Abstract
Piper sarmentosum Roxb. is a significant medicinal and edible plant, and its active compound piperlongumine (PL) has garnered attention due to its pharmacological activities, including anticancer and anti-inflammatory effects. However, the key enzymes and regulatory mechanisms of its biosynthetic pathway are not yet fully understood. In this study, we generated a chromosome-level genome assembly, with a contig N50 of 15.36 Mb and a scaffold N50 of 22.52 Mb. The BUSCO assessment indicated high completeness, with a score of 97.4%. Genome annotation revealed 39,154 protein-coding genes and identified three lineage-specific whole-genome duplication (WGD) events that expanded gene families associated with alkaloid biosynthesis. Metabolomic analysis identified 4,456 metabolites, including 238 alkaloids, and demonstrated that flowers and fruits are the primary organs for PL biosynthesis. Molecular docking and the correlation of gene expression with levels of PL suggest that PsHCT1 catalyzes the condensation of sinapoyl-CoA and 5,6-dihydropyridinone, while PsCCoAOMT1 is responsible for the final synthesis of PL. This study provides insights into the mechanism of alkaloid biosynthesis in P. sarmentosum and may help lay the groundwork for enhancing the production of medicinal compounds.
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Data availability
All the genome assembly and annotation files generated in this study have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under BioProject accession number PRJNA1289360. The genome assembly files have been deposited in NCBI under accession number PRJNA1265616. The complete genome annotation files have been uploaded to the public database Figshare and can be accessed via the following link: https://doi.org/10.6084/m9.figshare.30404920.v1. The untargeted metabolomics raw data used in this study are available in the MetaboLights database under accession number MTBLS13523, and can be accessed via the following link: https://www.ebi.ac.uk/metabolights/MTBLS13523. The source data for Fig. 1 has been uploaded to NCBI and can be accessed in the Methods section for reproducibility. The source files for Fig. 2A-D are in Supplementary Data S5-S9. The data for Fig. 2E, F can be obtained by downloading the genomic files shown in the figures. The source files for Fig. 3 are in Supplementary Data S10-S11, and those for Fig. 4 are in Supplementary Data S12. The WGCNA result files, uncropped images for subcellular localization, and molecular docking simulation data, which are included in Fig. 5, have been uploaded to the public database Figshare and can be accessed via the following link: https://doi.org/10.6084/m9.figshare.30404920.v1. The species protein data used in Fig. 2A is also available on Figshare.
Code availability
The software and parameters used in this study are described in the Methods section. No specific custom codes or scripts were utilized. Data processing was conducted according to the manuals and protocols provided with the respective software.
References
Shao, J., Liu, Y., Lv, J. & Chen, X. Alkaloid components in piper sarmentosum leaves. Nat. Prod. Res. Dev. 35, 231–235 (2023).
Sun, X. et al. Piper sarmentosum Roxb.: a review on its botany, traditional uses, phytochemistry, and pharmacological activities. J. Ethnopharmacol. 263, 112897 (2020).
de Azevedo, R. A. et al. Mastoparan induces apoptosis in B16F10-Nex2 melanoma cells via the intrinsic mitochondrial pathway and displays antitumor activity in vivo. Peptides 68, 113–119 (2015).
Bokesch, H. R. et al. A new hypoxia inducible factor-2 inhibitory pyrrolinone alkaloid from roots and stems of Piper sarmentosum. Chem. Pharm. Bull. (Tokyo) 59, 1178–1179 (2011).
Jyothi, D. et al. Diferuloylmethane augments the cytotoxic effects of piplartine isolated from Piper chaba. Toxicol. Vitr. 23, 1085–1091 (2009).
Gundala, S. R. et al. Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis. Toxicol. Appl Pharm. 280, 86–96 (2014).
Salehi, B. et al. Piper species: a comprehensive review on their phytochemistry, biological activities and applications. Molecules 24, 1364 (2019).
A Dictionary of the Economic Products of the Malay Peninsula. Nature 137, 255–255 (1936).
Abdul Rahman, N., Ku Mohd Noor, K. M., Suhaimi, F., Kutty, M. & Sinor, M. Z. Piper sarmentosum influences the oxidative stress involved in experimental diabetic rats. Internet J. Herb. Plant Med. 1, 1203 (2011).
Chaveerach, A., Mokkamul, P., Sudmoon, R. & Tanee, T. Ethnobotany of the Genus Piper (Piperaceae) in Thailand. Ethnobot. Res. Appl. 4, 233 (2006).
Othman, L., Sleiman, A. & Abdel-Massih, R. M. Antimicrobial activity of polyphenols and alkaloids in middle eastern plants. Front Microbiol 10, 911 (2019).
Cai, Y.-Z. et al. Structure-radical scavenging activity relationships of phenolic compounds from traditional Chinese medicinal plants. Life Sci. 78, 2872–2888 (2006).
Ware, I. et al. Comparative metabolite analysis of Piper sarmentosum organs approached by LC–MS-based metabolic profiling. Nat. Prod. Bioprospect. 14, 30 (2024).
Baliza, I. R. S. et al. Ruthenium complexes with piplartine cause apoptosis through MAPK Signaling by a p53-dependent pathway in human colon carcinoma cells and inhibit tumor development in a xenograft model. Front Oncol. 9, 582 (2019).
Niu, M. et al. Piperlongumine is a novel nuclear export inhibitor with potent anticancer activity. Chem. Biol. Interact. 237, 66–72 (2015).
Parama, D. et al. The promising potential of piperlongumine as an emerging therapeutics for cancer. Explor Target Antitumor Ther. 2, 323–354 (2021).
Choudhary, N. & Singh, V. A census of P. longum’s phytochemicals and their network pharmacological evaluation for identifying novel drug-like molecules against various diseases, with a special focus on neurological disorders. PLoS One 13, e0191006 (2018).
Bezerra, D. P. et al. Overview of the therapeutic potential of piplartine (piperlongumine). Eur. J. Pharm. Sci. 48, 453–463 (2013).
Fincher, R. M. et al. Inter- and intraspecific comparisons of antiherbivore defenses in three species of rainforest understory shrubs. J. Chem. Ecol. 34, 558–574 (2008).
Peeck, L. H., Leuthäusser, S. & Plenio, H. Switched stereocontrol in Grubbs−Hoveyda complex catalyzed ROMP utilizing proton-switched NHC ligands. Organometallics 29, 4339–4345 (2010).
Rosen, E. L., Sung, D. H., Chen, Z., Lynch, V. M. & Bielawski, C. W. Olefin metathesis catalysts containing acyclic diaminocarbenes. Organometallics 29, 250–256 (2010).
Blum, A. P., Ritter, T. & Grubbs, R. H. Synthesis of N-heterocylic carbene-containing metal complexes from 2-(Pentafluorophenyl)imidazolidines. Organometallics 26, 2122–2124 (2007).
Sanford, M. S., Love, J. A. & Grubbs, R. H. A versatile precursor for the synthesis of new ruthenium olefin metathesis catalysts. Organometallics 20, 5314–5318 (2001).
Süssner, M. & Plenio, H. pi-Face donor properties of N-heterocyclic carbenes. Chem. Commun. 21, 5417–5419 (2005).
Hua, D. H., Miao, S. W., Bharathi, S. N., Katsuhira, T. & Bravo, A. A. Selective nucleophilic addition reactions of alkyllithium reagents with N-(trimethylsilyl)lactams. Synthesis of cyclic ketimines. J. Org. Chem. 55, 3682–3684 (1990).
Tian, D.-S., Zhang, X. & Cox, R. J. Comparing total chemical synthesis and total biosynthesis routes to fungal specialized metabolites. Nat. Prod. Rep. 42, 720–738 (2025).
Liu, W., Hong, B., Wang, J. & Lei, X. New strategies in the efficient total syntheses of polycyclic natural products. Acc. Chem. Res. 53, 2569–2586 (2020).
Sivaranjani, R., George, J. K. & Saji, K. V. Evaluation of chemo-diversity in major Piper species for three piperamides using validated RP-HPLC method. Genet. Resour. Crop Evol. 66, 1635–1641 (2019).
Parmar, V. S. et al. Phytochemistry of the genus Piper. Phytochemistry 46, 597–673 (1997).
Hu, L. et al. The chromosome-scale reference genome of black pepper provides insight into piperine biosynthesis. Nat. Commun. 10, 4702 (2019).
Mathew, D. & Valsalan, R. The draft genome of Piper longum L. Crop Sci. 64, 2854–2862 (2024).
Zhang, L. et al. A high-quality apple genome assembly reveals the association of a retrotransposon and red fruit colour. Nat. Commun. 10, 1494 (2019).
Zhang, S.-J., Liu, L., Yang, R. & Wang, X. Genome size evolution mediated by gypsy retrotransposons in brassicaceae. Genomics, Proteom. Bioinforma. 18, 321–332 (2020).
Zhou, S.-S. et al. A comprehensive annotation dataset of intact LTR retrotransposons of 300 plant genomes. Sci. Data 8, 174 (2021).
Clark, J. W. & Donoghue, P. C. J. Whole-Genome Duplication and Plant Macroevolution. Trends Plant Sci. 23, 933–945 (2018).
Qin, L. et al. Insights into angiosperm evolution, floral development and chemical biosynthesis from the Aristolochia fimbriata genome. Nat. Plants 7, 1239–1253 (2021).
Wang Y. Metabolomics-based Resource Identification of Six Piper spp. Resources Quality Identification. (Hainan University, 2023).
Lv, Y. et al. Metabolome profiling and transcriptome analysis filling the early crucial missing steps of piperine biosynthesis in Piper nigrum L. Plant J. 117, 107–120 (2024).
Schnabel, A. et al. Identification and characterization of piperine synthase from black pepper, Piper nigrum L. Commun. Biol. 4, 1–10 (2021).
Jäckel, L. et al. The terminal enzymatic step in piperine biosynthesis is co-localized with the product piperine in specialized cells of black pepper (Piper nigrum L. Plant J. 111, 731–747 (2022).
Liu, C. et al. Genome-wide comparative analysis of the BAHD superfamily in seven Rosaceae species and expression analysis in pear (Pyrus bretschneideri). BMC Plant Biol. 20, 14 (2020).
Liu, Z. et al. Uncovering the role of hydroxycinnamoyl transferase in boosting chlorogenic acid accumulation in carthamus tinctorius cells under methyl jasmonate elicitation. Int J. Mol. Sci. 25, 2710 (2024).
Bassard, J.-E. et al. Protein–protein and protein–membrane associations in the lignin pathway. Plant Cell 24, 4465–4482 (2012).
Adib, A. M. et al. The metabolites of Piper sarmentosum and their biological properties: a recent update. Phytochem Rev. 23, 1443–1475 (2024).
Wang, J. et al. DNA gains and losses in gigantic genomes do not track differences in transposable element-host silencing interactions. Commun. Biol. 8, 704 (2025).
Cheng, L. et al. Genome assembly of Stewartia sinensis reveals origin and evolution of orphan genes in Theaceae. Commun. Biol. 8, 354 (2025).
Galindo-González, L., Mhiri, C., Deyholos, M. K. & Grandbastien, M.-A. LTR-retrotransposons in plants: engines of evolution. Gene 626, 14–25 (2017).
Grandbastien, M.-A. LTR retrotransposons, handy hitchhikers of plant regulation and stress response. Biochimica et. Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1849, 403–416 (2015).
Sun, W., Xu, Z., Song, C. & Chen, S. Herbgenomics: decipher molecular genetics of medicinal plants. Innovation 3, 100322 (2022).
Vs, G. Pepper - chemistry, technology, and quality evaluation. CRC Crit. Rev. Food Sci. Nutri. 9, 115–225 (1977).
Rajopadhye, A. A., Namjoshi, T. P. & Upadhye, A. S. Rapid validated HPTLC method for estimation of piperine and piperlongumine in root of Piper longum extract and its commercial formulation. Rev. Bras. Farmacogn. 22, 1355–1361 (2012).
Vanholme, R., De Meester, B., Ralph, J. & Boerjan, W. Lignin biosynthesis and its integration into metabolism. Curr. Opin. Biotechnol. 56, 230–239 (2019).
Yamada, Y. & Sato, F. Transcription factors in alkaloid engineering. Biomolecules 11, 1719 (2021).
Xu, W., Dubos, C. & Lepiniec, L. Transcriptional control of flavonoid biosynthesis by MYB–bHLH–WDR complexes. Trends Plant Sci. 20, 176–185 (2015).
Hao, Y., Zong, X., Ren, P., Qian, Y. & Fu, A. Basic Helix-Loop-Helix (bHLH) transcription factors regulate a wide range of functions in arabidopsis. Int. J. Mol. Sci. 22, 7152 (2021).
Prakash, P. et al. 199–217 (Academic Press, 2023). https://doi.org/10.1016/B978-0-323-90613-5.00019-4.
Zhou, Z. et al. Targeting cofactors regeneration in methylation and hydroxylation for high level production of Ferulic acid. Metab. Eng. 73, 247–255 (2022).
Chen, R. et al. De novo biosynthesis of plant lignans by synthetic yeast consortia. Nat. Chem. Biol. 21, 1487–1496 (2025).
Fisyuk, A. & Poendaev, N. Synthesis of 5,6-Dihydropyridin-2(1H)-ones, 1,5,6,8,8a-Hexahydroisoquinolin-3(2H)-ones and 4a,5,6,7,8,8a-Hexahydroquinolin-2(1H)-ones by intramolecular wittig reaction. Molecules 7, 124–128 (2002).
Fisyuk, A. S., Poendaev, N. V. & Bundel, Y. G. A new route for the synthesis of 5,6-dihydropyridin-2(1H)-ones, 2-pyridones and (4-hydroxy-2-oxopiperid-3-yl) pyridinium chlorides by intramolecular cyclization of N-3-oxoalkylchloroacetamide derivatives. Mendeleev Commun. 8, 12–13 (1998).
Cui, J. et al. Telomere-to-telomere Phragmites australis reference genome assembly with a B chromosome provides insights into its evolution and polysaccharide biosynthesis. Commun. Biol. 8, 73 (2025).
Zhang, X.-Y. et al. Optimization and quality control of genome-wide Hi-C library preparation. Yi Chuan 39, 847–855 (2017).
Sim, S. B., Corpuz, R. L., Simmonds, T. J. & Geib, S. M. HiFiAdapterFilt, a memory efficient read processing pipeline, prevents occurrence of adapter sequence in PacBio HiFi reads and their negative impacts on genome assembly. BMC Genomics 23, 157 (2022).
Marçais, G. & Kingsford, C. A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics 27, 764–770 (2011).
Vurture, G. W. et al. GenomeScope: fast reference-free genome profiling from short reads. Bioinformatics 33, 2202–2204 (2017).
Cheng, H., Concepcion, G. T., Feng, X., Zhang, H. & Li, H. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat. Methods 18, 170–175 (2021).
Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLOS ONE 9, e112963 (2014).
Roach, M. J., Schmidt, S. A. & Borneman, A. R. Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies. BMC Bioinforma. 19, 460 (2018).
Hu, J. et al. NextDenovo: an efficient error correction and accurate assembly tool for noisy long reads. Genome Biol. 25, 107 (2024).
Xu, M. et al. TGS-GapCloser: a fast and accurate gap closer for large genomes with low coverage of error-prone long reads. Gigascience 9, giaa094 (2020).
Durand, N. C. et al. Juicer provides a one-click system for analyzing loop-resolution Hi-C experiments. Cell Syst. 3, 95–98 (2016).
Loescher, S., Groeer, S. & Walther, A. 3D DNA origami nanoparticles: from basic design principles to emerging applications in soft matter and (bio-)nanosciences. Angew. Chem. Int Ed. Engl. 57, 10436–10448 (2018).
Robinson, J. T. et al. Juicebox.js Provides a Cloud-Based Visualization System for Hi-C Data. Cell Syst. 6, 256–258.e1 (2018).
Wolff, J. et al. Galaxy HiCExplorer 3: a web server for reproducible Hi-C, capture Hi-C and single-cell Hi-C data analysis, quality control and visualization. Nucleic Acids Res. 48, W177–W184 (2020).
Simão, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V. & Zdobnov, E. M. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31, 3210–3212 (2015).
Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018).
Ou, S. & Jiang, N. LTR_retriever: a highly accurate and sensitive program for identification of long terminal repeat retrotransposons. Plant Physiol. 176, 1410–1422 (2018).
Rhie, A., Walenz, B. P., Koren, S. & Phillippy, A. M. Merqury: reference-free quality, completeness, and phasing assessment for genome assemblies. Genome Biol. 21, 245 (2020).
Tarailo-Graovac, M. & Chen, N. Using RepeatMasker to identify repetitive elements in genomic sequences. Curr. Protoc. Bioinforma. Chapter 4, 1–4 (2009).
Flynn, J. M. et al. RepeatModeler2 for automated genomic discovery of transposable element families. Proc. Natl. Acad. Sci. USA 117, 9451–9457 (2020).
Zhang, R.-G. et al. TEsorter: an accurate and fast method to classify LTR-retrotransposons in plant genomes. Hortic. Res. 9, uhac017 (2022).
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2-approximately maximum-likelihood trees for large alignments. PLoS One 5, e9490 (2010).
Gabriel, L. et al. BRAKER3: fully automated genome annotation using RNA-seq and protein evidence with GeneMark-ETP, AUGUSTUS and TSEBRA. bioRxiv 2023.06.10.544449 https://doi.org/10.1101/2023.06.10.544449 (2024).
Burge, C. & Karlin, S. Prediction of complete gene structures in human genomic DNA. J. Mol. Biol. 268, 78–94 (1997).
Majoros, W. H., Pertea, M. & Salzberg, S. L. TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders. Bioinformatics 20, 2878–2879 (2004).
Keilwagen, J., Hartung, F. & Grau, J. GeMoMa: homology-based gene prediction utilizing intron position conservation and RNA-seq data. Methods Mol. Biol. 1962, 161–177 (2019).
Haas, B. J. et al. Automated eukaryotic gene structure annotation using evidencemodeler and the program to assemble spliced alignments. Genome Biol. 9, R7 (2008).
Riehl, K., Riccio, C., Miska, E. A. & Hemberg, M. TransposonUltimate: software for transposon classification, annotation and detection. Nucleic Acids Res. 50, e64 (2022).
Cantalapiedra, C. P., Hernández-Plaza, A., Letunic, I., Bork, P. & Huerta-Cepas, J. eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol. Biol. Evol. 38, 5825–5829 (2021).
Emms, D. M. & Kelly, S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 20, 238 (2019).
Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).
Puttick, M. N. MCMCtreeR: functions to prepare MCMCtree analyses and visualize posterior ages on trees. Bioinformatics 35, 5321–5322 (2019).
Kumar, S. et al. TimeTree 5: an expanded resource for species divergence times. Mol. Biol. Evol. 39, msac174 (2022).
Mendes, F. K., Vanderpool, D., Fulton, B. & Hahn, M. W. CAFE 5 models variation in evolutionary rates among gene families. Bioinformatics 36, 5516–5518 (2021).
Buchfink, B., Xie, C. & Huson, D. H. Fast and sensitive protein alignment using DIAMOND. Nat. Methods 12, 59–60 (2015).
Wang, Y. et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 40, e49 (2012).
Sun, P. et al. WGDI: A user-friendly toolkit for evolutionary analyses of whole-genome duplications and ancestral karyotypes. Mol. Plant 15, 1841–1851 (2022).
Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018).
Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).
Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinforma. 9, 559 (2008).
Wu, T. et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innov. (Camb.) 2, 100141 (2021).
Potter, S. C. et al. HMMER web server: 2018 update. Nucleic Acids Res. 46, W200–W204 (2018).
Acknowledgements
This work was supported by the Guangdong Provincial Key Research and Development Program (2022B0202110003, 2024B1212060007), the Natural Science Foundation of China (32171842), the National Key Research and Development Project of China (2019YFD1100403), and the Key Project of Central South University of Forestry and Technology (63223068). We also thank Guangzhou Yuda Biotechnology Co., Ltd. for providing sequencing and tech nical services. Special thanks to Luo Yongpeng for his assistance with language editing.
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Y.J.L. conducted writing–review, editing, writing original draft, software, methodology, and data curation; R.W. conducted writing–original draft, data curation, and conceptualization; Y.X.Z. conducted writing–original draft, data curation, and conceptualization; Y.J.W. conducted writing–original draft, data curation, and conceptualization; J.L. Writing review, editing, Writing– original draft, Investigation, Funding acquisition, Conceptualization. Y.Q.W. conducted writing–original draft, supervision, project administration, funding acquisition, and conceptualization.
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Luo, Y., Wang, R., Zhang, Y. et al. A chromosome-level genome assembly and multi-omics analyses provide insights into the biosynthesis of piperlongumine in Piper sarmentosum. Commun Biol (2026). https://doi.org/10.1038/s42003-025-09448-z
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DOI: https://doi.org/10.1038/s42003-025-09448-z
