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. 2025 Nov 25;13(3):uhaf321. doi: 10.1093/hr/uhaf321

The OfJAZ3-OfMYB21 complex mediates jasmonic acid signaling pathway to regulate linalool biosynthesis in Osmanthus fragrans

Yangang Lan 1,2,#, Xue Huang 3,#, Ziyi Li 4, Shunran Zhang 5, Yan Xiang 6,✉, Hongbo Zhao 7,✉
PMCID: PMC12963045  PMID: 41799738

Abstract

Osmanthus fragrans is a well-known ornamental tree species for its pleasing floral fragrance. Linalool, as the characteristic aromatic component of O. fragrans, holds significant potential for applications in the flavor and fragrance industry. Although jasmonic acid (JA) is well documented to regulate the biosynthesis and accumulation of various plant secondary metabolites, its role in linalool biosynthesis remains largely unclear. Here, we discovered a positive correlation between the endogenous JA levels and linalool accumulation during the flowering stage of O. fragrans. Exogenous JA treatment was shown to enhance linalool biosynthesis by activating the linalool synthase gene OfTPS2. Dual-LUC and EMSA assays demonstrated that the key protein in the JA signaling pathway, OfJAZ3, interacted with OfMYB21 and subsequently suppressed the transcriptional activation of OfTPS2 mediated by OfMYB21. Functional validation further revealed that overexpression of OfJAZ3 significantly inhibited linalool biosynthesis in O. fragrans, A. thaliana, and N. tabacum plants. In contrast, JA promoted the degradation of OfJAZ3, thereby disrupting the formation of the OfJAZ3-OfMYB21 complex and relieving its inhibitory effect on OfTPS2. Split-LUC, BiFC, and pull-down assays confirmed that OfJAZ3 interacted with the F-box protein OfCOI1 (a key component of the E3 ubiquitin ligase SCFCOI1 complex), and JA treatment enhanced the strength of this interaction. Moreover, OfCOI1 was found to participate in OfTPS2 regulation by facilitating the ubiquitination and degradation of OfJAZ3. In conclusion, our findings elucidate the molecular mechanism by which OfJAZ3-OfMYB21 complex mediates JA signaling to regulate linalool biosynthesis in O. fragrans.

Introduction

Osmanthus fragrans is extensively cultivated as an ornamental landscape species, valued for its prolific flowering and distinctive floral fragrance [1, 2]. Beyond its visual ornamental value, the floral volatile compounds of O. fragrans have significant commercial applications in the food, cosmetics, and phytotherapy industries [3, 4]. The monoterpenoid linalool has been identified as the dominant characteristic volatile organic compound in O. fragrans flowers, consistently present across different cultivars [5–8]. Moreover, as a high-value industrial and food additive, global linalool consumption exceeds 1000 metric tons annually [9]. Understanding the biosynthetic pathway and regulatory network of linalool could therefore offer innovative biotechnological approaches to improve floral fragrance characteristics in plants while boosting production efficiency of economically important terpenoid compounds.

Recent advances in multi-omics approaches have enabled the systematic investigation of linalool biosynthesis pathways in O. fragrans, facilitating the identification of key terpene synthase genes and their transcriptional regulators [8, 10, 11]. Zeng et al. identified four terpene synthase (TPS) genes in O. fragrans, among which OfTPS1 and OfTPS2 were functionally characterized as linalool synthases [12]. In a complementary study, Xiong et al. discovered two additional TPS genes in O. fragrans using integrated transcriptomic profiling and in vitro enzymatic assays; among them, one was molecularly validated as a linalool synthase and was similarly designated OfTPS2 [13]. Comparative studies demonstrated that aroma differences between the O. fragrans cultivars are driven by the differential expression of two genes: CCD4 and LIS1. Specifically, CCD4 mediates β-ionone production via carotenoid degradation, while LIS1 regulates the synthesis of linalool precursors [14]. Regarding transcriptional regulation, Ding et al. carried out genome-wide identification and expression profiling of WRKY transcription factors in O. fragrans [15]. Notably, the expression patterns of OfWRKY7/19/36/139 showed significant correlations with the emission levels of distinct monoterpenes. In a concurrent study, Li et al. identified 243 MYB transcription factors in O. fragrans [16]; of these, OfMYB19/20 expression levels were positively correlated with linalool oxide accumulation, while OfMYB51/65/8/121/137/144 exhibited negative correlations with multiple linalool oxides. Consistent with these findings, we previously found that the R2R3-MYB transcription factor OfMYB21 activates its expression by binding to the OfTPS2 promoter, actively regulating the biosynthesis of linalool, and preliminarily revealing the molecular mechanism of O. fragrans enrichment of linalool [17].

The jasmonic acid (JA) signaling pathway plays a pivotal role in modulating the biosynthesis of plant secondary metabolites [18]. In Gossypium arboreum, JA enhances the expression of GaWRKY1 and the sesquiterpene synthase gene CAD1-A, thereby promoting sesquiterpenoid biosynthesis [19]. Similarly, A. thaliana JAZ proteins and DELLA proteins physically interact with the transcription factor MYC2, forming a regulatory complex that suppresses MYC2-mediated transcriptional activation [20]. Upon exogenous JA or GA application, JAZ and DELLA proteins undergo degradation, releasing MYC2 to activate TPS genes and subsequently increasing sesquiterpenoid production. The bioactive JA-Ile is perceived by the SCFCOI1 ubiquitin ligase complex, comprising the F-box protein COI1 and JAZ repressors. This recognition triggers the ubiquitination and 26S proteasome-mediated degradation of JAZ proteins, leading to the release of downstream transcription factors and activation of JA-responsive genes [21]. In Salvia miltiorrhiza, JA regulates the biosynthesis of medicinal metabolites via the JAZ9-MYB76 complex [22]. Our preliminary studies have demonstrated that exogenous JA treatment enhances linalool biosynthesis in O. fragrans by up-regulating OfTPS2 expression [17]. Meanwhile, the key protein OfJAZ3 in the JA signaling pathway interacts with the upstream regulatory factor OfMYB21 of OfTPS2. However, the molecular mechanism of OfJAZ3 and the involved JA signaling pathway in the biosynthesis of linalool O. fragrans have not been elucidated.

In this study, we found that during the flowering process of O. fragrans, the JA levels are closely associated with the biosynthesis of linalool. Moreover, JA treatment activates the expression of OfTPS2 to promote linalool biosynthesis. The key repressor protein OfJAZ3 in the JA signaling pathway forms a transcriptional complex by interacting with OfMYB21, thereby inhibiting the OfMYB21-mediated activation of OfTPS2 and the subsequent biosynthesis of linalool. JA treatment facilitates the ubiquitination and degradation of OfJAZ3, which disrupts the OfJAZ3-OfMYB21 protein complex and releases OfMYB21 to activate OfTPS2. In vivo and in vitro protein–protein interaction assays revealed OfCOI1, an F-box protein of the E3 ubiquitin ligase complex in the JA signaling pathway, interacts with OfJAZ3. OfCOI1 participates in the regulation of OfTPS2 by facilitating ubiquitination-dependent degradation of OfJAZ3. Collectively, our results elucidate the molecular mechanism by which OfJAZ3 mediates JA signaling to regulate linalool biosynthesis in O. fragrans.

Results

JA positively regulates linalool biosynthesis in O. fragrans by activating OfTPS2

To investigate the regulatory role of endogenous hormones in linalool biosynthesis in O. fragrans flowers, this study selected three key flowering stages (designated as S1, S2, and S3) to detect the expression of key genes and the content of volatile compounds (Fig. 1A–C). Meanwhile, liquid chromatography-mass spectrometry (LC–MS) was used for quantitative determination of five endogenous hormones, including salicylic acid (SA), trans-zeatin (tZ), indole-3-acetic acid (IAA), abscisic acid (ABA), and JA (Fig. 1D). The results showed that during the transition from S1 to S3, both the expression level of the linalool synthase gene OfTPS2 and linalool biosynthesis were significantly up-regulated. Among the five endogenous hormones, only the content of JA increased significantly during this transition, which was consistent with the trend of linalool biosynthesis levels. To further validate the causal link between JA and linalool biosynthesis in O. fragrans, S1-stage flowers were exogenously treated with 100 μM methyl jasmonate (MeJA), a bioactive derivative of JA. MeJA treatment remarkably induced OfTPS2 transcription, and subsequent metabolic profiling demonstrated that linalool production was substantially enhanced compared to the mock-treated control group (Fig. 1E and F). Furthermore, Dual-LUC reporter assays coupled with Nicotiana benthamiana transient expression systems provided direct evidence for the activating effect of MeJA on OfTPS2 expression (Fig. 1G). Collectively, these findings establish that JA modulates linalool biosynthesis in O. fragrans through a regulatory pathway targeting the linalool synthase gene OfTPS2, highlighting the critical role of JA signaling in mediating terpenoid metabolism during floral development.

Figure 1.

Figure 1

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JA plays a crucial regulatory role in linalool biosynthesis in O. fragrans. (A) Three flowering stages of O. fragrans: S1 (bud stage), S2 (initial flowering stage), and S3 (full flowering stage). (B) Expression levels of the linalool synthase gene OfTPS2 across the three flowering stages of O. fragrans. (C) Linalool biosynthesis levels in O. fragrans at the three flowering stages. (D) Quantitative determination of five endogenous hormones (SA, tZ, IAA, ABA, and JA) at three key flowering stages of O. fragrans. (E) Determination of OfTPS2 expression levels in O. fragrans flowers under exogenous MeJA treatment; flowers treated with the solvent used to dissolve MeJA served as the control. (F) Determination of linalool biosynthesis levels in O. fragrans flowers under exogenous MeJA treatment. (G) Relative LUC to REN ratios from transient expression assays. These represent the activity of the OfTPS2 promoter in the absence/presence of MeJA. Error bars indicate the SD of three biological replicates, and the asterisk represents the significant difference, evaluated by the Student’s t-test (*  P < 0.05, **  P < 0.01). Different letters above the bars indicate significant differences between groups (P < 0.05).

OfJAZ3, a JA signaling repressor, inhibits OfTPS2 transcriptional activation mediated by OfMYB21

The role of JA in linalool biosynthesis in O. fragrans prompted us to further explore the underlying molecular regulatory mechanisms. In our previous study, we identified that the transcription factor OfMYB21 positively regulates linalool biosynthesis in O. fragrans by activating OfTPS2 [17]. Moreover, overexpression of OfMYB21 significantly enhanced linalool biosynthesis in A. thaliana and N. tabacum, which confirms the conserved function and core role of OfMYB21 in linalool biosynthesis (Supplementary Fig. S1). Concurrently, our preliminary work also demonstrated that the JAZ protein OfJAZ3 interacts with OfMYB21; however, the detailed regulatory mechanism of this interaction remains elusive. Given that OfJAZ3 serves as a key repressor in the JA signaling pathway, which motivated us to further investigate the regulatory role of OfJAZ3 in linalool biosynthesis in O. fragrans.

To further clarify the regulatory role of the OfJAZ3-OfMYB21 transcriptional complex, we employed a Dual-LUC assay to investigate the effect of OfJAZ3 on the promoter activity of OfTPS2. The results showed that when OfMYB21 was co-transformed with the reporter gene driven by the OfTPS2 promoter into N. benthamiana plants, OfMYB21 exhibited an activating effect on the OfTPS2 promoter; in contrast, the sole expression of OfJAZ3 failed to activate the OfTPS2 promoter. However, when OfJAZ3 and OfMYB21 were co-transformed, the transcriptional activation of OfTPS2 by OfMYB21 was significantly attenuated (Fig. 2A). The EMSA results demonstrated that when GST-OfJAZ3 fusion protein was incubated with the biotin-labeled OfTPS2 promoter sequence probe and subjected to gel electrophoresis, no retarded bands were observed (Fig. 2B). On the contrary, a shifted band was detected when the GST-OfMYB21 recombinant protein was used instead. Furthermore, when the GST-OfMYB21 and GST-OfJAZ3 recombinant proteins were co-incubated with the biotin-labeled probe, the intensity of the shifted band was reduced, and this reduction became more pronounced with the increase in the concentration of the GST-OfJAZ3 recombinant protein, indicating a weakened binding affinity of OfMYB21 to the OfTPS2 promoter. Subsequently, using a transient expression system in O. fragrans flowers, we further found that when OfJAZ3 and OfMYB21 were co-overexpressed in O. fragrans flowers (Supplementary Fig. S2), the expression level of OfTPS2 was significantly lower than that when OfMYB21 was overexpressed alone (Fig. 2C). Collectively, these findings demonstrate that OfJAZ3 interacts with OfMYB21, thereby repressing the transcriptional regulation of OfTPS2 by OfMYB21.

Figure 2.

Figure 2

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OfJAZ3 inhibits the transcriptional activation of the linalool synthase gene OfTPS2 by OfMYB21. (A) Dual-luciferase reporter gene assay validation shows that co-transformation of OfJAZ3/OfMYB21 significantly suppresses the promoter activity of OfTPS2 compared to transformation with OfMYB21 alone. (B) EMSA validation demonstrates that OfJAZ3 inhibits the binding of OfMYB21 to the MYB-binding site on the OfTPS2 promoter. (C) The transient expression system in O. fragrans flowers was used to validate the regulatory effect of the OfJAZ3-OfMYB21 transcriptional complex on OfTPS2 expression. Different letters above the bars indicate significant differences between groups (P < 0.05, Student’s t-test).

OfJAZ3 inhibits the biosynthesis of linalool in O. fragrans, A. thaliana, and N. tabacum plants

Given that the JA signaling repressor OfJAZ3 is involved in the regulation of OfTPS2, we further analyzed the function of OfJAZ3 in linalool biosynthesis. We constructed a gene co-expression network containing OfJAZ3 (Fig. 3A) and performed KEGG pathway enrichment analysis on genes co-expressed with OfJAZ3. KEGG enrichment results revealed that genes co-expressed with OfJAZ3 were significantly enriched in pathways such as ‘Biosynthesis of various plant secondary metabolites’ and ‘Ubiquitin mediated proteolysis’ (Fig. 3B), suggesting that OfJAZ3 plays a role in terpenoid biosynthesis and its protein may be modified by ubiquitination. To verify the regulatory function of OfJAZ3 in linalool biosynthesis, we transiently overexpressed OfJAZ3 in O. fragrans flowers using the transient transformation technology. The results indicated that transient overexpression of OfJAZ3 in O. fragrans flowers significantly down-regulated the expression of OfTPS2 (Fig. 3C). Furthermore, GC–MS analysis revealed that compared with the control group, transient overexpression of OfJAZ3 in O. fragrans flowers reduced the abundance of linalool (Fig. 3D). In the A. thaliana, the overexpression of OfJAZ3 markedly repressed the expression of the AtTPS14 (Fig. 3E), subsequently resulting in a significant decrease in the linalool abundance among the volatiles emitted by the flowers of this transgenic line (Fig. 3F). Analogously, in OfJAZ3-overexpressing transgenic N. tabacum plants, the expression level of the NtLIS, a key enzyme for linalool synthesis, was also notably suppressed (Fig. 3G). This alteration directly manifested as a pronounced reduction in linalool accumulation in the leaves of the transgenic N. tabacum plants (Fig. 3H), robustly demonstrating that the overexpression of OfJAZ3 effectively diminished the linalool synthetic capacity in these transgenic plants.

Figure 3.

Figure 3

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OfJAZ3 inhibits the biosynthesis of linalool. (A) Gene co-expression network involving OfJAZ3. (B) KEGG pathway enrichment analysis for genes co-expressed with OfJAZ3. (C) Transient overexpression of OfJAZ3 in O. fragrans flowers significantly up-regulates OfTPS2 expression. (D) Comparison of linalool abundance between flowers expressing 35S::GFP and 35S::OfJAZ3-GFP in O. fragrans. (E) Detection of the expression levels of the linalool synthase gene AtTPS14 in WT and OfJAZ3-overexpressing transgenic A. thaliana plants. (F) Comparison of linalool abundance between WT and OfJAZ3-overexpressing transgenic A. thaliana plants. (G) Detection of the expression levels of the linalool synthase gene NtLIS in WT and OfJAZ3-overexpressing transgenic N. tabacum plants. (H) Comparison of linalool abundance between WT and OfJAZ3-overexpressing transgenic N. tabacum plants. Error bars indicate the SD of three biological replicates, and the asterisk represents the significant difference, evaluated by the Student’s t-test (*  P < 0.05, **  P < 0.01).

JA restored the transcriptional activity of OfMYB21 by inducing degradation of OfJAZ3

Here, the Dual-LUC assays revealed that when OfJAZ3 and OfMYB21 were co-transformed into N. benthamiana leaves, OfJAZ3 significantly suppressed the activating effect of OfMYB21 on the OfTPS2 (Fig. 4A). Notably, under exogenous MeJA treatment, the inhibitory effect of OfJAZ3 was abrogated (Fig. 4A). Split LUC assays demonstrated that N. benthamiana leaves co-transformed with OfJAZ3-cLuc and OfMYB21-nLuc exhibited robust LUC fluorescence signals in the co-transformation region (Fig. 4B and Supplementary Fig. S3). However, exogenous treatment with MeJA solution led to a significant reduction in LUC activity, implying that MeJA attenuates the interaction between OfJAZ3 and OfMYB21. As key regulators in the JA signaling pathway, the stability of JAZ proteins is modulated by JA signals. To examine whether the stability of the OfJAZ3 protein is regulated by JA, we examined the GFP fluorescence in the roots of 7-day-old transgenic seedlings (35S::OfJAZ3-GFP transgenic A. thaliana plants) under both MeJA-treated (2 h) and untreated conditions (Fig. 4C). In the absence of MeJA, a nuclear-localized GFP signal was observed in all 35S::OfJAZ3-GFP transgenic plants. However, treating the seedlings with 100 μM MeJA resulted in the loss of nuclear 35S::OfJAZ3-GFP signal, although a diffuse fluorescence pattern was still detected in the roots. Furthermore, pretreatment of seedlings with MG132, a specific 26S proteasome inhibitor, hindered the MeJA-induced degradation of OfJAZ3-GFP, suggesting that the 26S proteasome pathway mediates the JA-induced degradation of OfJAZ3. To investigate the hormone-dependent stability of OfJAZ3, we employed a cell-free assay system to assess whether the protein levels of OfJAZ3 are regulated by JA, through transient expression analysis of N. benthamiana leaves expressing GFP-tagged OfJAZ3. The results demonstrated that treatment with MeJA facilitated the degradation of OfJAZ3-GFP protein (Fig. 4D).

Figure 4.

Figure 4

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JA restored the transcriptional activity of OfMYB21 by inducing degradation of OfJAZ3. (A) Dual-luciferase reporter gene assay analysis of the regulatory effect of OfJAZ3-OfMYB21 on the OfTPS2 promoter under exogenous MeJA treatment. (B) Split luciferase (Split LUC) assay combined with exogenous MeJA treatment to examine the modulation of JA on the interaction intensity between OfJAZ3 and OfMYB21. (C) Detection of GFP fluorescence signals in root tips of 35S::OfJAZ3-GFP transgenic A. thaliana plants under exogenous MeJA treatment. (D) Cell-free degradation assay to assess the impact of JA on the stability of the OfJAZ3 protein. Error bars indicate the SD of three biological replicates, and the asterisk represents the significant difference, evaluated by the Student’s t-test (*  P < 0.05, **  P < 0.01). Different letters above the bars indicate significant differences between groups (P < 0.05).

OfJAZ3 interacts with the F-box protein OfCOI1, a key component of the E3 ubiquitin ligase SCFCOI1 complex

Previous studies have shown that the F-box protein CORONATINE INSENSITIVE1 (COI1) is a component of the E3 ubiquitin ligase complex Skip-Cullin-F-box (SCFCOI1). JA and its active form JA-Ile can trigger the formation of a complex between COI1 and JAZ, thereby inducing the degradation of JAZ in a COI1-dependent manner via the 26S proteasome. Here, we identified an F-box protein in O. fragrans that exhibits high homology to A. thaliana COI1 using multiple sequence alignment, and named it OfCOI1 (Fig. 5A). Subcellular localization experiments revealed that OfCOI1 is localized to the nucleus, consistent with the localization of OfJAZ3 (Fig. 5B). Split-LUC assays demonstrated that OfCOI1 interacts with OfJAZ3 (Fig. 5C). To further validate the interaction between OfCOI1 and OfJAZ3, we performed BiFC assays (Fig. 5D). The results showed that co-transformation of OfCOI1-nYFP and OfJAZ3-cYFP resulted in yellow fluorescent protein (YFP) signals in the nuclei of N. benthamiana leaf cells. Next, we performed in vitro pull-down assays using a GST-OfCOI1 recombinant protein and a His-OfJAZ3 fusion protein. When incubated with GST-OfCOI1, the anti-GST antibody pulled down His-OfJAZ3, but not when incubated with GST alone (Fig. 5E). Split LUC assays revealed that co-expression of OfCOI1-nLUC and OfJAZ3-cLUC in N. benthamiana leaves produced strong luciferase activity (Fig. 5F). Interestingly, exogenous MeJA treatment further enhanced the interaction strength between OfCOI1 and OfJAZ3, which was manifested by a significant increase in luminescent signals (Fig. 5F). Taken together, these results confirm that there is a physical interaction between OfCOI1 and OfJAZ3, and that MeJA treatment enhances the strength of their interaction.

Figure 5.

Figure 5

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The E3 ubiquitin ligase OfCOI1 interacts with OfJAZ3. (A) Protein domain analysis of OfCOI1. (B) Subcellular localization of the OfCOI1 and OfJAZ3 protein. (C) Split luciferase assay confirmation of the interaction between OfCOI1 and OfJAZ3 in N. benthamiana leaf cells. (D) Bimolecular fluorescence complementation (BiFC) assay validation of the interaction between OfCOI1 and OfJAZ3 in N. benthamiana leaf cells. (E) GST pull-down assay verification of the interaction between OfCOI1 and OfJAZ3. (F) Split LUC assays confirmed that MeJA treatment enhances the interaction strength between OfCOI1 and OfJAZ3. Error bars indicate the SD of three biological replicates, and the asterisk represents the significant difference, evaluated by the Student’s t-test (*  P < 0.05, **  P < 0.01).

OfCOI1 mediates the ubiquitination and degradation of OfJAZ3, thereby restoring the transcriptional activation of OfTPS2 by OfMYB21

To further investigate whether OfCOI1 regulates the stability of the OfJAZ3 protein, we conducted in vitro protein degradation assays (Fig. 6A). Immunoblot analysis revealed that the OfJAZ3 protein degraded more rapidly in total protein extracts from 35S::OfCOI1-GFP N. benthamiana leaves compared to those from 35S::GFP N. benthamiana leaves. Moreover, MG132 reduced the degradation of OfJAZ3 in the total protein extracts from 35S::OfCOI1-GFP N. benthamiana leaves. These results indicate that OfCOI1 interacts with OfJAZ3 and promotes its degradation via a proteasome-mediated degradation pathway. To investigate whether OfCOI1 promotes the ubiquitination of OfJAZ3, we transformed the constructed 35S::OfJAZ3-GFP or 35S::OfJAZ3-GFP/35S::OfCOI1-GFP vectors into N. benthamiana leaves to express OfJAZ3-GFP and OfJAZ3-GFP/OfCOI1-GFP proteins, respectively. After extracting total proteins, immunoprecipitation was performed using anti-GFP and anti-ubiquitin antibodies. As shown in the figure, the immunoprecipitated samples were analyzed with anti-ubiquitin and anti-GFP antibodies. The results revealed that the level of polyubiquitination was significantly enhanced in the OfJAZ3-GFP/OfCOI1-GFP N. benthamiana leaf samples, indicating that OfCOI1 promote the ubiquitination of OfJAZ3 (Fig. 6B). Subsequently, we further employed the dual-LUC assays to investigate whether OfCOI1 could influence the regulatory effect of OfJAZ3 on OfTPS2. The results demonstrated that OfJAZ3 inhibited the transcriptional activation of OfTPS2 by OfMYB21, whereas OfCOI1 rescued the activation of OfTPS2 by OfMYB21 through the degradation of OfJAZ3 (Fig. 6C).

Figure 6.

Figure 6

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OfCOI1 mediates the ubiquitination of OfJAZ3 to restore the transcriptional activation of OfTPS2 by OfMYB21. (A) OfCOI1 promotes proteasome-mediated degradation of OfJAZ3. Using an anti-His antibody, the levels of His-OfJAZ3 in total crude extracts from 35S::GFP and 35S::OfCOI1-GFP transgenic N. benthamiana leaves, incubated at 4°C for the indicated time periods and treated with or without 50 μM MG132, were analyzed. (B) In vivo ubiquitination analysis of OfJAZ3. The recombinant vector 35S::OfJAZ3-GFP was transiently expressed alone or co-expressed with 35S::OfCOI1-GFP in N. benthamiana leaves. The leaves were treated with MG132 for 12 h prior to sampling. Immunoprecipitation (IP) products obtained using an anti-GFP antibody, as well as crude extracts (Input), were analyzed by immunoblotting with anti-GFP and anti-ubiquitin antibodies, respectively. (C) The dual-luciferase reporter gene assay demonstrates that OfCOI1 restores the transcriptional activation of OfTPS2 by OfMYB21. Error bars indicate the SD of three biological replicates, and the asterisk represents the significant difference, evaluated by the Student’s t-test (*  P < 0.05, **  P < 0.01).

Discussion

Terpenoids are a class of natural products synthesized by plants with significant biological functions and economic value [23]. As a representative monoterpene, linalool serves as a core component of floral volatile organic compounds (VOCs) in aromatic plants, and exhibits enormous application potential in industries such as food, pharmaceuticals, and household chemicals [24–26]. Therefore, elucidating the biosynthetic and regulatory mechanisms of linalool is crucial for improving floral aroma quality and advancing research on terpenoid metabolic engineering. Studies have demonstrated that R2R3-MYB transcription factors regulate terpenoid metabolism by directly binding to the promoters of terpene synthase (TPS) genes [27, 28]. Previous studies from our group identified OfTPS2 as the linalool synthase gene in O. fragrans and revealed that its upstream regulator OfMYB21 activates OfTPS2 expression by binding to MYB motifs within its promoter. In this study, heterologous overexpression of OfMYB21 in A. thaliana and N. tabacum further confirmed that OfMYB21 enhances linalool biosynthesis by up-regulating endogenous TPS homologs. This regulatory mechanism is conserved across different species. For instance, FhMYB21L2 in Freesia hybrida activates monoterpene synthesis by binding to MYBCORE motifs in the FhTPS1 promoter [9], while AmMYB24 in Antirrhinum majus regulates the expression of ocimene synthase gene AmOCS via MYBCOREATCYCB1 motifs [29].

Phytohormones have emerged as key regulators of linalool biosynthesis [30]. However, it remains unclear whether the biosynthesis of linalool in O. fragrans is regulated by its endogenous hormones. Our study identified JA as a critical signaling molecule modulating linalool production in O. fragrans. During the early stage of O. fragrans flower development, the endogenous JA content exhibited a consistent trend with the expression level of OfTPS2 and the linalool accumulation. Furthermore, exogenous JA treatment was found to regulate linalool biosynthesis by activating the expression of OfTPS2, though the underlying molecular mechanism requires further investigation. As key proteins in the JA signaling pathway, JAZ proteins in A. thaliana inhibit floral organ development by interacting with AtMYB21/AtMYB24 [20]. In contrast, overexpression of the JAZ transcription factor OsJAZ8 in rice suppresses linalool synthesis and increases the plant’s susceptibility to bacterial leaf streak [31]. Using Dual-LUC assays and EMSA experiments, we demonstrated for the first time that OfJAZ3 in O. fragrans specifically inhibits the transcriptional activation of OfTPS2 by forming a repressive complex with OfMYB21. This negative regulatory mechanism is homologous to the JAZ-MYB21/24 module involved in A. thaliana floral development, suggesting a conserved regulatory network for terpenoid metabolism across plants. Furthermore, transient overexpression of OfJAZ3 in O. fragrans flowers, as well as stable overexpression in transgenic A. thaliana and N. tabacum plants, significantly down-regulated TPS gene expression and reduced linalool accumulation. These findings are consistent with the functional role of OsJAZ8 in rice, further confirming the cross-species conservation of JAZ-mediated terpenoid regulation. However, how JA regulates linalool biosynthesis through modulating OfJAZ3 remains unknown.

The mechanism by which JA signaling regulates JAZ protein stability via the ubiquitin-proteasome system (UPS) has been characterized in multiple species [32]. For example, in Salvia miltiorrhiza, JA induces 26S proteasome-dependent degradation of SmJAZ9, thereby relieving its inhibition on tanshinone biosynthesis [22]. In apple, JAZ proteins suppress anthocyanin synthesis by disrupting the MBW complex, while exogenous JA treatment promotes JAZ degradation and activates pigment accumulation [33]. Our study revealed that exogenous JA accelerates OfJAZ3 degradation and weakens its interaction with OfMYB21, whereas the proteasome inhibitor MG132 blocks this process. These results clarify the molecular pathway by which JA signaling regulates OfJAZ3 stability via UPS. This discovery mechanistically links with JA-induced terpenoid synthesis in Camellia sinensis [34] and Sindora glabra [35], providing novel insights into the universal regulatory principles of JA in terpenoid metabolism.

Here, we identified OfCOI1 in O. fragrans, a homolog of A. thaliana COI1, and confirmed its in vivo interaction with OfJAZ3. This finding directly connects the canonical JA signaling pathway with terpenoid metabolism, laying the foundation for elucidating the molecular mechanism of SCFOfCOI1-mediated OfJAZ3 degradation. Notably, E3 ligase-mediated targeting of transcription factors holds significant value for metabolic engineering. For example, the apple E3 ligase MdMIEL1 inhibits anthocyanin synthesis by ubiquitinating MdMYB308L for degradation [36], while the banana E3 ligase MaXB3 delays fruit ripening by degrading MaNAC029 [37]. These examples highlight the potential of future research to investigate OfCOI1-mediated ubiquitination of OfJAZ3 and its regulatory effects on linalool synthesis, which may offer new targets for metabolic engineering in aromatic plants. Based on the above results, we propose a hypothetical model diagram (Fig. 7) for JA-mediated linalool biosynthesis: JA treatment promotes the interaction between OfCOI1, a key protein of the E3 ubiquitin ligase, and OfJAZ3, which subsequently induces the degradation of OfJAZ3 via the ubiquitination pathway to release OfMYB21. Consequently, OfMYB21 activates OfTPS2 to facilitate linalool biosynthesis.

Figure 7.

Figure 7

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A hypothetical model diagram illustrating the involvement of the OfJAZ3-OfMYB21 complex in the regulation of linalool biosynthesis through the jasmonic acid signaling pathway.

Conclusion

In summary, based on the results of this study, we propose a regulatory model illustrating how the OfJAZ3-OfMYB21 module mediates the JA signaling pathway to regulate linalool biosynthesis in O. fragrans (Fig. 7). Specifically, when OfJAZ3 interacts with the transcription factor OfMYB21 to form a protein complex, it attenuates the transcriptional activation of the OfTPS2 gene by OfMYB21, thereby inhibiting linalool biosynthesis. In contrast, increased endogenous or exogenous JA levels induce the interaction between the E3 ubiquitin ligase SCFCOI1 complex and OfJAZ3, which promotes the ubiquitination and degradation of OfJAZ3 via the 26S proteasome pathway. This disruption of the OfJAZ3–OfMYB21 interaction ultimately restores the transcriptional activation of OfTPS2 by OfMYB21.

Materials and methods

Plant materials and treatments

Osmanthus fragrans (O. fragrans cultivar ‘Liuyejingui’), Arabidopsis (A. thaliana Col-0), and Nicotiana (N. benthamiana and N. tabacum) were used in this study. The flowers of O. fragrans at three flowering stages (bud stage (S1), initial blossoming stage (S2), and full blossoming stage (S3)) were collected for the determination of endogenous hormones, analysis of volatile compounds, and extraction of total RNA. The blank control buffer and MeJA (100 μM; Yuanye, Shanghai, China) solutions containing 0.02% Silwett-L77 (Yuanye, Shanghai, China) were used for the treatment of O. fragrans at the S1 stage. MeJA was dissolved in ethanol, mixed with water, and used as the culture solution. Water containing 0.02% Silwett-L77 and ethanol was used as the control buffer. Endogenous hormone contents in flowers at different developmental stages were quantified using liquid chromatography–tandem mass spectrometry. For volatile compound analysis, headspace collection combined with gas chromatography–mass spectrometry (GC–MS) was employed. Total RNA was extracted via the Trizol method, followed by reverse transcription into complementary DNA (cDNA). The resulting cDNA was used for subsequent gene cloning and quantitative Real-Time-PCR (qRT-PCR) analysis.

Determination of linalool content

The flowers (0.5 g) of O. fragrans was collected and placed into a 20 mL capped solid-phase microextraction (SPME) vial. Volatile compounds were collected using a 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber for 50 min. Post-extraction, the SPME fiber was inserted into the GC–MS injection port and desorbed at 250°C for 5 min. The analysis employed splitless injection mode with an injector temperature of 250°C. The oven temperature program was: initial 40°C (2 min hold), 3°C/min ramp to 100°C, followed by 5°C/min ramp to 245°C (5 min hold). Mass spectra were acquired in electron ionization (EI) mode at 70 eV, scanning m/z 50–400. Compound identification was performed using the NIST11 database [38].

Co-expression network and functional enrichment analysis

The online O. fragrans genome data analysis website (https://yanglab.hzau.edu.cn/OfIR/home/) was used to construct the gene co-expression network of OfMYB21 and OfJAZ3 to determine the molecular regulatory network involved in OfMYB21 and OfJAZ3. All genes (Supplementary Tables S1 and S2) co-expressed with OfMYB21 and OfJAZ3 were screened out through the tips of the website, and the KEGG enrichment of these genes were analyzed according to the tools provided by the online website [7].

Plant transformation

Using the cDNA isolated from the flowers of O. fragrans as a template, the coding sequences of OfMYB21 and OfJAZ3 were cloned separately. The cloned sequences were then ligated into the overexpression vector containing the CaMV35S promoter. The constructed recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101 and used to transform A. thaliana via floral dip infiltration. The transformed A. thaliana seeds were collected and planted on medium containing hygromycin B (Hyg) to screen for resistant plants [39]. In addition, the overexpression of transgenic N. tabacum plants were obtained (Supplementary Fig. S4) using the leaf disc transformation method, and the method was based on published papers [40]. All primers used for the experiment are included in the supplementary document (Supplementary Table S3). Flowers (1 g each) were collected from WT and transgenic A. thaliana plants, respectively; similarly, leaves (1 g each) were collected from WT and transgenic N. tabacum plants, respectively. The collected flowers or leaves were placed in a mortar, rapidly frozen with liquid nitrogen, and then ground into a fine powder. This powder was transferred to a 20-mL capped solid-phase microextraction (SPME) vial, and volatile compounds were collected using headspace sampling coupled with solid-phase microextraction (SPME) with a 50/30 μm DVB/CAR/PDMS fiber.

Transient transformation of O. fragrans flowers

Transient transformation of O. fragrans flowers was performed via vacuum infiltration [41]. O. fragrans flowers were placed in a conical flask containing A. tumefaciens resuspension and subjected to vacuum infiltration for approximately 10 min. The flowers were then rinsed 2–3 times with 1/2 MS liquid medium to remove residual bacterial solution, transferred to petri dishes, and fully immersed in fresh 1/2 MS liquid medium. After 12 h of dark incubation, the petals were moved to light conditions. Samples were collected 36–48 h post-infiltration to assess target gene expression and volatile compound.

qRT-PCR assays

Total RNA was extracted from plants using the Trizol method [42]. The extracted RNA was reverse-transcribed into cDNA using the HiScript III All-in-one RT SuperMix Perfect for qPCR kit. Specific primers for qRT-PCR were designed using Primer 5 software, with OfACT, Atactin8 and NtEF1-α selected as the reference gene. qRT-PCR analysis was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The coding sequences of all genes can be downloaded from the supplementary files (Supplementary Table S4).

Dual-luciferase assay

The coding sequence of OfMYB21 or OfJAZ3 was cloned into the pGreenII 62-SK vector, and the promoter sequence of OfTPS2 was amplified and ligated into the pGreenII 0800-LUC vector [17]. The constructed effector vector and reporter vector were separately transformed into A. tumefaciens GV3101 strain for cultivation. Subsequently, the A. tumefaciens cells carrying the effector and reporter vectors were mixed at a 1:1 ratio and resuspended in infiltration buffer to a final OD600 of 1.0, followed by 3 h of dark incubation for N. benthamiana infiltration. After 3 days of cultivation, the transformed N. benthamiana leaves were assayed using a firefly luciferase potassium salt solution, and the activities of luciferase (LUC) and Renilla luciferase (REN) were measured.

Electrophoretic mobility shift assay

The biotin-labeled probe containing the OfTPS2 promoter fragment was synthesized [17]. GST-tagged pGEX-6P-1-OfMYB21 and pGEX-6P-1-OfJAZ3 proteins were expressed in Escherichia coli strain BL21 and purified using GST Sep Glutathione Magnetic Beads (Yesheng Biotech, Cat. 20562ES03). Electrophoretic mobility shift assay (EMSA) experiments were performed using the chemiluminescent EMSA kit (Beyotime Biotechnology, China) following the manufacturer’s protocol.

Subcellular localization

The coding sequence of OfCOI1 or OfJAZ3 was cloned and inserted into the pCAMBIA1305 vector containing a GFP tag. The constructed expression vector was then transformed into N. benthamiana leaves using A. tumefaciens-mediated transient transformation. After 40–44 h post-transformation, GFP fluorescence signals were observed using a confocal laser scanning microscope system.

Split luciferase assays

The coding sequences of OfMYB21 and OfCOI1 were cloned and individually inserted into the pCAMBIA1300-nLUC vector, while the OfJAZ3 coding sequence was cloned into the pCAMBIA1300-cLUC vector. Subsequently, the constructed recombinant plasmids were separately transformed into A. tumefaciens strain GV3101 and cultured. The A. tumefaciens cultures carrying OfMYB21-nLUC and OfCOI1-nLUC recombinant plasmids were each mixed 1:1 with A. tumefaciens harboring OfJAZ3-cLUC, resuspended in infiltration buffer (10 mM MES, 10 mM MgCl2, and 150 μM AS) to a final OD600 of 1.0, and incubated in the dark for 3 h before N. benthamiana leaves infiltration. Luciferase activity was detected using a potassium luciferin solution and a chemiluminescence imaging system (Tanon 5200, Tanon, China).

Bimolecular fluorescence complementation assay

The coding sequences of OfCOI1 and OfJAZ3 were cloned into cYFP and nYFP vectors, respectively. The resulting recombinant plasmids were transformed into A. tumefaciens strain GV3101 and cultured. Transient expression in N. benthamiana leaves was performed using the method described above, and YFP fluorescence signals were detected by confocal laser scanning microscopy.

GST pull-down assay

The coding sequence of OfCOI1 was inserted into the pGEX-6P-1 vector containing a GST tag. Similarly, the OfJAZ3 coding sequence was cloned into the pET-30a vector with a His-tag. The constructed recombinant plasmids were individually transformed into E. coli BL21 cells, and fusion protein expression was induced by adding 0.5 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture medium. The GST-tagged fusion protein was purified using commercially available GSTSep Glutathione MagBeads (Yisheng Biotechnology, Shanghai, China) according to the manufacturer’s protocol. Pull-down assays were performed following the kit instructions.

Protein degradation assay

The N. benthamiana leaves were infiltrated with A. tumefaciens strains harboring 35S::OfJAZ3-GFP. After infiltration 50 h, 3 g of A. tumefaciens-infiltrated leaves was collected. The total protein was extracted and incubated at 22°C for indicated time periods without or with 100 μM MeJA, then separated by SDS-PAGE, transferred to PVDF membrane, and detected with GFP antibody. For the detection of GFP fluorescence signals in root tip cells of 35S::OfJAZ3-GFP transgenic A. thaliana plants under JA solution treatment, as well as the processing of experimental materials, we followed the detailed procedures described in previously published literature [43].

To investigate the regulatory role of OfCOI1 on OfJAZ3 protein abundance, total proteins were extracted from N. benthamiana leaves transiently expressing 35S::OfCOI1-GFP. The recombinant His-OfJAZ3 protein obtained was added to equal amounts of total protein extracts and incubated for 0, 1, 2, 3, 4, and 5 h. Equal volumes of cell lysates were treated with 50 μM MG132. Finally, 2 × SDS loading buffer was added, and samples were boiled at 100°C for 10 min to terminate the reactions. The amount of His-OfJAZ3 protein was then assessed by immunoblotting using His antibody.

Ubiquitination assay

The ubiquitination assay was conducted with reference to published papers [44]. Using the Agrobacterium-mediated transformation method, the constructed 35S::OfJAZ3-GFP recombinant vector was expressed either alone or co-expressed with the 35S::OfCOI1-GFP recombinant vector in N. benthamiana leaves. After dark-culturing the transformed tobacco plants for 12 h, they were transferred to light conditions and cultured for 2 days. Leaf samples were collected following a 12-h treatment with 50 μM MG132. For immunoprecipitation, GFP-tagged protein magnetic beads were incubated with total protein extracted from N. benthamiana leaves for 2–3 h. Non-specific proteins bound to the magnetic beads were washed away using washing buffer, and the proteins were then eluted with 1 × SDS loading buffer. Subsequently, immunoblot analysis was performed using anti-ubiquitin antibodies and anti-GFP antibodies, respectively.

Supplementary Material

Web_Material_uhaf321
web_material_uhaf321.zip (1.1MB, zip)

Acknowledgments

We thank the members of the Zhejiang Agriculture and Forestry University School of Landscape Architecture and Anhui Province Key Laboratory of Forest Resources and Silviculture for their assistance in this study. This work was supported by National Natural Science Foundation of China (nos 32501723 and 32472782), Scientific Research Foundation of Zhejiang A&F University (no. 2024LFR117), Anhui Provincial Education Department (no. 2022AH050903), and Leading Talent Project for Scientific and Technological Innovation of Zhejiang Province (no. 2022R52026).

Contributor Information

Yangang Lan, School of Landscape Architecture, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China; Anhui Provincial Key Laboratory of Forest Resources and Silviculture, Anhui Agricultural University, Hefei 230036, China.

Xue Huang, School of Landscape Architecture, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China.

Ziyi Li, School of Landscape Architecture, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China.

Shunran Zhang, Anhui Provincial Key Laboratory of Forest Resources and Silviculture, Anhui Agricultural University, Hefei 230036, China.

Yan Xiang, Anhui Provincial Key Laboratory of Forest Resources and Silviculture, Anhui Agricultural University, Hefei 230036, China.

Hongbo Zhao, School of Landscape Architecture, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China.

Author contributions

Y.G. L designed and performed the experiments. Y.G. L and X. H wrote the manuscript and handled figures and tables. S.R. Z, Z.Y. L and H.B. Z helped to revise the manuscript. Y. X, H.B. Z, and Y.G. L provided financial support for the article and designed the way and frame of this study. All the authors read and approved the final manuscript.

Data availability

The data supporting the findings of this study are available within the paper figures and the Supplementary Information.

Conflicts of interest statement

The authors declare no conflict of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Web_Material_uhaf321
web_material_uhaf321.zip (1.1MB, zip)

Data Availability Statement

The data supporting the findings of this study are available within the paper figures and the Supplementary Information.

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