Apple SINA11‐JAZ2 module is involved in jasmonate signaling response

Jasmonic acid (JA) is an important phytolipid hormone involved in regulating many processes of plant growth and development, and plays a key role in plant resistance to biotic and abiotic stresses. Since the first discovery of methyl jasmonate (MeJA) from jasminum in 1962, the JA signaling pathway have been dissected. Two JA biosynthesis pathways have been demonstrated in Arabidopsis, namely, the octadecane pathway starting with linolenic acid and the cetane pathway starting with cetane trienoic acid. The identification of the F-box protein CORONATINE-INSENSITIVE 1 (COI1) has promoted the investigation of JA signal transduction. COI1 is a JA receptor that functions by binding to Arabidopsis SKP1-LIKE PROTEIN1/2 (ASK1/2), CULLIN 1 (CUL1), and RING-box PROTEIN 1 (RBX1) to form the SCFCOI1 complex. The JASMONATE ZIM-DOMAIN (JAZ) proteins act as JA co-receptors and repressors of JA signaling. The perception of bioactive JA-Ile facilitates the formation of the JA-COI1-JAZ complex and ubiquitin-dependent degradation of JAZ proteins, thereby initiating the JA signaling response (Major et al., 2017). Degradation of JAZs is the key to the JA signaling response. Several studies have reported that JAZ proteins are regulated by post-translational modification. In addition to the well studied SCFCOI ubiquitin ligase complex mediating degradation of JAZs, the RING E3 ubiquitin ligases KEEP ON GOING (KEG) and ASRF1, F-box protein SKP1-INTERACTING PARTNER 31 (SKIP31), and plant U-box E3 ubiquitin ligase PUB22 have been reported to mediate the stability of JAZs (Pauwels et al., 2015; Koh et al., 2023; Varshney et al., 2023; Wu et al., 2024). Small Ubiquitin-like MOdifier (SUMO)-deconjugating proteases OVERLY TOLERANT TO SALT 1 (OTS1) and OTS2 regulate the SUMOylation and stability of JAZs (Srivastava et al., 2018). BRASSINOSTEROID INSENSITIVE 2 (BIN2), a negative regulator of the brassinolide (BR) signaling pathway, phosphorylates JAZ1 and promotes its turnover (Song et al., 2021). In apple, the protein kinase SNF1-related kinase 1.1 (MdSnRK1.1) phosphorylates and promotes MdJAZ18 degradation, resulting in the accumulation of anthocyanin and proanthocyanidin (Liu et al., 2017). Apple BTB and TAZ DOMAIN PROTEIN 2 (MdBT2) inhibit JA-triggered leaf senescence by enhancing the stability of MdJAZ2 and accelerating ubiquitin-dependent degradation of MdMYC2 (An et al., 2021). However, the JAZ protein turnover mechanism remains largely unknown. In this study, we revealed that the E3 ubiquitin ligase SEVEN IN ABSENTIA 11 (MdSINA11) was involved in the ubiquitination regulation of the MdJAZ2 protein. SINA belongs to the RING-domain E3 ubiquitin ligase. Since the SINA protein was first identified in Drosophila, great progress has been made in dissecting the biological functions of SINA. Extensive studies have demonstrated that SINA E3 ubiquitin ligases play a multifunctional role in regulating plant growth and development and response to diverse stresses (Zhang et al., 2019). We noted that MeJA treatment reduced the abundance of MdJAZ2 protein, while the application of proteasome inhibitor MG132 greatly restored the inhibitory effect of MeJA on MdJAZ2 (Figures 1A, S1A), suggesting that MdJAZ2 undergoes ubiquitin-dependent degradation in response to JA. To understand how MeJA regulates the stability of the MdJAZ2 protein, we searched for E3 ubiquitin ligases that interact with MdJAZ2 using a liquid chromatography-tandem mass spectrometry system. As a result, MdSINA11, an E3 ubiquitin ligase encoding a positive regulator of anthocyanins (Li et al., 2023), was screened out as a potential interaction partner for MdJAZ2. We verified the interaction between MdSINA11 and MdJAZ2 through four parallel assays. MdSINA11 interacted with the N-terminal of MdJAZ2 containing the ZIM domain in yeast cells (Figures 1B, S2). In parallel, the interaction between MdSINA11 and MdJAZ2 was confirmed using pull-down, coimmunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC) assays (Figures S3A–C, S4A). In vitro and in vivo ubiquitination assays were conducted to test the effect of MdSINA11 on the ubiquitination of MdJAZ2. Adding E1, E2, and MdSINA11-GST in vitro resulted in the ubiquitination band of the MdJAZ2 protein (Figure 1C). Overexpression of MdSINA11 increased the ubiquitination level of MdJAZ2 in vivo (Figures 1D, S5). Next, we investigated the effect of MdSINA11 on MdJAZ2 stability in the in vitro cell-free protein degradation assay. MdSINA11 accelerated the degradation of MdJAZ2, while inhibiting MdSINA11 showed the opposite effect, and adding MG132 abolished the degradation of MdJAZ2 in the reaction solution (Figure 1E). GUS staining and western blotting analysis also indicated that MdSINA11 reduced the protein abundance of MdJAZ2 (Figure 1F). Moreover, we noted that inhibition of MdSINA11 expression significantly reduced the ubiquitination and degradation of MdJAZ2 exacerbated by MeJA (Figure 1G, H), indicating that MdSINA11 is essential for JA-induced ubiquitination and degradation of MdJAZ2. Taken together, these results illustrate that MdJAZ2 is a ubiquitinated substrate of MdSINA11. MdSINA11 promotes ubiquitination and degradation of MdJAZ2 (A) GUS staining and protein abundance determination of MdJAZ2-GUS transgenic apple callus showing the effects of MeJA and MG132 on MdJAZ2 protein stability. −MeJA, no MeJA treatment; +MeJA, MeJA treatment for 2 h; +MeJA/MG132, MeJA and MG132 treatments for 2 h. (B) Y2H assay showing the interaction between MdSINA11 and the full length and segment of MdJAZ2. −T/−L, SD/−Trp/−Leu; −T/−L/−H/−A, SD −Trp/−Leu/−His/−Ade. (C) Ubiquitination assay in vitro. MdJAZ2-HIS was tested for E3 ubiquitin ligase activity in the presence and absence of ATP, ubiquitin, E1, E2, MdSINA11-GST, and MdJAZ2-HIS. (D) Ubiquitination analysis in vivo. MdJAZ2-GUS was immunoprecipitated from MdJAZ2-GUS and MdJAZ2-GUS/MdSINA11 transgenic apple callus. (E) Protein degradation assay in vitro. Total proteins extracted from wild-type and transgenic apple callus with overexpression or repressed expression of MdSINA11 were incubated with the purified MdJAZ2-HIS fusion protein. (F) GUS staining and protein abundance determination of MdJAZ2-GUS, MdJAZ2-GUS/MdSINA11, and MdJAZ2-GUS/asMdSINA11 transgenic apple callus. (G, H) Evaluation of the role of MdSINA11 in MeJA-induced ubiquitination and degradation of MdJAZ2. (I) Evaluation of the relationship between MdJAZ2 and MdSINA11 in regulating anthocyanin biosynthesis via transient expression in apple fruits. pIR+TRV, IL60-1 + IL60-2 + TRV1 + TRV2; MdSINA11-pIR, IL60-1 + MdSINA11-IL60-2; MdJAZ2-TRV, TRV1 + MdJAZ2-TRV2; MdSINA11-pIR+MdJAZ2-TRV, IL60-1 + MdSINA11-IL60-2 + TRV1 + MdJAZ2-TRV2. The value of pIR+TRV was set to 1. Each treatment was performed in triplicate and each replicate contained 8–10 fruits. Representative photographs are shown here. Error bars denote standard deviations. Different lowercase letters indicate a significant difference at P < 0.05 based on one-way ANOVA. (J) Model of the MdSINA11–MdJAZ2 module regulating JA-mediated anthocyanin biosynthesis in apple. JA induces anthocyanin biosynthesis. Accordingly, we used anthocyanin accumulation as an indicator phenotype to investigate the regulatory relationship between MdSINA11 and MdJAZ2. The quantitative real-time-polymerase chain reaction (qRT-PCR) analysis showed that the MeJA treatment induced MdSINA11 transcription (Figure S6; Table S1). Suppressing MdSINA11 expression disrupted the anthocyanin accumulation induced by MeJA (Figures S7A, B, S8A, B), suggesting that MdSINA11 positively regulates JA-mediated anthocyanin biosynthesis. To address whether the role of MdSINA11 in anthocyanin biosynthesis depends on the presence of MdJAZ2, we conducted the transient transformation assay in apple fruits. The infiltration of MdSINA11-pIR and MdJAZ2-TRV did not increase anthocyanin accumulation compared with the infiltration of MdJAZ2-TRV alone (Figures 1I, S1B, S4B), indicating that the combination of overexpression of MdSINA11 and suppression of MdJAZ2 has no additive effect in regulating anthocyanin biosynthesis. A similar result was observed in the stable transformation assay in apple callus (Figure S9), suggesting that MdSINA11 regulates anthocyanin biosynthesis through ubiquitin-dependent degradation of MdJAZ2. Extensive interactions were detected between MdJAZs and MdSINAs (Figure S10A). In the MdJAZ2–MdSINAs interaction, we found that MdSINA4, MdSINA7, MdSINA9, MdSINA10, and MdSINA11 all interacted with the N-terminal of MdJAZ2 containing the ZIM domain (Figure S10B). The discovery of the SINA-JAZ module will undoubtedly provide a reference for further exploration of the JA response pathway. In two recent reports, SINA proteins were involved in gibberellin and auxin signaling responses by regulating the turnover of DELLA and auxin/indole-3-acetic acid (AUX/IAA) proteins, respectively (An et al., 2023; Li et al., 2023). Combined with the current study on the regulation of JAZ protein stability by SINA, we speculated that SINA proteins may be involved in various hormone signaling responses by mediating the homeostasis of repressors. In summary, we discovered a new ubiquitination pathway in JA signal transduction. A proposed model can describe the role of the MdSINA11–MdJAZ2 module in regulating the JA signaling response and JA-triggered anthocyanin biosynthesis in apple (Figure 1J). MdSINA11 promotes the ubiquitination and degradation of MdJAZ2 through the 26S proteasome pathway in response to JA signaling, thereby releasing MdMYC2, an important regulator of anthocyanin biosynthesis. MdMYC2 promotes anthocyanin biosynthesis by activating the expression of genes associated with anthocyanin biosynthesis. The ubiquitin regulation of MdSINA11 on MdJAZ2 is an important discovery in JA signal transduction research, which will provide a reference for the post-translational regulatory mechanism of the JA signaling pathway. We thank Professor Yanru Hu of Hubei University for their support on experimental design. This work was financially supported by grants from the Taishan Scholars Program (tsqn202312147), Natural Science Foundation of Shandong Province (ZR2022YQ24), Development Plan of the Youth Innovation Team of the Higher Education Institutions in Shandong Province (2022KJ326), and Wuhan Botanical Garden Scientific Research Support Project (E3559901). The authors declare no conflicts of interest. Jian-Ping An conceived and designed the experiments. Jian-Ping An, Di Ai, and Lei Zhao performed the research. Chun-Xiang You, and Y.H. analyzed the data. Jian-Ping An wrote the paper. All authors read and approved the contents of this paper. All the data generated or analyzed during this study are included in this published article. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13713/suppinfo Dataset S1. Materials and methods used in this study Dataset S2. Uncropped western blot images in this study Figure S1. Identification of MdJAZ2 transgenic apple callus and fruits Figure S2. Schematic diagram of the MdJAZ2 protein Figure S3. Interaction between MdSINA11 and MdJAZ2 Figure S4. Identification of MdSINA11 transgenic apple callus and fruits Figure S5. Evaluation of the roles of MdSINA4/7/9/10/11 in regulating the ubiquitination activity of MdJAZ2 via apple callus transient expression system Figure S6. Relative expression levels of MdSINA4/7/9/10/11 in apple fruits exposed to 20 µM MeJA for 4 h Figure S7. The phenotypes and relative anthocyanin contents of wild-type and MdSINA11 transgenic apple callus in response to the 20 µM MeJA treatment Figure S8. Identification of the role of MdSINA4/7/9/10/11 in regulating anthocyanin biosynthesis via apple callus transient expression system Figure S9. Evaluation of the relationship between MdJAZ2 and MdSINA11 in regulating anthocyanin biosynthesis via stable expression in apple callus Figure S10. Y2H assay showing the interaction between JAZ and SINA proteins Table S1. Primers used for vector construction and expression analysis 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.