The transcription factor MYC2 positively regulates terpene trilactone biosynthesis through activating GbGGPPS expression in Ginkgo biloba

Abstract

Terpene trilactones (TTLs) have important medicinal value, but their low content in Ginkgo biloba leaves makes their exploitation extremely costly, thereby limiting the development of TTL-related industries. It was found that exogenous methyl jasmonate (MeJA) treatment increased the accumulation of TTLs, but the molecular mechanism is still unclear. Here, we identified two bHLH transcription factors in G. biloba, with the protein subcellular localizations in the nucleus. Expression of GbMYC2s was strongly induced by MeJA treatment, and the interactions between GbJAZs and GbMYC2s were demonstrated by yeast two-hybrid and bimolecular fluorescence complementation experiments. Overexpression of GbMYC2_4 and GbMYC2_5 enhanced Arabidopsis root sensitivity and significantly increased TTL content. In addition, GbGGPPS was found to be a common target of GbMYC2_4 and GbMYC2_5 by yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase reporter assays and DAP-seq, and they achieved regulation of GbGGPPS by binding to the G-box. Further findings revealed that GbMYC2_4 and GbMYC2_5 bind the G-box not universally but selectively. Our study revealed that jasmonic acid signaling mediates TTL biosynthesis through the GbJAZ-GbMYC2-GbGGPPS module, which enriches the terpenoid biosynthesis regulatory networks and provides a research basis and target genes for enhancing TTL content through genetic engineering.

Acknowledgements

The study was supported by the National Natural Science Foundation of China (No. 31901344 and No. 31971693).

Author contributions

F.X., J.Z., and Y.L. conceived and designed the research; J.Z. and J.Y. performed the experiments; J.Y. and X.H. analyzed part of the data; Z.C., X.Z., Y.X., and Y.Y. constructed some of the vectors; Q.C. and J.S. contributed to subcellular localization experiments. J.Z. wrote the manuscript. L.W. and W.Z. revised the manuscript. All authors approved the final version.

Data availability

All relevant data can be found within the manuscript and its supporting materials. The RNA-seq data have been deposited in the China National GeneBank DataBase: CNP0004970. DAP-seq data have been deposited in the China National GeneBank DataBase: CNP0004964.

Conflict of interest

The authors declare that they have no competing interests.

Supplementary data

Supplementary data are available at Horticulture Research online.

References

[1] Tan JP, Han MX, Mao D. et al. Comparative transcriptomic analysis reveals the regulatory mechanism of terpene trilactones improvement by exogenous methyl jasmonate in Ginkgo biloba. Plant Mol Biol Report.. 2022; 40:81-94

[2] Ye J, Zhang X, Tan J. et al. Global identification of Ginkgo biloba microRNAs and insight into their role in metabolism regulatory network of terpene trilactones by high-throughput sequencing and degradome analysis. Ind Crop. Prod. 2020; 148:112289

[3] Zheng J, Zhang X, Fu M. et al. Effects of different stress treatments on the total terpene trilactone content and expression levels of key genes in Ginkgo biloba leaves. Plant Mol Biol Rep. 2020; 38:521-30

[4] Chandrasekaran K, Mehrabian Z, Spinnewyn B. et al. Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia. Brain Res. 2001; 922:282-92

[5] Strømgaard K, Saito DR, Shindou H. et al. Ginkgolide derivatives for photolabeling studies: preparation and pharmacological evaluation. J Med Chem. 2002; 45:4038-46

[6] Liu XW, Yang JL, Niu W. et al. Human pharmacokinetics of ginkgo terpene lactones and impact of carboxylation in blood on their platelet-activating factor antagonistic activity. Acta Pharmacol Sin. 2018; 39:1935-46

[7] Li Y, Wu Y, Yao X. et al. Ginkgolide A ameliorates LPS-induced inflammatory responses in vitro and in vivo. Int J Mol Sci. 2017; 18:794

[8] Strømgaard K, Nakanishi K. Chemistry and biology of terpene trilactones from Ginkgo biloba. Angew Chem.. 2004; 43:1640-58

[9] Tholl D. Biosynthesis and biological functions of terpenoids in plants. Adv Biochem Eng Biotechnol. 2015; 148:63-106

[10] Rodríguez-Concepción M, Boronat A. Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol. 2002; 130:1079-89

[11] Forman V, Luo D, Geu-Flores F. et al. A gene cluster in Ginkgo biloba encodes unique multifunctional cytochrome P450s that initiate ginkgolide biosynthesis. Nat Commun. 2022; 13:5143

[12] Ye J, Yang K, Li Y. et al. Genome-wide transcriptome analysis reveals the regulatory network governing terpene trilactones biosynthesis in Ginkgo biloba. Tree Physiol.. 2022; 42:2068-85

[13] Kumar SR, Rai A, Bomzan DP. et al. A plastid-localized bona fide geranylgeranyl diphosphate synthase plays a necessary role in monoterpene indole alkaloid biosynthesis in Catharanthus roseus. Plant J.. 2020; 103:248-65

[14] Shi M, Luo X, Ju G. et al. Enhanced diterpene tanshinone accumulation and bioactivity of transgenic Salvia miltiorrhiza hairy roots by pathway engineering. J Agric Food Chem. 2016; 64:2523-30

[15] Du M, Zhao J, Tzeng DTW. et al. MYC2 orchestrates a hierarchical transcriptional cascade that regulates jasmonate-mediated plant immunity in tomato. Plant Cell. 2017; 29:1883-906

[16] Goossens J, Swinnen G, Vanden BR. et al. Change of a conserved amino acid in the MYC 2 and MYC 3 transcription factors leads to release of JAZ repression and increased activity. New Phytol. 2015; 206:1229-37

[17] Zhou X, Liao Y, Kim SU. et al. Genome-wide identification and characterization of bHLH family genes from Ginkgo biloba. Sci Rep.. 2020; 10:13723

[18] Fernández-Calvo P, Chini A, Fernández-Barbero G. et al. The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses. Plant Cell. 2011; 23:701-15

[19] Godoy M, Franco-Zorrilla JM, Pérez-Pérez J. et al. Improved protein-binding microarrays for the identification of DNA-binding specificities of transcription factors: protein-binding microarrays for DNA-binding specificities of transcription factors. Plant J. 2011; 66:700-11

[20] Hong GJ, Xue XY, Mao YB. et al. Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression. Plant Cell. 2012; 24:2635-48

[21] Shoji T, Hashimoto T. Tobacco MYC2 regulates jasmonate-inducible nicotine biosynthesis genes directly and by way of the NIC2-locus ERF genes. Plant Cell Physiol. 2011; 52:1117-30

[22] 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

[23] Huo Y, Zhang J, Zhang B. et al. MYC2 transcription factors TwMYC2a and TwMYC2b negatively regulate triptolide biosynthesis in hairy roots. Plants (Basel). 2021; 10:679

[24] Sui X, Singh SK, Patra B. et al. Cross-family transcription factor interaction between MYC2 and GBFs modulates terpenoid indole alkaloid biosynthesis. J Exp Bot. 2018; 69:4267-81

[25] Yang N, Zhou W, Su J. et al. Overexpression of SmMYC2 increases the production of phenolic acids in Salvia miltiorrhiza. Front. Plant Sci.. 2017; 8:1804

[26] Zhang M, Jin X, Chen Y. et al. TcMYC2a, a basic helix-loop-helix transcription factor, transduces JA-signals and regulates taxol biosynthesis in Taxus chinensis. Front Plant Sci.. 2018; 9:863

[27] Montiel G, Zarei A, Körbes AP. et al. The jasmonate-responsive element from the ORCA3 promoter from Catharanthus roseus is active in Arabidopsis and is controlled by the transcription factor AtMYC2. Plant Cell Physiol. 2011; 52:578-87

[28] Ma C, Li R, Sun Y. et al. ZmMYC2s play important roles in maize responses to simulated herbivory and jasmonate. J Integr Plant Biol. 2023; 65:1041-58

[29] Shen Q, Lu X, Yan T. et al. The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua. New Phytol.. 2016; 210:1269-81

[30] Bartlett A, O’Malley RC, Huang SC. et al. Mapping genome-wide transcription-factor binding sites using DAP-seq. Nat Protoc. 2017; 12:1659-72

[31] Shi J, Zhao B, Jin R. et al. A phosphate starvation response-regulated receptor-like kinase, OsADK1, is required for mycorrhizal symbiosis and phosphate starvation responses. New Phytol. 2022; 236:2282-93

[32] Dombrecht B, Xue GP, Sprague SJ. et al. MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis. Plant Cell.. 2007; 19:2225-45

[33] Zhuo M, Sakuraba Y, Yanagisawa S.A jasmonate-activated MYC2-Dof2.1-MYC 2 transcriptional loop promotes leaf senescence in Arabidopsis. Plant Cell.. 2020; 32:242-62

[34] Horbowicz M, Wiczkowski W, Góraj-Koniarska J. et al. Effect of methyl jasmonate on the terpene trilactones, flavonoids, and phenolic acids in Ginkgo biloba L. leaves: relevance to leaf senescence. Molecules. 2021; 26:4682

[35] Kazan K, Manners JM. MYC2: The master in action. Mol Plant. 2013; 6:686-703

[36] Zhou Y, Sun W, Chen J. et al. SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza. Sci Rep.. 2016; 6:22852

[37] Zhang H, Hedhili S, Montiel G. et al. The basic helix-loop-helix transcription factor CrMYC2 controls the jasmonate-responsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus: CrMYC2 controls JA-responsive ORCA gene expression. Plant J. 2011; 67:61-71

[38] Min T, Yin X, Shi Y. et al. Ethylene-responsive transcription factors interact with promoters of ADH and PDC involved in persimmon (Diospyros kaki) fruit de-astringency. J Exp Bot. 2012; 63:6393-405

[39] Yin X, Xie X, Xia X. et al. Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening. Plant J. 2016; 86:403-12

[40] Li X, Xu Y, Shen S. et al. Transcription factor CitERF71 activates the terpene synthase gene CitTPS16 involved in the synthesis of E-geraniol in sweet orange fruit. J Exp Bot. 2017; 68:4929-38

[41] De Geyter N, Gholami A, Goormachtig S. et al. Transcriptional machineries in jasmonate-elicited plant secondary metabolism. Trends Plant Sci. 2012; 17:349-59

[42] Pei T, Ma P, Ding K. et al. SmJAZ8 acts as a core repressor regulating JA-induced biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots. J Exp Bot. 2018; 69:1663-78

[43] Chico JM, Chini A, Fonseca S. et al. JAZ repressors set the rhythm in jasmonate signaling. Curr Opin Plant Biol. 2008; 11:486-94

[44] Chini A, Fonseca S, Fernández G. et al. The JAZ family of repressors is the missing link in jasmonate signalling. Nature. 2007; 448:666-71

[45] Wu F, Deng L, Zhai Q. et al. Mediator subunit MED25 couples alternative splicing of JAZ genes with fine-tuning of jasmonate signaling. Plant Cell. 2020; 32:429-48

[46] Zhang C, Lei Y, Lu C. et al. MYC2, MYC3, and MYC4 function additively in wounding-induced jasmonic acid biosynthesis and catabolism. J Integr Plant Biol. 2020; 62:1159-75

[47] He K, Du J, Han X. et al. PHOSPHATE STARVATION RESPONSE1 (PHR1) interacts with JASMONATE ZIM-DOMAIN (JAZ) and MYC2 to modulate phosphate deficiency-induced jasmonate signaling in Arabidopsis. Plant Cell.. 2023; 35:2132-56

[48] Liu Y, Du M, Deng L. et al. MYC2 regulates the termination of jasmonate signaling via an autoregulatory negative feedback loop. Plant Cell. 2019; 31:106-27

[49] Min D, Li F, Zhang X. et al. SlMYC2 involved in methyl jasmonate-induced tomato fruit chilling tolerance. J Agric Food Chem. 2018; 66:3110-7

[50] Wang H, Li Y, Pan J. et al. The bHLH transcription factors MYC2, MYC3, and MYC4 are required for jasmonate-mediated inhibition of flowering in Arabidopsis. Mol Plant.. 2017; 10:1461-4

[51] Uji Y, Taniguchi S, Tamaoki D. et al. Overexpression of OsMYC2 results in the up-regulation of early JA-responsive genes and bacterial blight resistance in rice. Plant Cell Physiol. 2016; 57:1814-27

[52] Fu J, Liu L, Liu Q. et al. ZmMYC2 exhibits diverse functions and enhances JA signaling in transgenic Arabidopsis. Plant Cell Rep.. 2020; 39:273-88

[53] Qi T, Song S, Ren Q. et al. The jasmonate-ZIM-domain proteins interact with the WD-Repeat/bHLH/MYB complexes to regulate jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana. Plant Cell.. 2011; 23:1795-814

[54] Chini A, Fonseca S, Chico JM. et al. The ZIM domain mediates homo- and heteromeric interactions between Arabidopsis JAZ proteins. Plant J. 2009; 59:77-87

[55] Lu J, Xie L, Liu K. et al. Bilobalide: a review of its pharmacology, pharmacokinetics, toxicity, and safety. Phytother Res. 2021; 35:6114-30

[56] Samandar F, Tehranizadeh ZA, Saberi MR. et al. CB1 as a novel target for Ginkgo biloba’s terpene trilactone for controlling chemotherapy-induced peripheral neuropathy (CIPN). J Mol Model. 2022; 28:283

[57] Xu F, Huang XH, Li LL. et al. Molecular cloning and characterization of GbDXS and GbGGPPS gene promoters from Ginkgo biloba. Gen Mol Res.. 2013; 12:293-301

[58] Schepmann HG, Pang J, Matsuda SP. Cloning and characterization of Ginkgo biloba levopimaradiene synthase which catalyzes the first committed step in ginkgolide biosynthesis. Arch Biochem Biophys. 2001; 392:263-9

[59] Zhang N, Han Z, Sun G. et al. Molecular cloning and characterization of a cytochrome P 450 taxoid 9α-hydroxylase in Ginkgo biloba cells. Biochem Biophys Res Commun. 2014; 443:938-43

[60] López-Vidriero I, Godoy M, Grau J. et al. DNA features beyond the transcription factor binding site specify target recognition by plant MYC2-related bHLH proteins. Plant Commun. 2021; 2:100232

[61] De Boer K, Tilleman S, Pauwels L. et al. APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis. Plant J. 2011; 66:1053-65

[62] Yu ZX, Li JX, Yang CQ. et al. The jasmonate-responsive AP2/ERF transcription factors AaERF1 and AaERF2 positively regulate artemisinin biosynthesis in Artemisia annua L. Mol Plant. 2012; 5:353-65

[63] Zheng J, Yang X, Ye J. et al. Multiomics analysis provides new insights into the regulatory mechanism of carotenoid biosynthesis in yellow peach peel. Mol Hortic. 2023; 3:23

[64] Zhou Y, Ma Y, Zeng J. et al. Convergence and divergence of bitterness biosynthesis and regulation in Cucurbitaceae. Nat Plants. 2016; 2:16183

[65] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001; 25:402-8

[66] Zheng J, He X, Zhou X. et al. The Ginkgo biloba microRNA160-ERF4 module participates in terpene trilactone biosynthesis. Plant Physiol. 2024; 195:1446-60

[67] Fang Y, Wang D, Xiao L. et al. Allelic variation in transcription factor PtoWRKY68 contributes to drought tolerance in Populus. Plant Physiol.. 2023; 193:736-55

[68] Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9:357-9

[69] Machanick P, Bailey TL. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics. 2011; 27:1696-7

[70] Yu G, Wang LG, He QY. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics. 2015; 31:2382-3

[71] Ming R, Zhang Y, Wang Y. et al. The JA-responsive MYC2-BADH-like transcriptional regulatory module in Poncirus trifoliata contributes to cold tolerance by modulation of glycine betaine biosynthesis. New Phytol. 2021; 229:2730-50