High-quality Lindera megaphylla genome analysis provides insights into genome evolution and allows for the exploration of genes involved in terpenoid biosynthesis

Abstract

Lindera megaphylla, a Lauraceae species, is valued for timber, horticulture, landscape architecture, and traditional medicine. Here, a high-quality genome of L. megaphylla was obtained at the chromosome level. A total of 96.77% of genomic sequences were mapped onto 12 chromosomes, with a total length of 1309.2 megabase (Mb) and an N50 scaffold of 107.75 Mb. Approximately, 75.91% of genome consists of repetitive sequences and 7004 ncRNAs were predicted. We identified 29 482 genes, and 28 657 genes were annotated. Gene family analysis showed expanded gene families were mainly involved in energy metabolism and cellular growth, while contracted ones were associated with carbohydrate metabolism and signal transduction. Our analysis revealed that L. megaphylla has undergone two rounds of whole-genome duplication (WGD). Our results revealed that volatile compounds in L. megaphylla leaves inhibited the growth of several fungi and bacteria. Fifty-two terpene synthase (TPS) genes were identified and classified into six subfamilies, with significant expansion observed in the TPS-b, TPS-f, and TPS-g subfamilies in L. megaphylla. Transcriptomic and metabolomic co-analysis revealed that 43 DEGs were correlated with 117 terpenoids. Further analysis revealed that LmTPS1 was significantly correlated with caryophyllene oxide content. The overexpression of LmTPS1 in transgenic tomato lines significantly increased the contents of β-caryophyllene and humulene, which further improved the resistance of transgenic tomato plants to common fungal and bacterial diseases. The integrated analysis of genome, metabolome, and transcriptome provides comprehensive insights into the evolution of L. megaphylla and clarifies the molecular mechanisms underlying the protective effects of caryophyllene against biotic stress.

Acknowledgements

This study was founded by the NSFC (No. 32201615) and the Key Technology R&D Program of He’nan Province (No. 232102111106).

Author contributions

H.Y.L., X.S., H.L, and X.J.L conceived and designed the project. H.L., J.L., P.C., and R.L. conducted these experiments and analyze data. Y.B., Y.F., Y.L, Q.J., X.Z., and X.S. performed data generation and bioinformatic analyses. H.Y.L., X.S, X.J.L, H.L., J.L., W.X., Y.B, and Y.F. organized, written and revised the manuscript. All authors will approve the submission of the manuscript.

Data availability

The raw sequences from the RNA-seq of L. megaphylla and the whole-genome sequence data reported in this paper have been deposited in the Genome Warehouse at the National Genomics Data Center (NGDC), Beijing Institute of Genomics, Chinese Academy of Sciences/China National Center for Bioinformation, with accession numbers CRA019464 and PRJCA031378, respectively.

Conflict of interests

The authors declare no competing interests.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Nawaz M, Sun JF, Shabbir S. et al. A review of plants strategies to resist biotic and abiotic environmental stressors. Sci Total Environ. 2023; 900:165832

[2] Li S, Yan L, Venuste M. et al. A critical review of plant adap-tation to environmental boron stress: uptake, utilization, and interplay with other abiotic and biotic factors. Chemosphere. 2023; 338:139474

[3] Liu Q, Zhang CH, Fang HY. et al. Indispensable biomolecules for plant defense against pathogens: NBS-LRR and "nitrogen pool" alkaloids. Plant Sci. 2023; 334:111752

[4] Anjali KS, Korra T. et al. Role of plant secondary metabolites in defence and transcriptional regulation in response to biotic stress. Plant Stress. 2023; 8:100154

[5] Abbas F, Ke YG, Yu RC. et al. Volatile terpenoids: multiple func-tions, biosynthesis, modulation and manipulation by genetic engineering. Planta. 2017; 246:803-16

[6] Wang Q, Quan S, Xiao H. Towards efficient terpenoid biosyn-thesis: manipulating IPP and DMAPP supply. Bioresour Bioprocess. 2019; 6:6

[7] Lanier ER, Andersen TB, Hamberger B. Plant terpene specialized metabolism: complex networks or simple linear pathways? Plant J. 2023; 114:1178-201

[8] Li C, Zha W, Li W. et al. Advances in the biosynthesis of ter-penoids and their ecological functions in plant resistance. Int J Mol Sci. 2023; 24:11561

[9] Zhou YJ. Expanding the terpenoid kingdom. Nat Chem Biol. 2018; 14:1069-70

[10] Fidyt K, Fiedorowicz A, Strzadala L. et al. Beta-caryophyllene and beta-caryophyllene oxide-natural compounds of anticancer and analgesic properties. Cancer Med. 2016; 5:3007-17

[11] Ullah A, Sun L, Wang FF. et al. Eco-friendly bioactive β-caryophyllene/halloysite nanotubes loaded nanofibrous sheets for active food packaging. Food Packag Shelf Life. 2023; 35:101028

[12] Dong YM, Wei ZL, Zhang WY. et al. LaMYC7, a positive regulator of linalool and caryophyllene biosynthesis, confers plant resistance to pseudomonas syringae. Hortic Res. 2024;11: uhae044

[13] Ting HM, Delatte TL, Kolkman P. et al. SNARE-RNAi results in higher terpene emission from ectopically expressed caryophyl-lene synthase in Nicotiana benthamiana. Mol Plant. 2015; 8: 454-66

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

[15] Tian XC, Guo JF, Yan XM. et al. Unique gene duplications and conserved microsynteny potentially associated with resis-tance to wood decay in the Lauraceae. Front Plant Sci. 2023; 14: 1122549

[16] Rendón-Anaya M, Ibarra-Laclette E, Méndez-Bravo A. et al. The avocado genome informs deep angiosperm phylogeny, highlights introgressive hybridization, and reveals pathogen-influenced gene space adaptation. Proc Natl Acad Sci USA. 2019; 116:17081-9

[17] Chen SP, Sun WH, Xiong YF. et al. The Phoebe genome sheds light on the evolution of magnoliids. Hortic Res. 2020; 7:146

[18] Chen YC, Li Z, Zhao YX. et al. The Litsea genome and the evolution of the laurel family. Nat Commun. 2020; 11:1675

[19] Chaw SM, Liu YC, Wu YW. et al. Stout camphor tree genome fills gaps in understanding of flowering plant genome evolution. Nat Plants. 2019; 5:63-73

[20] Cao Y, Xuan BF, Peng B. et al. The genus Lindera: a source of structurally diverse molecules having pharmacological signifi-cance. Phytochem Rev. 2016; 15:869-906

[21] Lin HF, Huang HL, Liao JF. et al. Dicentrine analogue-induced G2/M arrest and apoptosis through inhibition of topoisomerase II activity in human cancer cells. Planta Med. 2015; 81:830-7

[22] Huang RL, Chen CC, Huang YL. et al. Anti-tumor effects of d-dicentrine from the root of Lindera megaphylla. Planta Med. 1998; 64:212-5

[23] Liu HL, Liu J, Chen P. et al. Selection and validation of optimal RT-qPCR reference genes for the normalization of gene expression under different experimental conditions in Lindera megaphylla. Plan Theory. 2023; 12:2185

[24] Zhao ML, Song Y, Ni J. et al. Comparative chloroplast genomics and phylogenetics of nine Lindera species (Lauraceae). Sci Rep. 2018; 8:8844

[25] Jiang SY, Jin J, Sarojam R. et al. A comprehensive survey on the terpene synthase gene family provides new insight into its evolutionary patterns. Genome Biol and Evol. 2019; 11:2078-98

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

[27] Song XM, Wang JP, Li N. et al. Deciphering the high-quality genome sequence of coriander that causes controversial feel-ings. Plant Biotechnol J. 2020; 18:1444-56

[28] Liu ZK, Fu YH, Wang H. et al. The high-quality sequencing of the Brassica rapa ’XiangQingCai’ genome and exploration of genome evolution and genes related to volatile aroma. Hortic Res. 2023;10:uhad187

[29] 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

[30] Lemoine F, Entfellner JBD, Wilkinson E. et al. Renewing Felsen-stein’s phylogenetic bootstrap in the era of big data. Nature. 2018; 556:452-6

[31] Li S, Jiang SX, Jia WT. et al. Natural antimicrobials from plants: recent advances and future prospects. Food Chem. 2024; 432: 137231

[32] Frank L, Wenig M, Ghirardo A. et al. Isoprene and β-caryophyllene confer plant resistance via different plant internal signalling pathways. Plant Cell Environ. 2021; 44:1151-64

[33] Yamagiwa Y, Inagaki Y, Ichinose Y. et al. Talaromyces wortman-nii FS2 emits β-caryphyllene, which promotes plant growth and induces resistance. J Gen Plant Pathol. 2011; 77:336-41

[34] Aguilar-Avila DS, Flores-Soto ME, Tapia-Vázquez C. et al. Beta-caryophyllene, a natural sesquiterpene, attenuates neuropathic pain and depressive-like behavior in experimental diabetic mice. J Med Food. 2019; 22:460-8

[35] Fan M, Yuan SQ, Li LS. et al. Application of terpenoid compounds in food and pharmaceutical products. Fermentation. 2023; 9:119

[36] Tita O, Constantinescu MA, Rusu L. et al. Natural polymers as carriers for encapsulation of volatile oils: applications and perspectives in food products. Polymers (Basel). 2024; 16:1026

[37] Feng K, Kan XY, Yan YJ. et al. Identification and characteri-zation of terpene synthase OjTPS1 involved in β-caryophyllene biosynthesis in Oenanthe javanica (Blume) DC. Ind Crop Prod. 2023; 192:115998

[38] HongGJ, XueXY, MaoYB. et al. Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression. Plant Cell. 2012; 24:2635-48

[39] Song XM, Liu HB, Shen SQ. et al. Chromosome-level Pepino genome provides insights into genome evolution and antho-cyanin biosynthesis in Solanaceae. Plant J. 2022; 110:1128-43

[40] Song XM, Sun PC, Yuan JQ. et al. The celery genome sequence reveals sequential paleo-polyploidizations, karyotype evolution and resistance gene reduction in apiales. Plant Biotechnol J. 2021; 19:731-44

[41] Marcais G, Kingsford C. A fast, lock-free approach for effi-cient parallel counting of occurrences of k-mers. Bioinformatics. 2011; 27:764-70

[42] Song XM. The Orychophragmus violaceus genome. Vegetable Res. 2023; 3:1-2

[43] Cheng HY, Concepcion GT, Feng XW. et al. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat Methods. 2021; 18:170-5

[44] Wingett S, Philip E, Mayra F-M. et al. HiCUP: pipeline for mapping and processing hi-C data. F1000Res. 2015; 4:1310

[45] Zhang X, Zhang S, Zhao Q. et al. Assembly of allele-aware, chromosomal-scale autopolyploid genomes based on hi-C data. Nat Plants. 2019; 5:833-45

[46] Shen SQ, Li N, Wang YJ. et al. High-quality ice plant reference genome analysis provides insights into genome evolution and allows exploration of genes involved in the transition from C3 to CAM pathways. Plant Biotechnol J. 2022; 20:2107-22

[47] Durand NC, Shamim MS, Machol I. et al. Juicer provides a one-click system for analyzing loop-resolution hi-C experiments. Cell Syst. 2016; 3:95-8

[48] Parra G, Bradnam K, Korf I. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics. 2007; 23:1061-7

[49] Manni M, Berkeley MR, Seppey M. et al. BUSCO update: novel and streamlined workflows along with broader and deeper phyloge-netic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol. 2021; 38:4647-54

[50] Li H, Durbin R. Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics. 2009; 25:1754-60

[51] Ou S, Chen J, Jiang N. Assessing genome assembly quality using the LTR assembly index (LAI). Nucleic Acids Res. 2018; 46:e126

[52] Xu Z, Wang H. LTR_FINDER: an efficient tool for the pre-diction of full-length LTR retrotransposons. Nucleic Acids Res. 2007;35:W265-8

[53] Price AL, Jones NC, Pevzner PA. De novo identification of repeat families in large genomes. Bioinformatics. 2005;21Suppl 1: i351-8

[54] Edgar RC, Myers EW. PILER: identification and classification of genomic repeats. Bioinformatics. 2005;21Suppl 1:i152-8

[55] Bao WD, Kojima KK, Kohany O.Repbase update, a database of repetitive elements in eukaryotic genomes. Mob DNA. 2015; 6:11

[56] Tarailo-Graovac M, Chen NS.Using RepeatMasker to identify repetitive elements in genomic sequences. Curr Protoc Bioinform. 2009; Chapter 4:4.10.1-14

[57] Nawrocki EP, Eddy SR. Infernal 1.1: 100-fold faster RNA homol-ogy searches. Bioinformatics. 2013; 29:2933-5

[58] Chan PP, Lowe TM. tRNAscan-SE: searching for tRNA genes in genomic sequences. Methods Mol Biol. 2019; 1962:1-14

[59] Stanke M, Morgenstern B. AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Res. 2005;33:W465-7

[60] Korf I. Gene finding in novel genomes. BMC Bioinformatics. 2004; 5:59

[61] Camacho C, Coulouris G, Avagyan V. et al. BLAST plus: architec-ture and applications. BMC Bioinformatics. 2009; 10:421

[62] Birney E, Clamp M, Durbin R. GeneWise and Genomewise. Genome Res. 2004; 14:988-95

[63] Haas BJ, Salzberg SL, Zhu W. et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the program to assemble spliced alignments. Genome Biol. 2008;9:R7

[64] Haas BJ, Delcher AL, Mount SM. et al. Improving the Arabidopsis genome annotation using maximal transcript alignment assem-blies. Nucleic Acids Res. 2003; 31:5654-66

[65] Emms DM, Kelly S. OrthoFinder: phylogenetic orthology infer-ence for comparative genomics. Genome Biol. 2019; 20:238

[66] Liu Z, Zhang CH, He JH. et al. plantGIR: a genomic database of plants. Hortic Res. 2024;11:uhae342

[67] De Bie T, Cristianini N, Demuth JP. et al. CAFE: a computa-tional tool for the study of gene family evolution. Bioinformatics. 2006; 22:1269-71

[68] Katoh K, Rozewicki J, Yamada KD. et al. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2019; 20:1160-6

[69] Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009; 25:1972-3

[70] Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30: 1312-3

[71] Yang ZH. PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007; 24:1586-91

[72] Kumar S, Stecher G, Suleski M. et al. TimeTree: a resource for timelines, Timetrees, and divergence times. MolBiolEvol. 2017; 34: 1812-9

[73] Sun PC, Jiao BB, Yang YZ. et al. WGDI: a user-friendly toolkit for evolutionary analyses of whole-genome duplications and ancestral karyotypes. Mol Plant. 2022; 15:1841-51

[74] Liu Z, Shen SQ, Li CJ. et al. SoIR: a comprehensive Solanaceae information resource for comparative and functional genomic study. Nucleic Acids Res. 2024;53:D1623-32

[75] Wang XJ, Shen SQ, Fu YH. et al. High-quality reference genome decoding and population evolution analysis of prickly Sechium edule. Hortic Plant J. 2025; 11:827-38

[76] Vanneste K, Van de Peer Y, Maere S. Inference of genome dupli-cations from age distributions revisited. MolBiolEvol. 2013; 30: 177-90

[77] Cui L, Wall PK, Leebens-Mack JH. et al. Widespread genome duplications throughout the history of flowering plants. Genome Res. 2006; 16:738-49

[78] Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32:1792-7

[79] Haas BJ, Papanicolaou A, Yassour M. et al. De novo transcript sequence reconstruction from RNA-seq using the trinity plat-form for reference generation and analysis. Nat Protoc. 2013; 8: 1494-512

[80] Xie C, Mao XZ, Huang JJ. et al. KOBAS 2.0: a web server for anno-tation and identification of enriched pathways and diseases. Nucleic Acids Res. 2011;39:W316-22

[81] Mistry J, Chuguransky S, Williams L. et al.Pfam:the protein families database in 2021. Nucleic Acids Res. 2021;49:D412-9

[82] Liu J, Huang CC, Xing DS. et al. The genomic database of fruits: a comprehensive fruit information database for comparative and functional genomic studies. Agric Commun. 2024; 2:100041

[83] Wang XJ, Wei YF, Liu Z. et al. TEGR: a comprehensive Ericaceae genome resource database. J Integr Agric. 2023; 24:1140-51

[84] Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. MolBiolEvol. 2013; 30:772-80

[85] Price MN, Dehal PS, Arkin AP. FastTree 2-approximately maximum-likelihood trees for large alignments. PLoS One. 2010; 5:e9490

[86] Bailey TL, Baker ME, Elkan CP. An artificial intelligence approach to motif discovery in protein sequences: application to steriod dehydrogenases. J Steroid Biochem Mol Biol. 1997; 62:29-44

[87] Wu T, Liu Z, Yu T. et al.Flowering genes identification, network analysis, and database construction for 837 plants. Hortic Res. 2024;11:uhae013

[88] Zhang Y, Zhang YC, Li B. et al. Polyploidy events shaped the expansion of transcription factors in Cucurbitaceae and exploitation of genes for tendril development. Hortic Plant J. 2022; 8:562-74

[89] Letunic I, Bork P. Interactive tree of life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics. 2006; 23:127-8

[90] LiuZ, LiN, YuT. et al. The Brassicaceae genome resource (TBGR): a comprehensive genome platform for Brassicaceae plants. Plant Physiol. 2022; 190:226-37

[91] Feng S, Liu Z, Chen HL. et al. PHGD: an integrative and user-friendly database for plant hormone-related genes. iMeta. 2024; 3:e164

[92] Pfaffla M. A new mathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Res. 2001; 29:e45

[93] Wang YJ, Sun SQ, Feng XH. et al. Two lncRNAs of Chinese cabbage confer Arabidopsis with heat and drought tolerance. Vegetable Res. 2024; 4:e029

[94] Chen KL, Liu HL, Lou Q. et al. Ectopic expression of the grape hyacinth (Muscari armeniacum) R2R3-MYB transcription factor gene, MaAN2, induces anthocyanin accumulation in tobacco. Front Plant Sci. 2017; 8:965

[95] Xie JF, Zhang HS, Yang SH. et al. Comparison of bacteriostatic effects of twelve common garden plants leaves in different seasons in Zhengzhou. J Hebei Agric Sci. 2022; 26:60-63, 67

[96] Van Eck J, Keen P, Tjahjadi M. Agrobacterium tumefaciens-mediated transformation of tomato. Methods Mol Biol. 2019; 1864: 225-34

[97] Cui Z, Jiang Y, Chen Y., et al. Overexpressing peach PpePAO1 gene improves the nutrient value of tomato and enhances resistance against Botrytis cinerea. Postharvest Biol Tec. 2024; 211:112792