Genetic blueprint of herbaceous peony floral scent: evidence from terpene synthase, Nudix hydrolase, and prenyltransferase

Abstract

Volatile terpenes constitute a predominant class of floral scent emitted by Paeonia lactiflora. Despite their ecological and economical significance, the genetic blueprint of the underlying biosynthetic pathway remains poorly elucidated. Although a few terpene synthase (TPS) genes have been reported, the broader network of genes orchestrating terpene production in P. lactiflora is still largely unresolved. In this study, we attempted to address this gap by exploring the terpene biosynthetic pathway genes in P. lactiflora ‘Zifeng yu’. β-caryophyllene, geraniol, citronellol, and 1,8-cineole were identified as the dominant floral terpenes, and catalytic functions of key proteins-terpene synthase (PlTPS), Nudix hydrolase (PlNUDX), and prenyltransferase (PlPT) were comprehensively characterized. Briefly, biochemical analyses revealed that six of the nine identified PlTPS proteins utilized diverse prenyl diphosphates to generate both monoterpenes and sesquiterpenes, while their products specificity were determined by plastidic or cytosolic localizations in planta. In particular, PlTPS4, PlTPS5, and PlTPS9 catalyzed the production of β-caryophyllene, 1,8-cineole, and geraniol, respectively. Besides, two amino acid residues were found to drive catalytic activity and product profiles in PlTPS4 and PlTPS5. Markedly, PlNUDX hydrolyzed GPP and NPP to yield geraniol and nerol thereby providing a plastid-independent pathway for monoterpene biosynthesis, and prenyltransferases were further functionally characterized to clarify the supply of prenyl diphosphates feeding into volatile terpenes. Collectively, these findings not only provide a mechanistic framework for understanding floral terpene biosynthesis in P. lactiflora but also reveal alternative metabolic routes that enrich its volatile profiles that could be utilized in scent improvement of ornamental plants.

References

[1] Chen ZY, Zhang XQ, Chen L, et al. Identification and expression pattern analysis of NUDIX gene family in Camellia sinensis. J Tea Sci Res. 2023b; 43: 159-72.

[2] Chelliah A, Sundararaju MP, Arumugam C, et al. Comprehensive characterization of Nudix hydrolase genes in Musa spp.: unveiling susceptibility factors to biotic stresses. Physiol Mol Plant Pathol. 2025; 139: 102749.

[3] Hsiao YY, Jeng MF, Tsai WC, et al. A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2-4D motif. Plant J. 2008; 55: 719-33.

[4] Rai A, Smita SS, Singh AK, et al. Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis. Mol Plant. 2013; 6: 1531-49.

[5] Schmidt A, Gershenzon J . Cloning and characterization of two different types of geranyl diphosphate synthases from Norway spruce (Picea abies). Phytochemistry. 2008; 69: 49-57.

[6] Schmidt A, Wächtler B, Temp U, et al. A bifunctional geranyl and geranylgeranyl diphosphate synthase is involved in terpene oleoresin formation in Picea abies. Plant Physiol. 2010; 152: 639-55.

[7] Burke C, Croteau R . Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate. J Biol Chem. 2002; 277: 3141-9.

[8] Tholl D, Kish CM, Orlova I, et al. Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases. Plant Cell. 2004; 16: 977-92.

[9] Wang G, Dixon RA . Heterodimeric geranyl (geranyl) diphosphate synthase from hop (Humulus lupulus) and the evolution of monoterpene biosynthesis. Proc Natl Acad Sci USA. 2009; 106: 9914-9.

[10] Chang TH, Hsieh FL, Ko TP, et al. Structure of a heterotetrameric geranyl pyrophosphate synthase from mint (Mentha piperita) reveals intersubunit regulation. Plant Cell. 2010; 22: 454-67.

[11] Burke CC, Wildung MR, Croteau R . Geranyl diphosphate synthase: cloning, expression, and characterization of this prenyltransferase as a heterodimer. Proc Natl Acad Sci. 1999; 96: 13062-7.

[12] Orlova I, Nagegowda DA, Kish CM, et al. The small subunit of snapdragon geranyl diphosphate synthase modifies the chain length specificity of tobacco geranylgeranyl diphosphate synthase in planta. Plant Cell. 2009; 21: 4002-17.

[13] Wei G, Tian P, Zhang F, et al. Integrative analyses of nontargeted volatile profiling and transcriptome data provide molecular insight into VOC diversity in cucumber plants (Cucumis sativus). Plant Physiol. 2016; 172: 603-18.

[14] Zhou F, Pichersky E . The complete functional characterisation of the terpene synthase family in tomato. New Phytol. 2020b; 226: 1341-60.

[15] Okada K, Saito T, Nakagawa T, et al. Five geranylgeranyl diphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis. Plant Physiol. 2000; 122: 1045-56.

[16] Thabet I, Guirimand G, Guihur A, et al. Characterization and subcellular localization of geranylgeranyl diphosphate synthase from Catharanthus roseus. Mol Biol Rep. 2012; 39: 3235-43.

[17] Suttiyut T, Auber RP, Ghaste M, et al. Integrative analysis of the shikonin metabolic network identifies new gene connections and reveals evolutionary insight into shikonin biosynthesis. Hortic Res. 2022; 9: uhab087.

[18] Ueoka H, Sasaki K, Miyawaki T, et al. A cytosollocalized geranyl diphosphate synthase from Lithospermum erythrorhizon and its molecular evolution. Plant Physiol. 2020; 182: 1933-45.

[19] Bergman ME . Monoterpenoid Volatile Biosynthesis in RoseScented Geranium (Pelargonium Graveolens). PhD thesis, University of Toronto (Canada); 2023.

[20] Kimani SK, Wang S, Xie J, et al. Integration of RNAseq and metabolite analysis reveals the key floral scent biosynthetic genes in herbaceous peony. Sci Hortic. 2024; 10: 617.

[21] Li Z, Zhang X, Li K, et al. Integrative analysis of transcriptomic and volatile compound profiles sheds new insights into the terpenoid biosynthesis in tree peony. Ind Crop Prod. 2022; 188: 115672.

[22] Song C, Wang Q, da Silva JAT, et al. Identification of floral fragrances and analysis of fragrance patterns in herbaceous peony cultivars. J Am Soc Hortic Sci. 2018; 143: 248-58.

[23] Zhang Y, Zhi H, Qu L, et al. Analysis of flower volatile compounds and odor classification of 17 tree peony cultivars. Sci Hortic. 2024; 338: 113665-4238.

[24] Feng L, Li Y, Sheng L, et al. Comparative analysis of headspace volatiles of different herbaceous peony (Paeonia lactiflora pall.) cultivars. J Essent Oil Bear Plants. 2016; 19: 167-75.

[25] Zhao Q, Zhang M, Gu LN, et al. Transcriptome and volatile compounds analyses of floral development provide insight into floral scent formation in Paeonia lactiflora ‘Wu Hua Long Yu’. Front Plant Sci. 2024a; 15: 1303156.

[26] Li S, Zhang L, Sun M, et al. Biogenesis of flavorrelated linalool is diverged and genetically conserved in tree peony (Paeonia × suffruticosa). Hortic Res. 2023; 10: uhac253.

[27] Ma X, Guo J, Ma Y, et al. Characterization of a monoterpene synthase from Paeonia lactiflora producing αpinene as its single product. Biotechnol Lett. 2016; 38: 1213-9.

[28] Zhao Q, Li YQ, Gu LN, et al. Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in the flower of Paeonia lactiflora. Postharvest Biol Technol. 2025b; 219: 113231.

[29] Chen C, Wu Y, Li J, et al. TBtoolsII: a "one for all, all for one" bioinformatics platform for biological bigdata mining. Mol Plant. 2023a; 16: 1733-42.

[30] Piechulla B, Bartelt R, Brosemann A, et al. The αterpineol to 1,8cineole cyclization reaction of tobacco terpene synthases. Plant Physiol. 2016; 172: 2120-31.

[31] Sheng L, Zang S, Wang J, et al. Overexpression of a Rosa rugosa Thunb. NUDX gene enhances biosynthesis of scent volatiles in petunia. PeerJ. 2021; 9: e11098.

[32] Salem BIA, Liao XP, Abduallah AMA, et al. Identification and comparison of 101 floral aroma components in 19 herbaceous peony (Paeonia lactiflora) cultivars. Sci Hortic. 2025; 347: 114200.

[33] Wang A, Luo Y, Niu T, et al. Identification and content analysis of volatile components in 100 cultivars of Chinese herbaceous peony. Ornam Plant Res. 2024; 4: 0.

[34] Köllner TG, Held M, Lenk C, et al. A maize (E)betacaryophyllene synthase implicated in indirect defense responses against herbivores is not expressed in most American maize varieties. Plant Cell. 2008; 20: 482-94.

[35] Xu H, Bohman B, Wong DCJ, et al. Complex sexual deception in an orchid is achieved by coopting two independent biosynthetic pathways for pollinator attraction. Curr Biol. 2017; 27: 1867-1877.e5.

[36] Chen F, Tholl D, Bohlmann J, et al. The family of terpene synthases in plants: a midsize family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 2011; 66: 212-29.

[37] Gao F, Liu B, Li M, et al. Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in flowers of Freesia x hybrida. J Exp Bot. 2018; 69: 4249-65.

[38] Kumar Y, Khan F, Rastogi S, et al. Genomewide detection of terpene synthase genes in holy basil (Ocimum sanctum L.). PLoS One. 2018; 13: e0207097.

[39] Muchlinski A, Ibdah M, Ellison S, et al. Diversity and function of terpene synthases in the production of carrot aroma and flavor compounds. Sci Rep. 2020; 10: 9989.

[40] Zhou HC, Shamala LF, Yi XK, et al. Analysis of terpene synthase family genes in Camellia sinensis with an emphasis on abiotic stress conditions. Sci Rep. 2020; 10: 933.

[41] Zhao YF, Cui GH, Wang J, et al. Functional identification of the terpene synthase family involved in biosynthesis in Paeonia lactiflora. Molecules. 2024b; 29: 4662.

[42] Zhao Q, Li YQ, Gu LN, et al. Methyl jasmonate activates an OPR gene that regulates the βcitronellol synthesis in herbaceous peony. Ind Crop Prod. 2025a; 232: 121294.

[43] Zhao Q, Li YQ, Gu LN, et al. The PlMYB73PlMYB70PlMYB108 complex regulates PlTPS1 to promote geraniol biosynthesis in Paeonia lactiflora. Hortic Res. 2025c; 12: uhaf141.

[44] Degenhardt J, Köllner TG, Gershenzon J . Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry. 2009; 70: 1621-37.

[45] Yang P, Chen YX, Wang TT, et al. Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa. Plant J. 2024; 118: 1218-31.

[46] Christianson DW . Structural and chemical biology of terpenoid cyclases. Chem Rev. 2017; 117: 11570-648.

[47] Köksal M, Hu H, Coates RM, et al. Structure and mechanism of the diterpene cyclase entcopalyl diphosphate synthase. Nat Chem Biol. 2011; 7: 431-3.

[48] Garms S, Chen F, Boland W, et al. A single amino acid determines the site of deprotonation in the active center of sesquiterpene synthases SbTPS1 and SbTPS2 from Sorghum bicolor. Phytochemistry. 2012; 75: 6-13.

[49] Köllner TG, Degenhardt J, Gershenzon J . The product specificities of maize terpene synthases TPS4 and TPS10 are determined both by active site amino acids and residues adjacent to the active site. Plants (Basel). 2020; 9: 552.

[50] Lu L, Tang Y, Xu H, et al. Selection and verification of reliable internal reference genes in stem development of herbaceous peony (Paeonia lactiflora Pall.). Physiol Mol Biol Plants. 2023; 29: 773-82.

[51] Martinelli L, Bihanic C, Bony A, et al. Citronellol biosynthesis in pelargonium is a multistep pathway involving progesterone 5βreductase and/or iridoid synthaselike enzymes. Plant Physiol. 2024; 194: 1006-23.

[52] Shang J, Feng D, Liu H, et al. Evolution of the biosynthetic pathways of terpene scent compounds in roses. Curr Biol. 2024; 34: 3550-3563.e8.

[53] Tan CS, Hassan M, Hussein ZAM, et al. Structural and kinetic studies of a novel nerol dehydrogenase from Persicaria minor, a nerolspecific enzyme for citral biosynthesis. Plant Physiol Biochem. 2018; 123: 359-68.

[54] Bönisch F, Frotscher J, Stanitzek S, et al. A UDPglucose:Monoterpenol glucosyltransferase adds to the chemical diversity of the grapevine metabolome. Plant Physiol. 2014; 165: 561-81.

[55] Rao S, Cao H, O’Hanna FJ, et al. Nudix hydrolase 23 posttranslationally regulates carotenoid biosynthesis in plants. Plant Cell. 2024; 36: 1868-91.

[56] Ge J, Zhao D, Han C, et al. Cloning and expression of floral organ developmentrelated genes in herbaceous peony (Paeonia lactiflora Pall.). Mol Biol Rep. 2014; 41: 6493-503.

[57] Shan X, Li Y, Yang S, et al. The spatiotemporal biosynthesis of floral flavonols is controlled by differential phylogenetic MYB regulators in Freesia hybrida. New Phytol. 2020; 228: 1864-79.

[58] Li Y, Shan X, Gao R, et al. MYB repressors and MBW activation complex collaborate to finetune flower coloration in Freesia hybrida. Commun Biol. 2020; 3: 396.

[59] Gao R, Han T, Xun H, et al. MYB transcription factors GmMYBA2 and GmMYBR function in a feedback loop to control pigmentation of seed coat in soybean. J Exp Bot. 2021; 72: 4401-18.

[60] Varadi M, Anyango S, Deshpande M, et al. AlphaFold protein structure database: massively expanding the structural coverage of proteinsequence space with highaccuracy models. Nucleic Acids Res. 2022; 50: D439-44.

[61] Kampranis SC, Ioannidis D, Purvis A, et al. Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function. Plant Cell. 2007; 19: 1994-2005.

[62] Noel JP, Dellas N, Faraldos JA, et al. Structural elucidation of cisoid and transoid cyclization pathways of a sesquiterpene synthase using 2fluorofarnesyl diphosphates. ACS Chem Biol. 2010; 5: 377-92.

[63] Sainsbury F, Thuenemann EC, Lomonossoff GP . pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J. 2009; 7: 682-93.

[64] Ma LJ, Feng Y, Jiang LP, et al. Modification of pFGC5941 and construction of RNAi vector of Brassica transparent Testa 1 gene (TT1) family. Agric Biotechnol. 2010; 18: 1189-96.