Integrated Multi-Omics Analysis Reveals the Regulatory Mechanisms of Lignans Biosynthesis in the Genus Polygonatum

Integrated Multi-Omics Analysis Reveals the Regulatory Mechanisms of Lignans Biosynthesis in the Genus Polygonatum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Integrated Multi-Omics Analysis Reveals the Regulatory Mechanisms of Lignans Biosynthesis in the Genus Polygonatum Guangdi Shi, Xiaolin Wan, Demiao Lan, Qiang Xiao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5450765/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract By integrating metabolomic and transcriptomic analyses, our study unravels the regulatory mechanisms underlying the biosynthesis of lignans in the plant genus Polygonatum. As a perennial herb of the Liliaceae family, Polygonatum boasts rhizomes that are rich in polysaccharides, saponins, flavonoids, sterols, and amino acids—micronutrients that contribute to its essence-nourishing, antioxidant, and anti-aging properties. These rhizomes have a positive impact on memory enhancement, blood glucose and lipid reduction, and immune system strengthening. Lignans, as secondary metabolites in plants, play a pivotal role in plant defense against pests and stress, and exhibit a range of pharmacological activities, including anti-tumor, anti-HIV, anti-inflammatory, hepatoprotective, and antioxidant effects.Focusing on Polygonatum sibiricum Red and Polygonatum kingianum var. grandifolium , our research delves into the transcriptional and metabolic mechanisms of lignan biosynthesis. We discovered that transcription factor families such as GARP-G2-like and SET may be crucial in regulating the lignan synthetic pathway within the rhizomes of Polygonatum. Additionally, we identified 17 lignans, with significant differences in the content of nine, particularly the marked expression variation of Cycloolivil-6-O-glucoside between the two Polygonatum species.Our findings not only fill a gap in the field but also offer guidance for molecular breeding, underscoring the significance of lignans in Polygonatum and providing theoretical support for enhancing the plant's stress tolerance and resistance to pests and diseases. We recommend further in-depth exploration of the metabolic and regulatory mechanisms of Polygonatum lignans to inform the development of new Polygonatum varieties with high quality and lignan content. Polygonatum sibiritum Red Polygonatum kingianum var. grandifolium Lignans Transcriptomics Metabolomics Transcription factor families Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Plants of the genus Polygonatum , belonging to the Liliaceae family, are perennial herbs (Yan et al., 2023 ). First recorded in the Han Dynasty's "Ming Yi Bie Lu," Polygonatum is a plant with both medicinal and dietary uses. The main part utilized is its rhizome, which is known for its benefits in replenishing vital energy and possessing antioxidant properties (Wan et al., 2024 ), It has the effect of delaying aging (Liu et al., 2011 ). In previous studies, it has been found that Polygonatum is rich in polysaccharides, saponins, flavonoids, sterols, amino acids, and other trace nutritional elements. It possesses effects such as improving memory, reducing blood sugar and lipid levels, and enhancing immune system function in the human body (Yang et al., 2024 ). Lignans are widely distributed in the xylem of plants and are a class of compounds formed by the polymerization of two molecules of phenylpropanoid derivatives. Researchers such as Kitts have found that lignans in flaxseed possess antioxidant properties and have potential anticancer effects on mammals (Kitts et al., 1999 ). Li-Xia Wang et al. have discovered that certain lignans have specific effects on particular protein targets and exhibit good antitumor capabilities and antiviral activity. To date, they have achieved promising clinical applications (Wang et al., 2022 ). Although lignan compounds have been reported to possess various pharmacological activities, a deep understanding of their mechanisms of action remains limited. The primary focus of this study is on the synthesis of lignans (Mori, 2018 )、Structural and Activity Studies (Marinov et al., 2023 )、Functional Activity Research and Studies on Anticancer Active Substances (Mukhija et al., 2022 ), However, there are few reports on the study of lignan biosynthetic mechanisms. In existing research, flaxseed (Zhu et al., 2024 ) is not the only source with high lignans, contentand Schisandra plants (Piao et al., 2018 ), Plants of the genus Polygonatum are also rich in lignans (Yang et al., 2024 )。 Polygonatum species have been reported to contain a variety of lignans, including Polygonatum lignans glycoside A, which is a benzofuran-type lignan, (+)-isolariciresinol-9′-O-β-D-glucoside, trans-N-p-coumaroyltyramine, 3-(4-hydroxyphenyl)-N-[2-(4-hydroxyphenyl)-2-methoxyethyl]acrylamide, and 3-(4-hydroxy-3-methoxyphenyl)-N-[2-(4-hydroxyphenyl)-2-methoxyethyl]acrylamide (Hui et al., 2020 ). Therefore, studying the regulatory mechanisms of lignan compound synthesis in Polygonatum and guiding the breeding of Polygonatum germplasm with higher lignans content holds significant theoretical and practical value. Transcription factors are multifunctional proteins that play a key role in perceiving stress signals, responding to the expression of stress-related genes, and transmitting stress signals(BARNES and ADCOCK, 1995). Among them, the TF family (transcription factor family) and the TR family (transcriptional regulator family) play crucial roles. TFs control cell growth, differentiation, and responses to environmental changes by directly or indirectly affecting gene transcriptional activity (Azofeifa et al., 2020 ). Members of the TR family can interact with various small molecule ligands to regulate a multitude of biological processes, including carbon metabolism and nitrogen metabolism (Sun et al., 2023 ). Studies have shown that the synthesis of lignans in Forsythia is associated with the R2R3-MYB class of transcription factor families (Shu-ping et al., 2021 ). Currently, studies on the biosynthesis of lignan compounds in the rhizomes of different Polygonatum varieties have been reported, but there is a lack of understanding of the transcription factor families involved in the regulatory mechanisms and differential pathways. This greatly limits the pharmaceutical industry's ability to extract specific lignan compounds from Polygonatum on a large scale. Moreover, the genetic modification of Polygonatum rhizomes and the breeding of varieties with high lignan content hold good prospects. This study uses the rhizomes of Polygonatum sibiritum Red and Polygonatum kingianum var. grandifolium as research materials to explore the transcriptional and metabolic mechanisms in the biosynthesis of lignan compounds in Polygonatum plants. The main objectives of this study are: (1) to determine the differences in lignans between the two Polygonatum species; (2) to screen for differentially expressed genes and differentially expressed metabolites related to lignan biosynthesis and metabolism; (3) to conduct a correlation analysis of the differential genes and differential metabolites associated with lignans. These findings will not only help to further reveal the molecular and metabolic mechanisms of lignan compound biosynthesis but also provide valuable information for the future breeding and cultivation of superior Polygonatum varieties and their related pharmaceutical industrial applications. Materials and Methods Plant Materials The experimental materials were selected from the two-year-old Polygonatum plants in the germplasm resource garden of Hubei Minzu University, specifically the rhizomes of Polygonatum sibiritum Red (HBES) and Polygonatum kingianum var. grandifolium (SXHZ). Three biological replicates were collected for each group of Polygonatum rhizomes. The sampled rhizomes were chopped, mixed evenly, and then placed into sterile centrifuge tubes. They were quickly frozen in liquid nitrogen and stored at -80°C until use. Metabolite Extraction and Analysis In this study, the analytical instruments used included an ultra-performance liquid chromatography system (UPLC) and a tandem mass spectrometer (MS/MS). The chromatographic system model was SHIMADZU Nexera X2, and the chromatographic column selected was an Agilent SB-C18 with a particle size of 1.8 micrometers, an inner diameter of 2.1 millimeters, and a length of 100 millimeters. The mobile phase consisted of two phases: Phase A was ultrapure water with 0.1% formic acid added; Phase B was acetonitrile, also with 0.1% formic acid added. The chromatographic conditions were set as follows: the initial proportion of Phase B was 5%, which was then linearly increased to 95% over 9 minutes, held for 1 minute, and then decreased to 5% over the next 1.1 minutes, and this proportion was maintained for 14 minutes to achieve equilibrium. The flow rate was set at 0.35 mL/minute, the column temperature was maintained at 40°C, and the injection volume was 4 microliters. The mass spectrometry analysis conditions were as follows: the ion spray voltage in negative ESI mode was set at -4500 volts, and in positive ESI+ mode it was 5500 volts; the pressures for ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set at 50, 60, and 25 psi, respectively; the collision-induced dissociation parameters were optimized for high efficiency mode, and the temperature of the electrospray ion source (ESI) was maintained at 550°C. Metabolomic analysis was conducted by Metware Biotechnology Co., Ltd. (Wuhan, China). RNA Extraction and Sequencing Analysis Total RNA was extracted from the rhizomes of Polygonatum using the Plant RNA Kit (product number R6827-02) provided by Omega Company, following the manufacturer's instructions. Subsequently, cDNA libraries were constructed based on the extracted RNA. High-quality raw data were obtained by sequencing the cDNA libraries on an Illumina high-throughput sequencing platform using the Sequencing By Synthesis technology. After filtering the raw data, checking the sequencing error rate, and examining the GC content distribution, clean reads were obtained for subsequent analysis. The clean reads were assembled using Trinity to generate transcript sequences, which served as the reference sequences for further analysis.。 Gene Expression and Differential Gene Analysis Basic gene annotation encompasses functional annotation of proteins, pathway annotation, COG/KOG functional annotation, and GO (Gene Ontology) annotation. To achieve detailed annotation of individual genes, we employed the BLASTx algorithm with an E-value threshold of 1e-5 for sequence alignment against the NCBI non-redundant protein database (Nr), Swiss-Prot, KEGG, and the COG/KOG databases. Based on the alignment results, we were able to provide functional annotations for the proteins. Furthermore, we utilized the BLASTp program to align the plant protein-encoding sequences of single genes with the Plant Transcription Factor Database (TFdb) to predict potential transcription factor family members. Employing the DESeq2 software, we conducted a differential gene expression analysis under two biological conditions. We set the False Discovery Rate (FDR) to be below 0.05 and required a fold change of at least 2 to identify differentially expressed genes. Transcriptomic and Metabolomic Association Analysis To identify key genes closely associated with the synthesis of lignan compounds, we conducted an in-depth analysis of differentially expressed genes in HBES and SXHZ samples, integrating the differential accumulation of lignan compounds. By calculating the Pearson correlation coefficients between the differential metabolites and these differentially expressed genes, we were able to reveal potential correlations between them. Furthermore, we constructed a co-expression network using Cytoscape software (version 3.9.1) to visualize the interactions and regulatory relationships among genes. This network analysis aids in our understanding of the complexity of gene regulation during the synthesis process of lignan compounds. Metabolomic Analysis of the Constituents in Rhizomes of Polygonatum sibiritum Red and Polygonatum kingianum var. grandifolium Through qualitative and quantitative detection of metabolites in the rhizomes of Polygonatum, we identified a total of 17 lignans. Principal component analysis (PCA) revealed significant differences in the overall metabolites between the HBES and SXHZ sample groups, with smaller variations among samples within the same group (Figure 1A). Further, this study employed orthogonal partial least squares discriminant analysis (OPLS-DA) for model validation and differential metabolite screening analysis (Figure 1B). Therefore, the results of this study are reliable. Metabolomic data revealed a total of 9 differentially expressed lignan metabolites (Figure 2). In order to gain a deeper understanding of the changes in lignan metabolites in the rhizomesof Polygonatum , we studied the differentially expressed metabolites involved in lignan biosynthesis and metabolism, and identified 9 differential metabolites, as shown in Figure 3. Differential Gene Analysis of Lignans This study identified 2,613 enzyme-coding genes associated with lignan biosynthesis. By annotating pathways ko00998, ko01100, and ko01110, the study selected 75 individual genes that could be categorized as related to the lignin biosynthesis pathway. These genes are divided into 12 categories, with details presented in Table 1. Table 1 Main lignan synthase genes in SXHZ vs HBES TF-Family number kegg SET 21 K11422 K11426 K11420 K11430 K23700 K11433 GARP-G2-like 18 K00975 K00008 K00915 Trihelix 5 K00641 K01051 PHD 2 K00031 K11422 C3H 2 K13832 K00963 WRKY 1 K03847 SWI/SNF-BAF60b 2 K01062 MYB-related 5 k22845 FAR1 3 K14760 C2H2 13 K12309 BES1 1 K01177 AP2/ERF-ERF 2 K00411 Transcription + Metabolism" Correlation Analysis of the Lignans Biosynthetic Pathway This study identified 75 differential genes related to lignin synthesis from the transcriptome data and selected 9 differential metabolites. In conjunction with metabolic pathways, these differential genes and metabolites are primarily involved in the biosynthesis of various secondary metabolites, Part 2 (ko00998). The relevant analysis results are depicted in the figures below. Discussion Lignans are important secondary metabolites in plants, playing a significant role in plant resistance to insects and stress-induced growth. Additionally, lignans possess important pharmacological activities such as antitumor, anti-HIV, anti-inflammatory, hepatoprotective, and antioxidant effects (Teponno et al., 2016 ). The regulation of lignans involves a variety of enzymes and metabolic pathways, with pinoresinol playing a crucial role in lignan biosynthesis. This study combines metabolomic and transcriptomic analyses to explore the synthesis mechanism of lignan compounds in the rhizomes of Polygonatum . It deepens our understanding of the regulatory network, the accumulation of lignan compounds during rhizome development, and the underlying molecular mechanisms, providing a valuable reference foundation for future research work. In the current study, it has been found that the TF (transcription factor) family may have an impact on the synthesis of lignans(Zhang et al., 2021 ), However, this study found that the SET family within the TR family may have an indirect effect on the synthesis of lignans. The analysis in this study revealed that transcripts related to lignans were classified into 12 families, with the GARP-G2-like family in the TF family and the SET family in the TR family being predominant. Tryptophan, phenylalanine, and tyrosine are three important aromatic amino acids that are not only used for protein synthesis in plants but also serve as metabolites participating in other biosynthetic pathways(Wu et al., 2024 ). This study found that these three substances were upregulated in the lignan synthesis pathway. Although these amino acids themselves do not directly participate in the biosynthesis of lignans, this suggests that they may indirectly affect the synthesis of lignans by regulating the metabolic balance within the plant. Metabolomic data analysis revealed that a total of 17 lignan compounds were detected in the rhizomes of HBES and SXHZ, with 9 being differential metabolites. Among them, Cycloolivil-6-O-glucoside is unique to Polygonatum . According to relevant studies, Cycloartol-6-O-glucoside is typically associated with plant growth and development as well as defense mechanisms. It may be involved in regulating the plant's response to environmental stress(Noctor et al., 2024 ), This indirectly suggests that the stress resistance of Polygonatum sibiritum Red might be higher than that of Polygonatum kingianum var. grandifolium. Eleutheroside E, which is unique to Polygonatum kingianum var. grandifolium , can reduce oxidative stress and NF-κB activation, and reprogram the metabolic response to hypoxia-reoxygenation injury. This indicates its ability to exert anti-inflammatory effects and protect cells from oxidative stress damage (Wang and Yang, 2020 ), Additionally, it can significantly improve the inflammatory response induced by SBA (sulfate butylated albumin)(Zhao et al., 2022 ), This provides a theoretical basis for the anti-inflammatory properties of Polygonatum kingianum var. grandifolium. Compared to HBES, in the rhizomes of SXHZ, the differential fold change of Cycloolivil-6-O-glucoside is the highest, reaching 2180.82 times; followed by Matairesinol-4,4'-di-O-glucoside, which is 43.94 times. The regulation of lignans biosynthesis is a complex process that involves multiple enzymes and metabolic pathways. Polygonatum sibiritum Red plays a central role in this process, while hormonal signaling, metabolism of aromatic amino acids, and cellular metabolic status may all indirectly affect the synthesis of lignans (Chen et al., 2021 ). This study found that multiple lignan compounds in Polygonatum kingianum var. grandifolium , including Matairesinol-4,4'-di-O-glucoside, 5'-Methoxyisolariciresinol-9'-O-glucoside, Syringaresinol, Syringaresinol-4'-O-glucoside, Isolariciresinol-9'-O-glucoside, and Dihydrodehydrodiconiferyl alcohol-4-O-glucoside, are all related to the synthesis of Polygonatum sibiritum Red. Relevant studies suggest that Matairesinol-4,4'-di-O-glucoside may possess immunomodulatory properties(Pierre et al., 2011 )、Inhibiting protein kinase activity (Yokoyama et al., 2003 ) and anti-angiogenic effects (Lee et al., 2012 ) and multiple biological activities. 5'-Methoxyisolariciresinol-9'-O-glucoside exhibits significant biological activities, including anti-inflammatory and antioxidant effects. In plants, it may serve to protect the plant from ultraviolet damage and participate in the plant's defense responses. Syringaresinol and Syringaresinol-4'-O-glucoside are known for their anti-inflammatory and anti-cancer activities. In plants, they may contribute to enhancing the plant's adaptability and survival capabilities, especially when facing adversities such as pathogen attacks(Wang et al., 2017 ). Isolariciresinol-9'-O-glucoside and Dihydrodehydrodiconiferyl alcohol-4-O-glucoside possess anti-inflammatory and antioxidant properties. In plants, they may assist in combating environmental stressors such as temperature fluctuations and drought conditions (Witvrouw et al., 2023 ). By analyzing the data, this study found that lignan compounds in the Polygonatum genus mainly possess functions of resisting pathogens and detoxification. This study explored the synthetic regulatory mechanisms of lignans in the Polygonatum genus, not only filling a research gap in this field but also identifying several key gene families and enzyme genes that affect lignan synthesis. These findings have significant implications for molecular breeding. Moreover, these discoveries highlight the importance of lignan compounds in the Polygonatum genus and provide theoretical support for enhancing the stress and pest resistance of Polygonatum plants. In the Metabolic pathways (ko01100) pathway and the Biosynthesis of secondary metabolites (ko01110), it was found that gene families such as C2H2, GARP-G2-like, and SET play significant roles in the regulation of lignan synthesis in the Polygonatum genus. Genes within these families have both positive and negative dual impacts on the synthesis of pinoresinol, thereby affecting lignan synthesis. This discovery also supports the current research perspective that the regulation of lignan synthesis may be influenced by pinoresinol. The study also found that substances such as Lariciresinol, Matairesinol, and Scoisolariciresinol are involved in the regulatory synthesis of lignans. Conclusion During the analysis of the Polygonatum metabolome data, we identified 17 lignans compounds, among which the content of nine showed significant differences. Notably, Cycloolivil-6-O-glucoside exhibited a significant difference in expression between the two Polygonatum species in the lignan biosynthetic pathway. Additionally, transcription factor families such as WRKY, Trihelix, SWI/SNF-BAF60b, SET, PHD, MYB-related, GARP-G2-like, FAR1, C3H, C2H2, BES1, and AP2/ERF-ERF were highly enriched in the transcriptome data, and these differences were significant. The differentially expressed genes in the lignan biosynthetic pathway mainly originated from the GARP-G2-like and SET families, suggesting that GARP-G2-like and SET family transcription factors may play a key role in regulating the lignan synthesis pathway in Polygonatum rhizomes. Furthermore, changes in enzyme genes or substances such as Pimaric alcohol, (+)-Larix pimaric alcohol reductase, (-)-Pimaric alcohol reductase, and (-)-Larix pimaric alcohol reductase may be one of the factors causing differences in the lignan synthesis pathway between the two Polygonatum rhizomes. Integrating these findings, this study provides new insights into the biosynthesis and accumulation of lignan compounds in Polygonatum rhizomes. The differential accumulation of various compounds may affect the medicinal value of different varieties of Polygonatum . Therefore, this study suggests further in-depth exploration of the metabolism and regulatory mechanisms of Polygonatum lignans, aiming to guide the cultivation of new high-quality varieties of Polygonatum with high lignan content. The synthesis of Polygonatum lignans is a complex process involving the joint regulation of multiple transcription factors and related enzymes. By combining transcriptome and metabolome analyses, we can gain a deeper understanding of the molecular mechanisms of this process, thereby providing a theoretical basis for the development and improvement of the medicinal value of Polygonatum plants. Declarations Data Declaration The experimental design for this study was completed by the team of mentors, and the data collection was produced by Metware Company in Wuhan, China, with the link being https://cloud.metware.cn . The account number is 1820231395, and the password is ]XjPzk. If the link is invalid, please contact me to obtain it. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Competing Interests The authors declare they have no competing interests. Funding This study was jointly funded by the National Natural Science Foundation of China( 31260057 ),Natural Science Foundation of Hubei Province(Joint Fund)(2023AFD077),and Hubei Provincial Department of Science and Technology Technology Innovation Major Special Funding Project(2019ACA120). Author Contribution GS is responsible for writing the paper and data processing, XW is responsible for assistance, DL manages experimental materials, and QX provides guidance on the design of the paper's experiments.The authors declare they have no competing interests. Acknowledgment We gratefully acknowledge the guidance and support provided by our mentor and team in supplying the experimental methods and materials. We also extend our thanks to MetWare Biotechnology Co., Ltd. in Wuhan, China, for providing the data. Data Availability The experimental design for this study was completed by the team of mentors, and the data collection was produced by Metware Company in Wuhan, China, with the link being https://cloud.metware.cn. The account number is 1820231395, and the password is ]XjPzk. If the link is invalid, please contact me to obtain it. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. 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Bmc Genomics 22:1-670. http://doi.org/10.1186/s12864-021-07980-w Zhao B, Fan Y, Li H, Zhang C, Han R, Che D (2022) Mitigative effects of eleutheroside e against the mechanical barrier dysfunction induced by soybean agglutinin in ipec‐j2 cell line. J Anim Physiol an N 106:664-670. http://doi.org/10.1111/jpn.13677 Zhu D, Han J, Liu C, Zhang J, Qi Y (2024) Modeling of flaxseed protein, oil content, linoleic acid, and lignan content prediction based on hyperspectral imaging. Front Plant Sci 15:1344143. http://doi.org/10.3389/fpls.2024.1344143 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5450765","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":387529471,"identity":"e8caff5f-5256-4e1b-8cc3-8eeabdf07375","order_by":0,"name":"Guangdi Shi","email":"","orcid":"","institution":"Hubei Minzu University)","correspondingAuthor":false,"prefix":"","firstName":"Guangdi","middleName":"","lastName":"Shi","suffix":""},{"id":387529472,"identity":"21d65b8b-bd20-4118-b146-b9f923e7eb2a","order_by":1,"name":"Xiaolin Wan","email":"","orcid":"","institution":"Hubei Minzu University)","correspondingAuthor":false,"prefix":"","firstName":"Xiaolin","middleName":"","lastName":"Wan","suffix":""},{"id":387529473,"identity":"71722277-d84b-4e21-a84b-4cb843c8ab4a","order_by":2,"name":"Demiao Lan","email":"","orcid":"","institution":"Hubei Minzu University)","correspondingAuthor":false,"prefix":"","firstName":"Demiao","middleName":"","lastName":"Lan","suffix":""},{"id":387529474,"identity":"4c5e09a9-e4c4-4844-8347-5d2f3979b6c1","order_by":3,"name":"Qiang Xiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBACNvbmgw8SKmzkDCQgAowNhLTw8RxLNvhwJs2YeC1yEjlmkjPbDiduIFoLm0SOgTQPG3P6dunu5M88DDayGw4wP3uAVwvPswJjHh623J1zzm4w5mFIM95wgM3cAK8W9uQNyTwSPLkbbuQCGQxAFx7gYZPAq4UhweAwj4FEugFQy2Eehv9EaOFIMWyckWCQANSysZmH4QARWoCBzPDhQILhhjtnNzPOMUg2nnmYzQyvFvn25uM/Ev/9lze43bv5w5sKO9m+483P8GpBA6CgYiZB/SgYBaNgFIwC7AAALKBLnVA9xhAAAAAASUVORK5CYII=","orcid":"","institution":"Hubei Minzu University)","correspondingAuthor":true,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Xiao","suffix":""}],"badges":[],"createdAt":"2024-11-14 04:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5450765/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5450765/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71357667,"identity":"23a52ed9-b000-4d11-8a2c-1d41605e3076","added_by":"auto","created_at":"2024-12-13 15:46:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":100476,"visible":true,"origin":"","legend":"\u003cp\u003eMultivariate statistical analysis of HBES and SXHZ.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5450765/v1/e7ee40720f88a906a01998e5.png"},{"id":71357666,"identity":"4838a72a-d0f8-4fd4-a01a-1959b5e9ecb8","added_by":"auto","created_at":"2024-12-13 15:46:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91611,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of Differentially Expressed Metabolites\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5450765/v1/b63df75bbacbbcc8f55734b8.png"},{"id":71357668,"identity":"18f2fb68-42f9-4d1d-a216-b9afdd4f20db","added_by":"auto","created_at":"2024-12-13 15:46:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72221,"visible":true,"origin":"","legend":"\u003cp\u003eRegarding the differential metabolites of lignans, red represents upregulation, and green represents downregulation.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5450765/v1/96799f36d52d08d70947cb62.png"},{"id":71357669,"identity":"37029fc1-cce5-4279-ba38-0d9ba17169d1","added_by":"auto","created_at":"2024-12-13 15:46:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":202857,"visible":true,"origin":"","legend":"\u003cp\u003eA: Gene clustering heatmap of the SXHZ vs HBES group. B: Number of differentially expressed TF family genes related to lignans.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5450765/v1/550c240746939a75072e1a21.png"},{"id":71357671,"identity":"740f57b7-8412-4058-ae1b-c20cae5888a4","added_by":"auto","created_at":"2024-12-13 15:46:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":734455,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptome and metabolome correlation analysis:A: Correlation and chord diagram between the two omics groups.B: Correlation map between genes and metabolites.C: Combined correlation diagram.D: Correlation network diagram, top 20 genes.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5450765/v1/b270419a8b32aca87d153b62.png"},{"id":71357670,"identity":"cd16de03-7b4d-4185-85dd-cba59adeb717","added_by":"auto","created_at":"2024-12-13 15:46:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":95284,"visible":true,"origin":"","legend":"\u003cp\u003eStructural formulas of some metabolites.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5450765/v1/e1041da79a7fea463f0d78d8.png"},{"id":71358413,"identity":"0ad51c45-0ac0-435d-91b9-ea0375409b64","added_by":"auto","created_at":"2024-12-13 15:54:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":246273,"visible":true,"origin":"","legend":"\u003cp\u003eLignan biosynthesis pathway ko00998, where red represents upregulation and green represents downregulation.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5450765/v1/babfa4ab4935948d1f7c12dd.png"},{"id":74115192,"identity":"e79c4d6c-b36e-4b0f-a32b-ad3b41c0c107","added_by":"auto","created_at":"2025-01-18 02:46:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2110955,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5450765/v1/6204041d-c142-4eed-90e0-4d8f991144d2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated Multi-Omics Analysis Reveals the Regulatory Mechanisms of Lignans Biosynthesis in the Genus Polygonatum","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants of the genus \u003cem\u003ePolygonatum\u003c/em\u003e, belonging to the Liliaceae family, are perennial herbs (Yan et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). First recorded in the Han Dynasty's \"Ming Yi Bie Lu,\" Polygonatum is a plant with both medicinal and dietary uses. The main part utilized is its rhizome, which is known for its benefits in replenishing vital energy and possessing antioxidant properties (Wan et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), It has the effect of delaying aging (Liu et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In previous studies, it has been found that \u003cem\u003ePolygonatum\u003c/em\u003e is rich in polysaccharides, saponins, flavonoids, sterols, amino acids, and other trace nutritional elements. It possesses effects such as improving memory, reducing blood sugar and lipid levels, and enhancing immune system function in the human body (Yang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLignans are widely distributed in the xylem of plants and are a class of compounds formed by the polymerization of two molecules of phenylpropanoid derivatives. Researchers such as Kitts have found that lignans in flaxseed possess antioxidant properties and have potential anticancer effects on mammals (Kitts et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLi-Xia Wang et al. have discovered that certain lignans have specific effects on particular protein targets and exhibit good antitumor capabilities and antiviral activity. To date, they have achieved promising clinical applications (Wang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although lignan compounds have been reported to possess various pharmacological activities, a deep understanding of their mechanisms of action remains limited. The primary focus of this study is on the synthesis of lignans (Mori, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)、Structural and Activity Studies (Marinov et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)、Functional Activity Research and Studies on Anticancer Active Substances (Mukhija et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), However, there are few reports on the study of lignan biosynthetic mechanisms. In existing research, flaxseed (Zhu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) is not the only source with high lignans, contentand Schisandra plants (Piao et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), Plants of the genus \u003cem\u003ePolygonatum\u003c/em\u003e are also rich in lignans (Yang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)。\u003cem\u003ePolygonatum\u003c/em\u003e species have been reported to contain a variety of lignans, including \u003cem\u003ePolygonatum\u003c/em\u003e lignans glycoside A, which is a benzofuran-type lignan, (+)-isolariciresinol-9\u0026prime;-O-β-D-glucoside, trans-N-p-coumaroyltyramine, 3-(4-hydroxyphenyl)-N-[2-(4-hydroxyphenyl)-2-methoxyethyl]acrylamide, and 3-(4-hydroxy-3-methoxyphenyl)-N-[2-(4-hydroxyphenyl)-2-methoxyethyl]acrylamide (Hui et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, studying the regulatory mechanisms of lignan compound synthesis in \u003cem\u003ePolygonatum\u003c/em\u003e and guiding the breeding of \u003cem\u003ePolygonatum\u003c/em\u003e germplasm with higher lignans content holds significant theoretical and practical value.\u003c/p\u003e \u003cp\u003eTranscription factors are multifunctional proteins that play a key role in perceiving stress signals, responding to the expression of stress-related genes, and transmitting stress signals(BARNES and ADCOCK, 1995). Among them, the TF family (transcription factor family) and the TR family (transcriptional regulator family) play crucial roles. TFs control cell growth, differentiation, and responses to environmental changes by directly or indirectly affecting gene transcriptional activity (Azofeifa et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Members of the TR family can interact with various small molecule ligands to regulate a multitude of biological processes, including carbon metabolism and nitrogen metabolism (Sun et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Studies have shown that the synthesis of lignans in Forsythia is associated with the R2R3-MYB class of transcription factor families (Shu-ping et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrently, studies on the biosynthesis of lignan compounds in the rhizomes of different \u003cem\u003ePolygonatum\u003c/em\u003e varieties have been reported, but there is a lack of understanding of the transcription factor families involved in the regulatory mechanisms and differential pathways. This greatly limits the pharmaceutical industry's ability to extract specific lignan compounds from \u003cem\u003ePolygonatum\u003c/em\u003e on a large scale. Moreover, the genetic modification of \u003cem\u003ePolygonatum\u003c/em\u003e rhizomes and the breeding of varieties with high lignan content hold good prospects. This study uses the rhizomes of \u003cem\u003ePolygonatum sibiritum Red and Polygonatum kingianum var. grandifolium\u003c/em\u003e as research materials to explore the transcriptional and metabolic mechanisms in the biosynthesis of lignan compounds in \u003cem\u003ePolygonatum\u003c/em\u003e plants. The main objectives of this study are: (1) to determine the differences in lignans between the two \u003cem\u003ePolygonatum\u003c/em\u003e species; (2) to screen for differentially expressed genes and differentially expressed metabolites related to lignan biosynthesis and metabolism; (3) to conduct a correlation analysis of the differential genes and differential metabolites associated with lignans. These findings will not only help to further reveal the molecular and metabolic mechanisms of lignan compound biosynthesis but also provide valuable information for the future breeding and cultivation of superior \u003cem\u003ePolygonatum\u003c/em\u003e varieties and their related pharmaceutical industrial applications.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental materials were selected from the two-year-old \u003cem\u003ePolygonatum\u003c/em\u003e plants in the germplasm resource garden of Hubei Minzu University, specifically the rhizomes of \u003cem\u003ePolygonatum sibiritum Red\u0026nbsp;\u003c/em\u003e(HBES) and \u003cem\u003ePolygonatum kingianum var. grandifolium\u003c/em\u003e (SXHZ). Three biological replicates were collected for each group of \u003cem\u003ePolygonatum\u003c/em\u003e rhizomes. The sampled rhizomes were chopped, mixed evenly, and then placed into sterile centrifuge tubes. They were quickly frozen in liquid nitrogen and stored at -80\u0026deg;C until use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolite Extraction and Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the analytical instruments used included an ultra-performance liquid chromatography system (UPLC) and a tandem mass spectrometer (MS/MS). The chromatographic system model was SHIMADZU Nexera X2, and the chromatographic column selected was an Agilent SB-C18 with a particle size of 1.8 micrometers, an inner diameter of 2.1 millimeters, and a length of 100 millimeters. The mobile phase consisted of two phases: Phase A was ultrapure water with 0.1% formic acid added; Phase B was acetonitrile, also with 0.1% formic acid added. The chromatographic conditions were set as follows: the initial proportion of Phase B was 5%, which was then linearly increased to 95% over 9 minutes, held for 1 minute, and then decreased to 5% over the next 1.1 minutes, and this proportion was maintained for 14 minutes to achieve equilibrium. The flow rate was set at 0.35 mL/minute, the column temperature was maintained at 40\u0026deg;C, and the injection volume was 4 microliters. The mass spectrometry analysis conditions were as follows: the ion spray voltage in negative ESI mode was set at -4500 volts, and in positive ESI+ mode it was 5500 volts; the pressures for ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set at 50, 60, and 25 psi, respectively; the collision-induced dissociation parameters were optimized for high efficiency mode, and the temperature of the electrospray ion source (ESI) was maintained at 550\u0026deg;C. Metabolomic analysis was conducted by Metware Biotechnology Co., Ltd. (Wuhan, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Extraction and Sequencing Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the rhizomes of \u003cem\u003ePolygonatum\u003c/em\u003e using the Plant RNA Kit (product number R6827-02) provided by Omega Company, following the manufacturer\u0026apos;s instructions. Subsequently, cDNA libraries were constructed based on the extracted RNA. High-quality raw data were obtained by sequencing the cDNA libraries on an Illumina high-throughput sequencing platform using the Sequencing By Synthesis technology. After filtering the raw data, checking the sequencing error rate, and examining the GC content distribution, clean reads were obtained for subsequent analysis. The clean reads were assembled using Trinity to generate transcript sequences, which served as the reference sequences for further analysis.。\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene Expression and Differential Gene Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBasic gene annotation encompasses functional annotation of proteins, pathway annotation, COG/KOG functional annotation, and GO (Gene Ontology) annotation. To achieve detailed annotation of individual genes, we employed the BLASTx algorithm with an E-value threshold of 1e-5 for sequence alignment against the NCBI non-redundant protein database (Nr), Swiss-Prot, KEGG, and the COG/KOG databases. Based on the alignment results, we were able to provide functional annotations for the proteins. Furthermore, we utilized the BLASTp program to align the plant protein-encoding sequences of single genes with the Plant Transcription Factor Database (TFdb) to predict potential transcription factor family members. Employing the DESeq2 software, we conducted a differential gene expression analysis under two biological conditions. We set the False Discovery Rate (FDR) to be below 0.05 and required a fold change of at least 2 to identify differentially expressed genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptomic and Metabolomic Association Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify key genes closely associated with the synthesis of lignan compounds, we conducted an in-depth analysis of differentially expressed genes in HBES and SXHZ samples, integrating the differential accumulation of lignan compounds. By calculating the Pearson correlation coefficients between the differential metabolites and these differentially expressed genes, we were able to reveal potential correlations between them. Furthermore, we constructed a co-expression network using Cytoscape software (version 3.9.1) to visualize the interactions and regulatory relationships among genes. This network analysis aids in our understanding of the complexity of gene regulation during the synthesis process of lignan compounds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolomic Analysis of the Constituents in Rhizomes of \u003cem\u003ePolygonatum sibiritum Red and Polygonatum kingianum var. grandifolium\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough qualitative and quantitative detection of metabolites in the rhizomes of\u003cem\u003e\u0026nbsp;Polygonatum,\u0026nbsp;\u003c/em\u003ewe identified a total of 17 lignans. Principal component analysis (PCA) revealed significant differences in the overall metabolites between the HBES and SXHZ sample groups, with smaller variations among samples within the same group (Figure 1A). Further, this study employed orthogonal partial least squares discriminant analysis (OPLS-DA) for model validation and differential metabolite screening analysis (Figure 1B). Therefore, the results of this study are reliable.\u003c/p\u003e\n\u003cp\u003eMetabolomic data revealed a total of 9 differentially expressed lignan metabolites (Figure 2).\u003c/p\u003e\n\u003cp\u003eIn order to gain a deeper understanding of the changes in lignan metabolites in the rhizomesof \u003cem\u003ePolygonatum\u003c/em\u003e, we studied the differentially expressed metabolites involved in lignan biosynthesis and metabolism, and identified 9 differential metabolites, as shown in Figure 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential Gene Analysis of Lignans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study identified 2,613 enzyme-coding genes associated with lignan biosynthesis. By annotating pathways ko00998, ko01100, and ko01110, the study selected 75 individual genes that could be categorized as related to the lignin biosynthesis pathway. These genes are divided into 12 categories, with details presented in Table 1.\u003c/p\u003e\n\u003cp\u003eTable 1 Main lignan synthase genes in SXHZ vs HBES\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eTF-Family\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003enumber\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003ekegg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eSET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003eK11422 K11426 K11420 K11430 K23700 K11433\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eGARP-G2-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003eK00975 K00008 K00915\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eTrihelix\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003eK00641 K01051\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003ePHD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003eK00031 K11422\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eC3H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003eK13832 K00963\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eWRKY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003eK03847\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eSWI/SNF-BAF60b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003eK01062\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eMYB-related\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003ek22845\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eFAR1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003eK14760\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eC2H2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003eK12309\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eBES1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003eK01177\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003eAP2/ERF-ERF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 279px;\"\u003e\n \u003cp\u003eK00411\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTranscription + Metabolism\u0026quot; Correlation Analysis of the Lignans Biosynthetic Pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study identified 75 differential genes related to lignin synthesis from the transcriptome data and selected 9 differential metabolites. In conjunction with metabolic pathways, these differential genes and metabolites are primarily involved in the biosynthesis of various secondary metabolites, Part 2 (ko00998). The relevant analysis results are depicted in the figures below.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLignans are important secondary metabolites in plants, playing a significant role in plant resistance to insects and stress-induced growth. Additionally, lignans possess important pharmacological activities such as antitumor, anti-HIV, anti-inflammatory, hepatoprotective, and antioxidant effects (Teponno et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The regulation of lignans involves a variety of enzymes and metabolic pathways, with pinoresinol playing a crucial role in lignan biosynthesis. This study combines metabolomic and transcriptomic analyses to explore the synthesis mechanism of lignan compounds in the rhizomes of \u003cem\u003ePolygonatum\u003c/em\u003e. It deepens our understanding of the regulatory network, the accumulation of lignan compounds during rhizome development, and the underlying molecular mechanisms, providing a valuable reference foundation for future research work.\u003c/p\u003e \u003cp\u003eIn the current study, it has been found that the TF (transcription factor) family may have an impact on the synthesis of lignans(Zhang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), However, this study found that the SET family within the TR family may have an indirect effect on the synthesis of lignans. The analysis in this study revealed that transcripts related to lignans were classified into 12 families, with the GARP-G2-like family in the TF family and the SET family in the TR family being predominant. Tryptophan, phenylalanine, and tyrosine are three important aromatic amino acids that are not only used for protein synthesis in plants but also serve as metabolites participating in other biosynthetic pathways(Wu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This study found that these three substances were upregulated in the lignan synthesis pathway. Although these amino acids themselves do not directly participate in the biosynthesis of lignans, this suggests that they may indirectly affect the synthesis of lignans by regulating the metabolic balance within the plant.\u003c/p\u003e \u003cp\u003eMetabolomic data analysis revealed that a total of 17 lignan compounds were detected in the rhizomes of HBES and SXHZ, with 9 being differential metabolites. Among them, Cycloolivil-6-O-glucoside is unique to \u003cem\u003ePolygonatum\u003c/em\u003e. According to relevant studies, Cycloartol-6-O-glucoside is typically associated with plant growth and development as well as defense mechanisms. It may be involved in regulating the plant's response to environmental stress(Noctor et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), This indirectly suggests that the stress resistance of \u003cem\u003ePolygonatum sibiritum\u003c/em\u003e Red might be higher than that of \u003cem\u003ePolygonatum kingianum var. grandifolium.\u003c/em\u003e Eleutheroside E, which is unique to \u003cem\u003ePolygonatum kingianum var. grandifolium\u003c/em\u003e, can reduce oxidative stress and NF-κB activation, and reprogram the metabolic response to hypoxia-reoxygenation injury. This indicates its ability to exert anti-inflammatory effects and protect cells from oxidative stress damage (Wang and Yang, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Additionally, it can significantly improve the inflammatory response induced by SBA (sulfate butylated albumin)(Zhao et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), This provides a theoretical basis for the anti-inflammatory properties of \u003cem\u003ePolygonatum kingianum var. grandifolium.\u003c/em\u003e Compared to HBES, in the rhizomes of SXHZ, the differential fold change of Cycloolivil-6-O-glucoside is the highest, reaching 2180.82 times; followed by Matairesinol-4,4'-di-O-glucoside, which is 43.94 times.\u003c/p\u003e \u003cp\u003eThe regulation of lignans biosynthesis is a complex process that involves multiple enzymes and metabolic pathways. \u003cem\u003ePolygonatum sibiritum\u003c/em\u003e Red plays a central role in this process, while hormonal signaling, metabolism of aromatic amino acids, and cellular metabolic status may all indirectly affect the synthesis of lignans (Chen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This study found that multiple lignan compounds in \u003cem\u003ePolygonatum kingianum var. grandifolium\u003c/em\u003e, including Matairesinol-4,4'-di-O-glucoside, 5'-Methoxyisolariciresinol-9'-O-glucoside, Syringaresinol, Syringaresinol-4'-O-glucoside, Isolariciresinol-9'-O-glucoside, and Dihydrodehydrodiconiferyl alcohol-4-O-glucoside, are all related to the synthesis of \u003cem\u003ePolygonatum sibiritum\u003c/em\u003e Red. Relevant studies suggest that Matairesinol-4,4'-di-O-glucoside may possess immunomodulatory properties(Pierre et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e)、Inhibiting protein kinase activity (Yokoyama et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and anti-angiogenic effects (Lee et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and multiple biological activities. 5'-Methoxyisolariciresinol-9'-O-glucoside exhibits significant biological activities, including anti-inflammatory and antioxidant effects. In plants, it may serve to protect the plant from ultraviolet damage and participate in the plant's defense responses. Syringaresinol and Syringaresinol-4'-O-glucoside are known for their anti-inflammatory and anti-cancer activities. In plants, they may contribute to enhancing the plant's adaptability and survival capabilities, especially when facing adversities such as pathogen attacks(Wang et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Isolariciresinol-9'-O-glucoside and Dihydrodehydrodiconiferyl alcohol-4-O-glucoside possess anti-inflammatory and antioxidant properties. In plants, they may assist in combating environmental stressors such as temperature fluctuations and drought conditions (Witvrouw et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBy analyzing the data, this study found that lignan compounds in the \u003cem\u003ePolygonatum\u003c/em\u003e genus mainly possess functions of resisting pathogens and detoxification. This study explored the synthetic regulatory mechanisms of lignans in the \u003cem\u003ePolygonatum\u003c/em\u003e genus, not only filling a research gap in this field but also identifying several key gene families and enzyme genes that affect lignan synthesis. These findings have significant implications for molecular breeding. Moreover, these discoveries highlight the importance of lignan compounds in the \u003cem\u003ePolygonatum\u003c/em\u003e genus and provide theoretical support for enhancing the stress and pest resistance of \u003cem\u003ePolygonatum\u003c/em\u003e plants.\u003c/p\u003e \u003cp\u003eIn the Metabolic pathways (ko01100) pathway and the Biosynthesis of secondary metabolites (ko01110), it was found that gene families such as C2H2, GARP-G2-like, and SET play significant roles in the regulation of lignan synthesis in the \u003cem\u003ePolygonatum\u003c/em\u003e genus. Genes within these families have both positive and negative dual impacts on the synthesis of pinoresinol, thereby affecting lignan synthesis. This discovery also supports the current research perspective that the regulation of lignan synthesis may be influenced by pinoresinol. The study also found that substances such as Lariciresinol, Matairesinol, and Scoisolariciresinol are involved in the regulatory synthesis of lignans.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eDuring the analysis of the \u003cem\u003ePolygonatum\u003c/em\u003e metabolome data, we identified 17 lignans compounds, among which the content of nine showed significant differences. Notably, Cycloolivil-6-O-glucoside exhibited a significant difference in expression between the two \u003cem\u003ePolygonatum\u003c/em\u003e species in the lignan biosynthetic pathway. Additionally, transcription factor families such as WRKY, Trihelix, SWI/SNF-BAF60b, SET, PHD, MYB-related, GARP-G2-like, FAR1, C3H, C2H2, BES1, and AP2/ERF-ERF were highly enriched in the transcriptome data, and these differences were significant. The differentially expressed genes in the lignan biosynthetic pathway mainly originated from the GARP-G2-like and SET families, suggesting that GARP-G2-like and SET family transcription factors may play a key role in regulating the lignan synthesis pathway in \u003cem\u003ePolygonatum\u003c/em\u003e rhizomes. Furthermore, changes in enzyme genes or substances such as Pimaric alcohol, (+)-Larix pimaric alcohol reductase, (-)-Pimaric alcohol reductase, and (-)-Larix pimaric alcohol reductase may be one of the factors causing differences in the lignan synthesis pathway between the two \u003cem\u003ePolygonatum\u003c/em\u003e rhizomes. Integrating these findings, this study provides new insights into the biosynthesis and accumulation of lignan compounds in \u003cem\u003ePolygonatum\u003c/em\u003e rhizomes. The differential accumulation of various compounds may affect the medicinal value of different varieties of \u003cem\u003ePolygonatum\u003c/em\u003e. Therefore, this study suggests further in-depth exploration of the metabolism and regulatory mechanisms of \u003cem\u003ePolygonatum\u003c/em\u003e lignans, aiming to guide the cultivation of new high-quality varieties of \u003cem\u003ePolygonatum\u003c/em\u003e with high lignan content. The synthesis of \u003cem\u003ePolygonatum\u003c/em\u003e lignans is a complex process involving the joint regulation of multiple transcription factors and related enzymes. By combining transcriptome and metabolome analyses, we can gain a deeper understanding of the molecular mechanisms of this process, thereby providing a theoretical basis for the development and improvement of the medicinal value of \u003cem\u003ePolygonatum\u003c/em\u003e plants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eData Declaration\u003c/h2\u003e \u003cp\u003eThe experimental design for this study was completed by the team of mentors, and the data collection was produced by Metware Company in Wuhan, China, with the link being \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.metware.cn\u003c/span\u003e\u003cspan address=\"https://cloud.metware.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The account number is 1820231395, and the password is ]XjPzk. If the link is invalid, please contact me to obtain it. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was jointly funded by the National Natural Science Foundation of China( 31260057 ),Natural Science Foundation of Hubei Province(Joint Fund)(2023AFD077),and Hubei Provincial Department of Science and Technology Technology Innovation Major Special Funding Project(2019ACA120).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eGS is responsible for writing the paper and data processing, XW is responsible for assistance, DL manages experimental materials, and QX provides guidance on the design of the paper's experiments.The authors declare they have no competing interests.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eWe gratefully acknowledge the guidance and support provided by our mentor and team in supplying the experimental methods and materials. We also extend our thanks to MetWare Biotechnology Co., Ltd. in Wuhan, China, for providing the data.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe experimental design for this study was completed by the team of mentors, and the data collection was produced by Metware Company in Wuhan, China, with the link being https://cloud.metware.cn. The account number is 1820231395, and the password is ]XjPzk. If the link is invalid, please contact me to obtain it. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAzofeifa J, Basken J, Lai M, Langendorf R, Norris L, Read T, Robbins-Pianka A (2020) Abstract 1272: enhancer rna cell line database reveals new associations between tf activity and cancer subtype. 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Phytochemistry (Oxford) 202:113326. http://doi.org/10.1016/j.phytochem.2022.113326\u003c/li\u003e\n \u003cli\u003eWang S, Wu C, Li X, Zhou Y, Zhang Q, Ma F, Wei J, Zhang X, Guo P (2017) Syringaresinol-4-o-\u0026beta;-d-glucoside alters lipid and glucose metabolism in hepg2 cells and c2c12 myotubes. Acta Pharmaceutica Sinica. B 7:453-460. http://doi.org/10.1016/j.apsb.2017.04.008\u003c/li\u003e\n \u003cli\u003eWang S, Yang X (2020) Eleutheroside e decreases oxidative stress and nf-\u0026kappa;b activation and reprograms the metabolic response against hypoxia-reoxygenation injury in h9c2 cells. Int Immunopharmacol 84:106513. http://doi.org/10.1016/j.intimp.2020.106513\u003c/li\u003e\n \u003cli\u003eWitvrouw K, Kim H, Vanholme R, Goeminne G, Ralph J, Boerjan W, Vanholme B, Great Lakes Bioenergy Research Center GLBRC MWUS (2023) Reassessing the claimed cytokinin-substituting activity of dehydrodiconiferyl alcohol glucoside. 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Front Plant Sci 15:1344143. http://doi.org/10.3389/fpls.2024.1344143\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Polygonatum sibiritum Red, Polygonatum kingianum var. grandifolium, Lignans, Transcriptomics, Metabolomics, Transcription factor families","lastPublishedDoi":"10.21203/rs.3.rs-5450765/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5450765/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBy integrating metabolomic and transcriptomic analyses, our study unravels the regulatory mechanisms underlying the biosynthesis of lignans in the plant genus \u003cem\u003ePolygonatum.\u003c/em\u003e As a perennial herb of the Liliaceae family, \u003cem\u003ePolygonatum\u003c/em\u003e boasts rhizomes that are rich in polysaccharides, saponins, flavonoids, sterols, and amino acids\u0026mdash;micronutrients that contribute to its essence-nourishing, antioxidant, and anti-aging properties. These rhizomes have a positive impact on memory enhancement, blood glucose and lipid reduction, and immune system strengthening. Lignans, as secondary metabolites in plants, play a pivotal role in plant defense against pests and stress, and exhibit a range of pharmacological activities, including anti-tumor, anti-HIV, anti-inflammatory, hepatoprotective, and antioxidant effects.Focusing on \u003cem\u003ePolygonatum sibiricum\u003c/em\u003e Red and \u003cem\u003ePolygonatum kingianum var. grandifolium\u003c/em\u003e, our research delves into the transcriptional and metabolic mechanisms of lignan biosynthesis. We discovered that transcription factor families such as GARP-G2-like and SET may be crucial in regulating the lignan synthetic pathway within the rhizomes of \u003cem\u003ePolygonatum.\u003c/em\u003e Additionally, we identified 17 lignans, with significant differences in the content of nine, particularly the marked expression variation of Cycloolivil-6-O-glucoside between the two \u003cem\u003ePolygonatum\u003c/em\u003e species.Our findings not only fill a gap in the field but also offer guidance for molecular breeding, underscoring the significance of lignans in \u003cem\u003ePolygonatum\u003c/em\u003e and providing theoretical support for enhancing the plant's stress tolerance and resistance to pests and diseases. We recommend further in-depth exploration of the metabolic and regulatory mechanisms \u003cem\u003eof Polygonatum\u003c/em\u003e lignans to inform the development of new \u003cem\u003ePolygonatum\u003c/em\u003e varieties with high quality and lignan content.\u003c/p\u003e","manuscriptTitle":"Integrated Multi-Omics Analysis Reveals the Regulatory Mechanisms of Lignans Biosynthesis in the Genus Polygonatum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-13 15:46:04","doi":"10.21203/rs.3.rs-5450765/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"51aa88af-db00-4641-829c-548492ad6a99","owner":[],"postedDate":"December 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-18T02:38:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-13 15:46:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5450765","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5450765","identity":"rs-5450765","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}