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

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 SCF COI1 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.

Acknowledgements

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).

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.

References

[1] Qing H, Chen J, Jiang L. et al. Functional characterization of two lycopene cyclases from sweet Osmanthus (Osmanthus fragrans). Sci Hortic. 2022; 299:111062

[2] Zhou L, Tian Q, Ding W. et al. The OfMYB1R114-OfSDIR1-like-OfCCD4 module regulates β-ionone synthesis in Osmanthus fragrans. Ind Crop Prod. 2024; 217:118879

[3] Miao Y, Li W, Zhu H. et al. The roles of OfEXPA2 and OfEXPA4 on petal cell expansion during flower opening in Osmanthus fragrans. Sci Hortic. 2024; 338:113720-0

[4] Yang Z, Lu Y, Li T. et al. Osmanthus fragrans flavonoid extract inhibits adipogenesis and induces beiging in 3T3-L 1 adipocytes. Foods. 2024; 13:1894-4

[5] Chen H, Zeng X, Yang J. et al. Whole-genome resequencing of Osmanthus fragrans provides insights into flower color evolution. Hortic Res. 2021; 8:8

[6] Yang X, Yue Y, Li H. et al. The chromosome-level quality genome provides insights into the evolution of the biosynthesis genes for aroma compounds of Osmanthus fragrans. Hortic Res. 2018; 5:72

[7] Yu Z, Li Y, Song T. et al. OGRP: a comprehensive bioinformatics platform for the efficient empowerment of Oleaceae genomics research. Hortic Plant J. 2025; 11:1308-25

[8] Xi W, Jiang MY, Zhu LL. et al. OfWRKY33 binds to the promoter of key linalool synthase gene OfTPS7 to stimulate linalool synthesis in Osmanthus fragrans flowers. Hortic Res. 2025; 9:12

[9] Yang Z, Li Y, Gao F. et al. MYB21 interacts with MYC2 to control the expression of terpene synthase genes in flowers of Freesia hybrida and Arabidopsis thaliana. J Exp Bot. 2020; 71:4140-58

[10] Wei S, Wu J, Yu P. et al. Metabolomic and transcriptomic analysis of unique floral coloration in Osmanthus fragrans cultivars. Horticulturae. 2024; 10:801-1

[11] Yue Y, Shi T, Liu J. et al. Genomic, metabonomic and transcriptomic analyses of sweet osmanthus varieties provide insights into floral aroma formation. Sci Hortic. 2022; 3:111442-2

[12] Zeng X, Liu C, Zheng R. et al. Emission and accumulation of monoterpene and the key terpene synthase (TPS) associated with monoterpene biosynthesis in Osmanthus fragrans Lour. Front Plant Sci. 2016; 6:1232

[13] Xiong R, Chen Z, Wang W. et al. Combined transcriptome sequencing and prokaryotic expression to investigate the key enzyme in the 2-C-methylerythritol-4-phosphate pathway of Osmanthus fragrans. Funct Plant Biol. 2020; 47:945-58

[14] Han Y, Wang H, Wang X. et al. Mechanism of floral scent production in Osmanthus fragrans and the production and regulation of its key floral constituents, β-ionone and linalool. Hortic Res. 2019; 6:106

[15] Ding W, Ouyang Q, Li Y. et al. Genome-wide investigation of WRKY transcription factors in sweet osmanthus and their potential regulation of aroma synthesis. Tree Physiol. 2020; 40:557-72

[16] Li H, Yue Y, Ding W. et al. Genome-wide identification, classification, and expression profiling reveals R2R3-MYB transcription factors related to monoterpenoid biosynthesis in Osmanthus fragrans. Genes. 2020; 4:11

[17] Lan Y, Zhang K, Wang L. et al. The R2R3-MYB transcription factor OfMYB21 positively regulates linalool biosynthesis in Osmanthus fragrans flowers. Int J Biol Macromol. 2023; 249:126099

[18] Sato C, Aikawa K, Sugiyama S. et al. Distal transport of exogenously applied Jasmonoyl-isoleucine with wounding stress. Plant Cell Physiol. 2011; 52:509-17

[19] Xu YH, Wang JW, Wang S. et al. Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-δ-cadinene synthase-a. Plant Physiol. 2004; 135:507-15

[20] Song S, Qi T, Huang H. et al. The jasmonate-ZIM domain proteins interact with the R2R3-MYB transcription factors MYB21 and MYB24 to affect jasmonate-regulated stamen development in Arabidopsis. Plant Cell. 2011; 23:1000-13

[21] Liu Y, Wang H. JA modulates phytochrome a signaling via repressing FHY3 activity by JAZ proteins. Plant Signal Behav. 2020; 15:15

[22] Liu S, Gao X, Shi M. et al. Jasmonic acid regulates the biosynthesis of medicinal metabolites via the JAZ9-MYB76 complex in salvia miltiorrhiza. Hortic Res. 2023; 3:10

[23] Wei G, Chen Y, Wang J. et al. Molecular cloning and characterization of farnesyl diphosphate synthase from Rosa rugosa Thunb associated with salinity stress. PEERJ. 2024; 12:e16929

[24] Yuan M, Xu F, Zheng Y. et al. Diverse terpenoid glycosides with invitro cytotoxicity from Glechoma longituba. Phytochemistry. 2024; 217:113923

[25] Aytac Z, Yildiz ZI, Kayaci-Senirmak F. et al. Electrospinning of cyclodextrin/linalool-inclusion complex nanofibers: fast-dissolving nanofibrous web with prolonged release and antibacterial activity. Food Chem. 2017; 231:192-201

[26] Ying Z, Rufang D, Xinlan X. et al. Enzyme catalytic efficiencies and relative gene expression levels of (R)-linalool synthase and (S)-linalool synthase determine the proportion of linalool enantiomers in Camellia sinensis var. sinensis. J Agric Food Chem. 2020; 37:10109-17

[27] Ke Y, Abbas F, Zhou Y. et al. Auxin-responsive R2R3-MYB transcription factors HcMYB1 and HcMYB2 activate volatile biosynthesis in Hedychium coronarium flowers. Front Plant Sci. 2021; 12:710826

[28] Yin J, Sun L, Li Y. et al. Functional identification of BpMYB21 and BpMYB61 transcription factors responding to MeJA and SA in birch triterpenoid synthesis. BMC Plant Biol. 2020; 20:20

[29] Han J, Li T, Wang X. et al. AmMYB24 regulates floral terpenoid biosynthesis induced by blue light in snapdragon flowers. Front Plant Sci. 2022; 13:885168

[30] Shen Y, Wang J, Si X. et al. Revealing the molecular mechanism of biosynthesis and transcriptional regulation of PAs, caffeine and linalool globally under simulative stress in coffee plants. Int J Biol Macromol. 2025; 310:143103

[31] Taniguchi S, Hosokawa-Shinonaga Y, Tamaoki D. et al. Jasmonate induction of the monoterpene linalool confers resistance to rice bacterial blight and its biosynthesis is regulated by JAZ protein in rice. Plant Cell Environ. 2014; 37:451-61

[32] Kowalski L, Bragoszewski P, Chacinska A. Molecular and functional insights into mitochondrial intermembrane space proteins degradation by ubiquitin-proteasome system. FEBS J. 2017; 284:299-9

[33] Xing-Bin X, Shen L, Rui-Fen Z. et al. The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples. Plant Cell Environ. 2012; 11:1884-97

[34] Shi J, Ma C, Qi D. et al. Transcriptional responses and flavor volatiles biosynthesis in methyl jasmonate-treated tea leaves. BMC Plant Biol. 2015; 15:233

[35] Yu N, Chen Z, Yang J. et al. Integrated transcriptomic and metabolomic analyses reveal regulation of terpene biosynthesis in the stems of Sindora glabra. Tree Physiol. 2021; 41:1087-102

[36] An J, Wang X, Zhang X. et al. An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation. Plant Biotechnol J. 2020; 18:337-53

[37] Wei W, Yang Y, Chen J. et al. MaNAC029 modulates ethylene biosynthesis and fruit quality and undergoes MaXB3-mediated proteasomal degradation during banana ripening. J Adv Res. 2023; 53:33-47

[38] Lan Y, Xiong R, Zhang K. et al. Geranyl diphosphate synthase large subunits OfLSU1/2 interact with small subunit OfSSUII and are involved in aromatic monoterpenes production in Osmanthus fragrans. Int J Biol Macromol. 2024; 256:128328

[39] Li W, Yu S, Zhang K. et al. Characterization of the JAZ family in Osmanthus fragrans and the role of OfLJAZ 2 in flavonoid synthesis mediated regulation of salt and drought stress tolerance. Plant Physiol Biochem. 2025; 228:110235

[40] Wan Q, Lu M, Jiang G. et al. The characterization of OfRGA in regulation of flower size through tuning cell expansion genes. Front Plant Sci. 2024; 15:1502347

[41] Hai X, Liu G, Lu M. et al. The AP2/ERF transcription factor OfERF2 promotes flavonoid biosynthesis in Osmanthus fragrans. Tree Genet Genomes. 2025; 21:21

[42] Zhang K, Lan Y, Shi Y. et al. Systematic analysis and functional characterization of the PLATZ transcription factors in Moso bamboo (Phyllostachys edulis). J Plant Growth Regul. 2023; 42:218-36

[43] Chung H, Howe G. A critical role for the TIFY motif in repression of jasmonate signaling by a stabilized splice variant of the JASMONATE ZIM-domain protein JAZ 10 in Arabidopsis. Plant Cell. 2009; 21:131-45

[44] Yan J, Liu Q, Guo P. et al. Time-course transcriptome analysis unveils the CoFKF1-CoMYB4-CoFT1 regulatory module in flowering control of Camellia oleifera Abel. Plant Cell Environ. 2025; 48:6153-69