The MYC2–EBF1–JAZ2 module bridges jasmonate and ethylene signals in apple

MdEBF1, a negative regulator of ethylene signaling, promotes ubiquitination-mediated degradation of the jasmonate (JA) signaling repressor MdJAZ2 to release the key transcription factor MdMYC2 and initiate JA-enhanced disease resistance in apple, while JA-activated MdMYC2 upregulates MdEBF1 transcription, establishing a positive feedback loop that amplifies JA signaling. Jasmonates (JAs) are a class of lipid-derived plant hormones, with methyl jasmonate (MeJA) being the first member identified in 1962 from Jasminum plants. Functioning as a crucial signaling molecule, JA mediates plant responses to insect herbivory, pathogen infection, and various abiotic stresses. It also regulates key growth and developmental processes, including seed germination, trichome formation, root development, leaf senescence, and anthocyanin accumulation. Under normal conditions, JA levels in plants remain low. During this state, JASMONATE ZIM-DOMAIN (JAZ) proteins interact with MYC2 transcription factor and recruit additional transcriptional repressors, cooperatively suppressing the expression of JA-responsive genes. When the bioactive form jasmonyl–isoleucine (JA-Ile) is perceived by the F-box protein CORONATINE INSENSITIVE 1 (COI1), COI1 associates with S-PHASE KINASE-ASSOCIATED PROTEIN 1 (SKP1), CULLIN 1 (CUL1), and RING-BOX PROTEIN 1 (RBX1) to assemble the SCFCOI1 E3 ubiquitin ligase complex. This complex ubiquitinates JAZ proteins, targeting them for degradation by the 26S proteasome. The removal of JAZ repressors liberates MYC2, enabling the activation of downstream JA-responsive genes. Plants modulate protein activity or stability through post-translational modifications, enabling specific and rapid responses to environmental stimuli. Among these, ubiquitination serves as a key regulatory mechanism for controlling intracellular protein abundance. The post-translational regulation of JAZ repressors is crucial for maintaining their homeostasis and ensuring JA signal transduction. Beyond the classical SCFCOI1 ubiquitin ligase complex, several other factors have been reported to regulate JAZ protein turnover, including RING-domain E3 ubiquitin ligases KEEP ON GOING (KEG), ASRF1, and SEVEN IN ABSENTIA 11 (SINA11) (Pauwels et al., 2015; Koh et al., 2023; Ai et al., 2024); the plant U-box E3 ubiquitin ligase PUB22 (Wu et al., 2024); the F-box protein SKP1-INTERACTING PARTNER 31 (SKIP31) (Varshney et al., 2023); and BTB-TAZ DOMAIN PROTEIN 2 (BT2) (An et al., 2021). Although the JA signaling network has been extensively studied, the mechanisms by which other regulatory components interact with JAZ proteins and modulate their stability remain to be further elucidated. Hormonal interactions regulate diverse physiological functions in plants, aiding their adaptation to ever-changing environments. JA is no exception, engaging in complex signaling crosstalk with multiple hormones, including gibberellins, salicylic acid, abscisic acid, auxin, ethylene (ET), cytokinins, and brassinosteroids. The JAZ proteins and the MYC2 transcription factor serve as central hubs mediating JA crosstalk with other signaling pathways. Taking the interplay between JA and ET as an example, these two hormones exhibit both synergistic and antagonistic relationships in regulating plant development and stress responses. In terms of antagonism, the JA-activated MYC2 promotes the degradation of the ET signaling core factors ETHYLENE-INSENSITIVE 3 (EIN3)/EIN3-LIKE 1 (EIL1) by inducing the expression of EIN3-BINDING F-BOX PROTEIN 1 (EBF1), and MYC2 also directly interacts with EIN3/EIL1 to suppress their DNA-binding activity, thereby playing an antagonistic regulatory role in apical hook development (Zhang et al., 2014). Meanwhile, EIN3/EIL1 can impair MYC2 function, reducing plant resistance to insects (Song et al., 2014). During tomato (Solanum lycopersicum) fruit ripening, cytochrome P450 94C1 (CYP94C1) inactivates bioactive JA-Ile to attenuate the JA-mediated defense response, underscoring its pivotal role as a molecular link integrating ET-regulated ripening and JA-mediated defense (Yang et al., 2024). On the synergistic side, ET activates JA biosynthetic genes via the OsEBF1–OsEIL1 module, cooperatively enhancing rice (Oryza sativa L.) defense against infestation by Nilaparvata lugens (Ma et al., 2020). Despite existing knowledge, the mechanisms underlying the interplay between JA and ET signaling remain incompletely understood. Here, we investigated the molecular mechanism of the MdMYC2–MdEBF1–MdJAZ2 module in ET-mediated initiation and amplification of JA signaling during apple (Malus × domestica) disease defense responses. Treatment with the ET synthesis inhibitor aminoethoxyvinylglycine (AVG) significantly reduced MdJAZ2 protein levels, whereas the proteasome inhibitor MG132 blocked the AVG-induced destabilization of MdJAZ2 (Figure 1A; Table S1). This indicates that ET regulates MdJAZ2 stability through the proteasomal pathway. A direct interaction between MdEBF1 and MdJAZ2 was confirmed by yeast two-hybrid (Y2H) assay (Figures 1B, C, S1A), and this finding was further validated by subsequent pull-down, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (Co-IP) assays (Figure S1B–D). Overexpression of MdEBF1 significantly increased the ubiquitination of MdJAZ2 (Figure 1D), indicating that MdJAZ2 serves as a ubiquitination substrate of MdEBF1. A cell-free protein degradation assay in vitro was employed to assess the impact of MdEBF1 on MdJAZ2 protein stability. Overexpression of MdEBF1 shortened the half-life of MdJAZ2, while suppression of MdEBF1 (asMdEBF1) exhibited the opposite effect (Figure 1E). Moreover, the addition of the proteasome inhibitor MG132 interrupted the MdEBF1-mediated degradation of MdJAZ2 (Figure 1E), demonstrating that MdEBF1 promotes MdJAZ2 destabilization via the proteasome pathway. In vivo protein quantification further confirmed that MdEBF1 overexpression reduced MdJAZ2 accumulation, whereas MdEBF1 suppression (asMdEBF1) increased MdJAZ2 protein abundance (Figures 1F, S2). These results collectively indicate that MdEBF1 targets MdJAZ2 for ubiquitination and degradation. The MdMYC2–MdEBF1–MdJAZ2 module integrates ET and JA signals (A) Protein abundance determination of MdJAZ2-GUS transgenic apple callus showing the effects of AVG and MG132 on MdJAZ2 protein stability. −AVG, no AVG treatment; +AVG, 2 μM AVG treatment for 10 h; +AVG/MG132, co-treatment with 2 μM AVG and 100 μM MG132 for 10 h. (B) Schematic diagram of the MdJAZ2 protein. (C) Y2H assay. (D) In vivo ubiquitination analysis. (E) In vitro cell-free protein degradation assay. (F) In vivo protein abundance determination. (G) Effect of MdEBF1 on ET regulation of MdJAZ2 ubiquitination. (H) Effect of MdEBF1 on ET regulation of MdJAZ2 degradation. (I) Apple fruit pathogen inoculation assay showing the effect of MdEBF1 on the disease regulatory function of MdJAZ2. EV, the empty vector control; MdEBF1, overexpression of MdEBF1; MdJAZ2, overexpression of MdJAZ2; MdJAZ2/MdEBF1, simultaneous overexpression of MdJAZ2 and MdEBF1. Bars = 1 cm. Different lowercase letters indicated significant differences at P < 0.05 based on one-way ANOVA followed by Tukey's test. (J) EMSA. Mut represents the replacement of the CACGTT sequence in the MdEBF1 promoter with CCCGGG. (K) Dual-luciferase assay in Nicotiana benthamiana leaves. Different lowercase letters indicated significant differences at P < 0.05 based on one-way ANOVA followed by Tukey's test. (L) Working model. To evaluate the role of MdEBF1 in ET-regulated MdJAZ2 turnover, we treated MdJAZ2-GUS and MdJAZ2-GUS/MdEBF1 transgenic callus with 1-aminocyclopropane-1-carboxylic acid (ACC), an ET synthesis precursor. Consistent with previous findings, MdEBF1 enhanced MdJAZ2 ubiquitination and reduced its protein accumulation (Figure 1G, H). Notably, ACC treatment substantially alleviated the MdEBF1-mediated ubiquitination and degradation of MdJAZ2 (Figures 1G, H, S3), suggesting that ET regulates MdJAZ2 proteolysis in an MdEBF1-dependent manner. Ring rot, caused by the necrotrophic pathogen Botryosphaeria dothidea, is one of the most significant diseases threatening global apple production. Based on the experimental results, where MeJA treatment enhanced fruit resistance to Botryosphaeria dothidea and ACC treatment increased fruit susceptibility (Figure S4), we conclude that JA and ET exhibit antagonistic regulatory effects against this pathogen in apple fruits. Correspondingly, MdJAZ2 functioned as a negative regulator, and MdEBF1 as a positive regulator, of fruit ring rot (Figures 1I, S5). Compared with overexpression of MdJAZ2 alone, co-overexpression with MdEBF1 significantly mitigated the MdJAZ2-exacerbated disease symptoms (Figures 1I, S5), indicating that MdEBF1 negatively regulates MdJAZ2-mediated disease susceptibility. These results demonstrate that MdEBF1 inhibits MdJAZ2-aggravated fruit disease by targeting MdJAZ2 for ubiquitination and degradation. It is noteworthy that MdEBF1 could interact with multiple JAZ proteins (Figure S1A), suggesting that the EBF1-JAZ ubiquitination pathway may be broadly involved in plant defense and growth regulation. MdMYC2 acted as a positive regulator of fruit ring rot by directly activating the expression of the disease defense-responsive gene PATHOGENESIS-RELATED PROTEIN 1 (MdPR1) (Figures S6, S7; Table S2). Overexpression of MdMYC2 upregulated the transcription of MdEBF1 (Figure S8A, B), raising the possibility that MdEBF1 is a target gene of MdMYC2. Chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) and electrophoretic mobility shift (EMSA) assays confirmed that MdMYC2 directly binds to the promoter region of MdEBF1 in vitro and in vivo (Figures 1J, S8C, D; Table S3). Furthermore, dual‑luciferase assays and β‑glucuronidase activity measurements demonstrated that MdMYC2 activates the promoter activity of MdEBF1 (Figures 1K, S9). These results indicate that MdEBF1 is a direct transcriptional activation target of MdMYC2. Additionally, overexpression of MdMYC2 could restore the attenuated disease resistance phenotype observed in MdEBF1-silenced fruits (Figure S10). In summary, our findings demonstrate that the ET signaling repressor MdEBF1 targets MdJAZ2 for ubiquitination-mediated degradation, thereby releasing MdMYC2 and initiating JA-enhanced disease resistance. Simultaneously, JA-activated MdMYC2 promotes the transcription of MdEBF1, establishing a positive feedback loop that amplifies JA signaling (Figure 1L). We propose that this JA signal amplification mechanism, orchestrated by the MdMYC2–MdEBF1–MdJAZ2 module, may be crucial for plants to rapidly optimize resource allocation and adapt to dynamically changing environments. Similar to tomato, apple is also a climacteric fruit, and its JA and ET levels exhibit similar dynamic changes during ripening. Given the antagonistic roles of JA and ET signaling in regulating defense responses and fruit ripening in tomato (Yang et al., 2024), future research will focus on how the MdMYC2–MdEBF1–MdJAZ2 module balances the trade-off between ring rot resistance and fruit ripening in apple. We believe that identifying key disease regulators, such as MdEBF1, MdJAZ2, and MdMYC2, will provide important genetic resources for the future development of new apple germplasms resistant to Botryosphaeria dothidea. Overall, these findings advance our understanding of post-translational modifications of JAZ proteins, JA signal transduction, and cross-talk between JA and ET signaling pathways in plant disease defense. This work was financially supported by grants from the Hubei Provincial Young Top-notch Talent Program (E4399901), Natural Science Foundation of China (32572995), Taishan Scholars Program (tsqn202312147), Natural Science Foundation of Shandong Province (ZR2024MC214), and Chinese Academy of Sciences Pioneer Hundred Talents Program (E5299903). The authors declare no conflicts of interest. J.P.A. conceived and designed the experiments. X.W.Z., R.R.X., and L.Z. performed the research. J.P.A. and Y.H. analyzed the data. X.W.Z. and J.P.A. wrote the paper. All authors read and approved the content. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.70288/suppinfo Figure S1. The interaction between MdEBF1 and MdJAZ2. Figure S2. The expression of MdJAZ2 and MdEBF1 in apple callus is shown in Fig. 1F. Figure S3. Effect of ACC on the ubiquitination and degradation of MdJAZ2. Figure S4. The impacts of MeJA and ACC on the pathogenicity of Botryosphaeria dothidea in apple fruits. Figure S5. The expression of MdJAZ2 and MdEBF1 in apple fruits is shown in Fig. 1I. Figure S6. Apple fruit pathogen inoculation assay showing the regulatory roles of MdMYC2 and MdJAZ2 in disease response and the effect of MdJAZ2 on the disease regulatory function of MdMYC2. Figure S7. The binding and activation of MdMYC2 to the MdPR1 promoter. Figure S8. ChIP-PCR assay analysis of MdMYC2 binding to the MdEBF1 promoter. Figure S9. GUS staining and activity assay to analyze MdMYC2-mediated activation of the MdEBF1 promoter. Figure S10. Apple fruit pathogen inoculation assay showing the genetic relationship between MdEBF1 and MdMYC2 in disease response. Table S1. Primers used for vector construction and expression analysis. Table S2. The promoter sequence of MdPR1. Table S3. The promoter sequence of MdEBF1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.