fragments were detected in the nuclear fraction, whereas no fragments were present in the soluble cytoplasm (Figure 1K). It is likely that fragments C2, C4, C5 might associate with other non-nuclear organelles or insoluble particles. Collectively, these findings demonstrate that, in the presence of ethylene signal, EIN2-GFP is cleaved and the resulting C-terminal ends are selectively translocated from ER to the nucleus to activate downstream ethylene signaling.

Previous studies revealed that EIN2 is required for EIN3 and EIL1 protein accumulation6,10. We found that while EIN3 protein was undetectable in the ein2 mutant, expression of CEND in ein2 led to the re-accumulation of EIN3 protein (Supplementary information, Figure S3A). Genetic analysis further indicated that EIN3 and EIL1 are required for the action of CEND, as loss of EIN3/EIL1 function completely suppressed CEND activity in all ethylene response phenotypes examined, as well as ethylene-responsive gene expression (Supplementary information, Figure S3B–S3F). Therefore, CEND acts to activate EIN3-mediated transcription and ethylene responses. We are currently investigating how CEND stabilizes EIN3 protein in the nucleus.

In this study, we have demonstrated that EIN2 harboring a functional NLS is subjected to a hormone-induced cleavage event, followed by transporting its carboxyl end (CEND) into the nucleus, where CEND acts to stabilize EIN3 and activate ethylene responses (Supplementary information, Figure S4). This model uncovers a molecular mechanism to bridge two separate compartments (i.e., ER and nucleus) involved in ethylene signal transduction, wherein CEND serves as a trafficking molecule conveying ethylene signal from ER membrane to the nucleus. A recent study by Qiao et al.11 reported similar findings of EIN2 processing and subcellular translocation. In comparison with their results, we observed at least 5 ethylene-induced C-terminal cleavage fragments, and only 2 fragments are preferentially associated with nuclear fraction, including the one reported by Qiao et al. Meanwhile, we also observed that EIN2-GFP formed speckles in cytoplasm apart from nuclear localization (Figure 1G, Supplementary information, Figure S2B–S2D), implying that EIN2 might play additional roles in ethylene signaling other than activating EIN3-mediated transcription. Detailed methods are described in the Supplementary information, Data S1.