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.
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.
References
Guo H, Ecker JR . The ethylene signaling pathway: new insights. Curr Opin Plant Biol 2004; 7:40–49.
Roman G, Lubarsky B, Kieber JJ, Rothenberg M, Ecker JR . Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway. Genetics 1995; 139:1393–1409.
Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR . EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science 1999; 284:2148–2152.
Bisson MM, Bleckmann A, Allekotte S, Groth G . EIN2, the central regulator of ethylene signalling, is localized at the ER membrane where it interacts with the ethylene receptor ETR1. Biochem J 2009; 424:1–6.
Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR . Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell 1997; 89:1133–1144.
An F, Zhao Q, Ji Y, et al. 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. Plant Cell 2010; 22:2384–2401.
Cokol M, Nair R, Rost B . Finding nuclear localization signals. EMBO Rep 2000; 1:411–415.
Huang W, Perez-Garcia P, Pokhilko A, et al. Mapping the core of the Arabidopsis circadian clock defines the network structure of the oscillator. Science 2012; 336:75–79.
Lloyd AM, Schena M, Walbot V, Davis RW . Epidermal cell fate determination in Arabidopsis: patterns defined by a steroid-inducible regulator. Science 1994; 266:436–439.
Guo H, Ecker JR . Plant responses to ethylene gas are mediated by SCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor. Cell 2003; 115:667–677.
Qiao H, Shen Z, Huang SS, et al. Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas. Science 2012 Sep 25; doi:10.1126/science.1225974
Acknowledgements
We thank Drs D Guo and Y Wang for critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China (91017010), Ministry of Science and Technology (2009CB119101), and Ministry of Agriculture of China (2010ZX08010-002), and the 111 Project of Peking University to H G.
Author information
Authors and Affiliations
Corresponding author
Additional information
( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Nuclear localization of CEND is necessary and sufficient for ethylene response phenotypes. (PDF 281 kb)
Supplementary information, Figure S2
ACC promotes EIN2-GFP accumulation in the nucleusnucleus (PDF 269 kb)
Supplementary information, Figure S3
CEND activates ethylene responses in an EIN3/EIL1-dependent manner. (PDF 311 kb)
Supplementary information, Figure S4
A proposed model depicting the action of EIN2 in the ethylene signaling pathway. (PDF 172 kb)
Supplementary information, Data S1
Materials and Methods (PDF 201 kb)
Rights and permissions
About this article
Cite this article
Wen, X., Zhang, C., Ji, Y. et al. Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus. Cell Res 22, 1613–1616 (2012). https://doi.org/10.1038/cr.2012.145
Published:
Issue Date:
DOI: https://doi.org/10.1038/cr.2012.145
This article is cited by
-
Ethylene-triggered subcellular trafficking of CTR1 enhances the response to ethylene gas
Nature Communications (2023)
-
A translational regulator MHZ9 modulates ethylene signaling in rice
Nature Communications (2023)
-
The roles of abscisic acid and ethylene in cadmium accumulation and tolerance in plants
Plant and Soil (2023)
-
Overexpression of AHL9 accelerates leaf senescence in Arabidopsis thaliana
BMC Plant Biology (2022)
-
Seedling morphogenesis: when ethylene meets high ambient temperature
aBIOTECH (2022)