Science, Irving K. Barber Faculty of (Okanagan)
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Biology, Department of (Okanagan)
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International Journal of Molecular Sciences 26 (6): 2669 (2025)
Wang, Yumeng
Zhang, Jiaxin
Wang, Ping
Li, Yongkang
Wang, Yihan
Yan, Yan
Chi, Junwen
Chen, Jiankang
Lian, Junmei
Piao, Xiangmin
Lei, Xiujuan
Xiao, Ying
Murray, Jeremy
Deyholos, Micheal K.
Wang, Yingping
Di, Peng
Zhang, Jian (Professor of biology)
Panax quinquefolius is a globally valued medicinal plant rich in bioactive flavonoids, yet the molecular mechanisms underlying their biosynthesis remain poorly understood. In this study, we integrated transcriptomic and metabolomic analyses to investigate tissue-specific flavonoid accumulation and regulatory networks in roots, leaves, and flowers. Metabolomic profiling identified 141 flavonoid metabolites, with flavones, flavonols, and C-glycosylflavones predominantly enriched in aerial tissues (leaves and flowers), while specific glycosides like tricin 7-O-acetylglucoside showed root-specific accumulation. Transcriptome sequencing revealed 15,551–18,946 DEGs across tissues, and the reliability of the transcriptomic data was validated by qRT-PCR. KEGG and GO annotation analyses suggested that these DEGs may play a crucial role in the biosynthesis and metabolism of secondary metabolites. From the DEGs, UGTs and MYB TFs were identified and subjected to correlation analysis. Functional validation through in vitro enzymatic assays confirmed that PqUGT71A1 catalyzes apigenin and naringenin glycosylation at the 7-OH position. Additionally, subcellular localization and yeast one-hybrid assays demonstrated that PqMYB7 and PqMYB13 interact with the PqUGT71A1 promoter and activate its expression.. This study unveils the spatial dynamics of flavonoid metabolism in P. quinquefolius and establishes a MYB-UGT regulatory axis, providing critical insights for metabolic engineering and bioactive compound optimization in medicinal plants.
Academic Editor: Soulaiman SakrReceived: 26 February 2025Revised: 10 March 2025Accepted: 14 March 2025Published: 16 March 2025Citation: Wang, Y.; Zhang, J.; Wang,P.; Li, Y.; Wang, Y.; Yan, Y.; Chi, J.; Chen,J.; Lian, J.; Piao, X.; et al. IntegratedTranscriptomic and MetabolomicAnalysis Reveals Tissue-SpecificFlavonoid Biosynthesis andMYB-Mediated Regulation ofUGT71A1 in Panax quinquefolius. Int. J.Mol. Sci. 2025, 26, 2669. https://doi.org/10.3390/ijms26062669Copyright: © 2025 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license(https://creativecommons.org/licenses/by/4.0/).ArticleIntegrated Transcriptomic and Metabolomic Analysis RevealsTissue-Specific Flavonoid Biosynthesis and MYB-MediatedRegulation of UGT71A1 in Panax quinquefoliusYumeng Wang 1,† , Jiaxin Zhang 1,†, Ping Wang 1, Yongkang Li 2, Yihan Wang 1, Yan Yan 1, Junwen Chi 1,Jiankang Chen 1, Junmei Lian 1, Xiangmin Piao 1, Xiujuan Lei 1, Ying Xiao 2, Jeremy Murray 3,Micheal K. Deyholos 4 , Yingping Wang 1, Peng Di 1,* and Jian Zhang 4,5,*1 State Local Joint Engineering Research Center of Ginseng Breeding and Application, College of ChineseMedicinal Materials, Jilin Agricultural University, Changchun 130118, China; ping@mails.jlau.edu.cn (P.W.);20231693@mails.jlau.edu.cn (J.C.); chenjk@mails.jlau.edu.cn (J.C.); lian@mails.jlau.edu.cn (J.L.)2 The MOE Key Laboratory for Standardization of Chinese Medicines and the SHTCM Key Laboratory for NewResources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica,Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China3 CAS Center for Excellence in Molecular Plant Sciences, 300 Fenglin Road, Shanghai 200032, China4 Department of Biology, University of British Columbia, Okanagan, Kelowna, BC V1V 1V7, Canada5 Faculty of Agronomy, Jilin Agricultural University, Changchun 130118, China* Correspondence: di@jlau.edu.cn (P.D.); jian.zhang@ubc.ca (J.Z.)† These authors contributed equally to this work.Abstract: Panax quinquefolius is a globally valued medicinal plant rich in bioactiveflavonoids, yet the molecular mechanisms underlying their biosynthesis remain poorlyunderstood. In this study, we integrated transcriptomic and metabolomic analyses toinvestigate tissue-specific flavonoid accumulation and regulatory networks in roots, leaves,and flowers. Metabolomic profiling identified 141 flavonoid metabolites, with flavones,flavonols, and C-glycosylflavones predominantly enriched in aerial tissues (leaves andflowers), while specific glycosides like tricin 7-O-acetylglucoside showed root-specificaccumulation. Transcriptome sequencing revealed 15,551–18,946 DEGs across tissues, andthe reliability of the transcriptomic data was validated by qRT-PCR. KEGG and GO annota-tion analyses suggested that these DEGs may play a crucial role in the biosynthesis andmetabolism of secondary metabolites. From the DEGs, UGTs and MYB TFs were identifiedand subjected to correlation analysis. Functional validation through in vitro enzymaticassays confirmed that PqUGT71A1 catalyzes apigenin and naringenin glycosylation atthe 7-OH position. Additionally, subcellular localization and yeast one-hybrid assaysdemonstrated that PqMYB7 and PqMYB13 interact with the PqUGT71A1 promoter andactivate its expression.. This study unveils the spatial dynamics of flavonoid metabolism inP. quinquefolius and establishes a MYB-UGT regulatory axis, providing critical insights formetabolic engineering and bioactive compound optimization in medicinal plants.Keywords: flavonoid biosynthesis; American ginseng; Panax quinquefolius; transcriptomicand metabolomic integration study1. IntroductionPanax quinquefolius (P.quinquefolius), a species in the Araliaceae family, is one of theworld’s most popular medicinal herbs, known for its diverse pharmacological activities [1].Various tissues of P. quinquefolius, including roots, stems, leaves, and flowers, hold signifi-cant medicinal and economic value, being widely utilized in pharmaceutical formulations,Int. J. Mol. Sci. 2025, 26, 2669 https://doi.org/10.3390/ijms26062669Int. J. Mol. Sci. 2025, 26, 2669 2 of 18functional foods, and cosmetic products. P. quinquefolius has been traditionally used inherbal medicine for centuries and is now commonly incorporated into dietary supplements,functional foods, and cosmetics. Pharmacological studies have demonstrated its potentialtherapeutic applications in cardiovascular diseases [2], immune enhancement [3], cancercell inhibition [4], and cognitive function improvement [5]. Extensive phytochemical in-vestigations have revealed P. quinquefolius as a rich source of bioactive constituents [6,7],notably triterpenoid saponins (e.g., ginsenosides), flavonoid glycosides, essential minerals(zinc, iron), and phenolic acids.Flavonoids, a major class of polyphenolic compounds, function as signaling molecules,intermediates, and secondary metabolites in plants [8]. They are among the most bi-ologically active polyphenolic compounds, including flavones, isoflavones, flavonols,isoflavonols, flavanones, isoflavanones, and chalcones [9], and they are widely distributedacross the plant kingdom. To date, over 8000 flavonoid compounds [10] have been identifiedin plants. They are important not only in UV radiation [11] and inhibiting auxin trans-port [12] it is also important in mitigation of various biotic [13], abiotic stress [14]. Althoughthe biosynthetic pathways of flavonoids have been studied in model and medicinal plants,such as Arabidopsis thaliana [15], Ziziphus jujuba [16], Ginkgo biloba [17], and Phalaenopsisamabilis [18], the flavonoid biosynthesis pathways in P. quinquefolius remain unclear.Transcription factors play a crucial role in regulating flavonoid biosynthesis by modu-lating the expression of key enzyme genes, thereby influencing metabolic pathway activity.Transcriptomics is a powerful tool for investigating functional genes [19], offering valuableinsights into differential gene expression and regulatory networks through transcriptomesequencing [20,21]. Metabolomics, a relatively newer “omics” discipline emerging along-side genomics, transcriptomics, and proteomics, provides a direct reflection of biologicalresponses and serves as a key foundation for understanding phenotypic traits [22,23]. Itfocuses on identifying the types, quantities, and dynamic fluctuations of metabolites withinan organism in response to perturbations [24]. Due to the intricate regulatory mecha-nisms governing secondary metabolite biosynthesis, data from a single omics approachoften provide an incomplete picture of the underlying pathways. Multi-omics integration,however, facilitates a more comprehensive exploration of potential regulatory networkswithin organisms, thereby providing deeper insights into biological mechanisms [25,26].For instance, an approach that combined transcriptomic and metabolomic analyses wasused to reveal the complex metabolic genes involved in the biosynthesis of diterpenoidsin different organs of the Anoectochilus Roxburghii [27], transcriptomic and metabolomicanalyses have been used to reveal the relationship between metabolic processes and peelcolor in melons [28] and to elucidate flavonoid biosynthesis pathways in Areca Catechu [29].Glycosylation plays a crucial role in the biosynthesis of secondary metabolites byenhancing their water solubility, chemical stability, and influencing their bioactivity andintracellular distribution. UGTs are key enzymes mediating this process, catalyzing thetransfer of sugar moieties from donor molecules to acceptor compounds (e.g., flavonoids),thereby modulating their functional properties. In P. quinquefolius, flavonoids primarily existin glycosylated forms, which enhances their solubility and stability within the plant [30].Flavonoid glycosyltransferases responsible for these modifications remain unidentified inP. quinquefolius. In flavonoids, glycosylation typically occurs at hydroxyl groups at positions3, 5, 7, 3′, 4′, and 5′ [31]. Specific glycosyltransferases catalyze the formation of distinctflavonoid monoglycosides. For example, the pomegranate glycosyltransferase PgUGT95B2specifically glycosylates tricetin, a flavone with a trihydroxylated B-ring, at the 4′-OHposition [32]. Similarly, UGT73AC11, identified in Arabidopsis, transfers glucose or xylose tothe 7-OH or 4′-OH positions of flavonoids, leading to the formation of glycosides [33].Int. J. Mol. Sci. 2025, 26, 2669 3 of 18In this study, transcriptomic and metabolomic analyses were performed on P. quinque-folius roots, leaves, and flowers to elucidate flavonoid biosynthetic pathways. Additionally,key TFs involved in metabolite biosynthesis regulation were identified. Furthermore,specific members of the PqMYBs and PqUGTs families were identified as regulators offlavonoid biosynthesis in P. quinquefolius. These findings provide a foundation for furtherexploration of flavonoid biosynthetic mechanisms and metabolic regulation.2. Results2.1. Tissue-Specific Differential Accumulation of Flavonoid Metabolites in P. quinquefoliusLiquid chromatography-mass spectrometry (UPLC-MS/MS) was used to analyzethe composition of metabolites in the roots, leaves, and flowers of P. quinquefolius. PCAresults showed distinct differences among the three tissues, with each biological replicatedisplaying strong correlation within groups (Figure 1A). Flowers and leaves exhibited moresimilar metabolic profiles, while roots displayed a distinct metabolic pattern. A total of67 differentially accumulated metabolites (DAMs) were identified between flowers androots, with 63 upregulated and 4 downregulated in flowers. Between flowers and leaves,25 DAMs were identified, of which 3 were upregulated and 22 downregulated in flowers.When comparing leaves and roots, 90 DAMs were identified in roots, with 83 upregulatedand 7 downregulated (Figure 1B).Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 3 of 19 positions 3, 5, 7, 3′, 4′, and 5′ [31]. Specific glycosyltransferases catalyze the formation of distinct flavonoid monoglycosides. For example, the pomegranate glycosyltransferase PgUGT95B2 specifically glycosylates tricetin, a flavone with a trihydroxylated B-ring, at the 4′-OH position [32]. Similarly, UGT73AC11, identified in Arabidopsis, transfers glucose or xylose to the 7-OH or 4′-OH positions of flavonoids, leading to the formation of glyco-sides [33]. In this study, transcriptomic and metabolomic analyses were performed on P. quin-quefolius roots, leaves, and flowers to elucidate flavonoid biosynthetic pathways. Addi-tionally, key TFs involved in metabolite biosynthesis regulation were identified. Further-more, specific members of the PqMYBs and PqUGTs families were identified as regulators of flavonoid biosynthesis in P. quinquefolius. These findings provide a foundation for fur-ther exploration of flavonoid biosynthetic mechanisms and metabolic regulation. 2. Results 2.1. Tissue-Specific Differential Accumulation of Flavonoid Metabolites in P. quinquefolius Liquid chromatography-mass spectrometry (UPLC-MS/MS) was used to analyze the composition of metabolites in the roots, leaves, and flowers of P. quinquefolius. PCA results showed distinct differences among the three tissues, with each biological replicate dis-playing strong correlation within groups (Figure 1A). Flowers and leaves exhibited more similar metabolic profiles, while roots displayed a distinct metabolic pattern. A total of 67 differentially accumulated metabolites (DAMs) were id ntified b tween flowe s and roots, with 63 upregulated and 4 d wnregulated in flowers. Between flowers and leaves, 25 DAMs were identified, of which 3 were upregulated and 22 ownregulated in flowers. When comparing leaves and roots, 90 DAMs were identified in roots, with 83 upregulated and 7 downregulated (Figure 1B). Figure 1. Analysis of Metabolite Accumulation Characteristics Across Three Tissues of P. quinquefo-lius (A) PCA of flavonoid metabolites in different tissues of P. quinquefolius. (B) Bar chart displaying the number of differentially accumulated metabolites among the tissues. (C) Clustering heatmap of Figure 1. Analysis of Metabolite Accumulation Characteristics Across Three Tissues of P. quinquefolius(A) PCA of flavonoid metabolites in different tissues of P. quinquefolius. (B) Bar chart displayingthe number of differentially accumulate metabolites am ng the tiss s. (C) Cluste ing heatmapof flavonoid metabolites in different tissues of P. quinquefolius. Red indicates high abundance ofmetabolites, while blue represents relatively low abundance.A total of 141 flavonoid metabolites were identified across the roots, leaves, and flow-ers, including 47 flavones, 31 flavonols, 22 C-glycosylflavones, 17 flavanones, 13 isoflavones,and 11 anthocyanins (Table S2). Clustering analysis of the accumulation of six types offlavonoid metabolites across different tissues (Figure 1C, The respective clustering analysisInt. J. Mol. Sci. 2025, 26, 2669 4 of 18plots are shown in Figures S1–S6.) revealed significant differences in flavonoid metabolitelevels between leaves and roots. Specifically, flavonols and anthocyanins were highlyenriched in flowers, while flavones and C-glycosylflavones accumulated predominantly inleaves. In contrast, the roots exhibited a relatively low accumulation of flavonoids, with theexception of specific compounds such as tricin 7-O-acetylglucoside. The accumulation offlavonoid metabolites in the underground part (roots) was markedly lower than that in theaboveground part, with only a few flavonoid compounds exhibiting higher accumulationin the roots, which may be associated with the growth and development characteristics ofthe roots.2.2. Identification of Differentially Expressed Genes (DEGs) and Transcriptomic Analysis AmongThree TissuesTo further investigate the molecular basis of flavonoid biosynthesis, transcriptomeanalysis was conducted to identify DEGs across roots, leaves, and flowers of P. quinquefolius.The number of DEGs among these three tissues is presented in Figure 2A. In the flower vs.root comparison, 8402 genes were downregulated and 8202 were upregulated in flowersrelative to roots. In the leaf vs. root comparison, 8271 DEGs showed higher expressionin leaves than in roots, while 7280 DEGs had lower expression. In the flower vs. leafcomparison, 6307 genes were upregulated and 7594 were downregulated in flowers.Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 4 of 19 flavonoid metabolites in different tissues of P. quinquefolius. Red indicates high abundance of me-tabolites, while blue represents relatively low abundance. A total of 141 flavonoid metabolites were identified across the roots, leaves, and flow-ers, including 47 flavones, 31 flavonols, 22 C-glycosylflavones, 17 flavanones, 13 isofla-vones, and 11 anthocyanins (Table S2). Clustering analysis of the accumulation of six types of flavonoid metabolites across different tissues (Figure 1C, The respective clustering anal-ysis plots are shown in Figures S1–S6.) revealed significant differences in flavonoid me-tabolite levels between leaves and roots. Specifically, flavonols and anthocyanins were highly enriched in flowers, while flavones and C-glycosylflavones accumulated predom-inantly in leaves. In contrast, the roots exhibited a relatively low accumulation of flavo-noids, with the exception of specific compounds such as tricin 7-O-acetylglucoside. The accumulation of flavonoid metabolites in the underground part (roots) was markedly lower than that in the aboveground part, with only a few flavonoid compounds exhibiting higher accumulation in the roots, which may be associated with the growth and develop-ment characteristics of the roots. 2.2. Identification of Differentially Expressed Genes (DEGs) a d Transcriptomic A alysis Among Three Tissues To further investigate the molecular basis of flavonoid biosynthesis, transcriptome analysis was conducted to identify DEGs across roots, leaves, and flowers of P. quinquefo-lius. The number of DEGs among these three tissues is presented in Figure 2A. In the flower vs. root comparison, 8402 genes were downregulated and 8202 were upregulated in flowers relative to roots. In the leaf vs. root comparison, 8271 DEGs showed higher expression in leaves than in oots, while 7280 DEGs had lower expression. In the flower vs. leaf c mparison, 6307 genes were upregulated and 7594 were downregulated in flow-ers. Figure 2. Analysis of DEGs Among Three Tissues of P. quinquefolius (A) Volcano plots of DEGs incomparisons between Flower_vs._Root, Flower_vs._Leaf, and Leaf_vs._Root. (B) GO annotation ofDEGs. (C) KEGG enrichment analysis of DEGs.To understand their biological functions and gene interactions, DEGs involved inmetabolic pathways across different tissues were identified. Pairwise comparisons wereconducted between the roots, flowers, and leaves of Panax quinquefolius, classifying DEGsinto 30 functional groups. The three main categories of Gene Ontology (GO) terms areInt. J. Mol. Sci. 2025, 26, 2669 5 of 18“Biological Process”, “Cellular Component”, and “Molecular Function” (Figure 2B). Inthe “Biological Process” category for the flower vs. leaf (F_vs._L) comparison, geneswere highly enriched in “plastid organization” and “plant-type cell wall organization orbiogenesis”. For the “Cellular Component” category, the most enriched term was “anchoredcomponent of membrane”. Within “Molecular Function”, “oxidoreductase activity, actingon paired donors, with incorporation or reduction of molecular oxygen” was the mostabundant term. In the flower vs. root (F_vs._R) comparison, “secondary metabolitebiosynthetic process” was the most enriched term in “Biological Process”. The “CellularComponent” category showed significant enrichment in “plant-type cell wall” and “integralcomponent of plasma membrane”. The “Molecular Function” terms with the highestenrichment were “oxidoreductase activity, acting on paired donors, with incorporation orreduction of molecular oxygen” and “one donor and incorporation of one atom of oxygenanion transmembrane transporter activity”. For the leaf vs. root (L_vs._R) comparison,“plastid organization” and “secondary metabolic biosynthetic process” were the mostenriched terms within “Biological Process”, while “oxidoreductase activity, acting on paireddonors, with incorporation of reduction of molecular oxygen” and “anion transmembranetransporter activity” were prominent in “Molecular Function”.To further analyze the functions of DEGs across different tissues of P. quinquefolius,Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed(Figure 2C). The results indicated that among all DEGs, the pathway with the highestenrichment was “phenylpropanoid biosynthesis”, a metabolic activity related to plantdevelopment, with the most prominent enrichment observed in the flower vs. root (F_vs._R)comparison. Additionally, GO enrichment analysis showed that DEGs were primarilyenriched in the “secondary metabolic process”. These findings suggest that these DEGs aremainly involved in the biosynthesis and metabolism of secondary metabolites, potentiallyregulating the synthesis of flavonoid compounds in P. quinquefolius.2.3. Validation of DEGs in Flavonoid Biosynthesis of Panax quinquefolius by qRT-PCRTo validate the transcriptome data, qRT-PCR analysis was performed on DEGs relatedto flavonoid biosynthesis across three tissues of P. quinquefolius. As shown in Figure 3, theexpression trends of all selected genes were consistent between qRT-PCR and RNA-seqdata (Figure S7), thereby confirming the reliability of the transcriptomic data.Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 6 of 19 Figure 3. qRT-PCR Validation of DEGs randomly selected from three tissues of P. quinquefolius. 2.4. Correlation Analysis of Transcriptomic and Metabolomic Data Correlation analysis was conducted to explore potential regulatory networks be-tween genes and metabolites. Transcriptomic and metabolomic data revealed significant differences in metabolite accumulation and gene expression between the aerial tissues (leaves and flowers) and the underground tissue (roots) of P. quinquefolius. Therefore, tran-scriptomic data from the leaves, representing the aerial parts, and the roots, representing the underground parts, were selected for comparison, with 18,946 highly expressed genes (FPKM > 5) and 141 metabolites analyzed for correlations. As shown in the nine-quadrant plot (Figure 4A), genes and metabolites in the fifth quadrant, representing non-differentially expressed elements, were relatively few. Anal-ysis of the third and seventh quadrants identified 13 DEGs significantly correlated with 27 differentially accumulated metabolites (Table S3). Furthermore, a single metabolite can be regulated by multiple genes, while conversely, a single gene can be regulated by mul-tiple metabolites. Figure 4. Integrated metabolomic and transcriptomic analysis across three tissues of P. quinquefolius, (A) Nine-quadrant plot of genes and metabolites. (B) Correlation analysis of DEGs and DAMs se-lected from the third and seventh quadrants. Based on the results from the third and seventh quadrants, a correlation network analysis was conducted between the identified DEGs and DAMs (Figure 4B), which re-Figure 3. qRT-PCR Validation of DEGs randomly selected from three tissues of P. quinquefolius.Int. J. Mol. Sci. 2025, 26, 2669 6 of 182.4. Correlation Analysis of Transcriptomic and Metabolomic DataCorrelation analysis was conducted to explore potential regulatory networks betweengenes and metabolites. Transcriptomic and metabolomic data revealed significant differ-ences in metabolite accumulation and gene expression between the aerial tissues (leavesand flowers) and the underground tissue (roots) of P. quinquefolius. Therefore, transcrip-tomic data from the leaves, representing the aerial parts, and the roots, representing theunderground parts, were selected for comparison, with 18,946 highly expressed genes(FPKM > 5) and 141 metabolites analyzed for correlations.As shown in the nine-quadrant plot (Figure 4A), genes and metabolites in the fifthquadrant, representing non-differentially expressed elements, were relatively few. Anal-ysis of the third and seventh quadrants identified 13 DEGs significantly correlated with27 differentially accumulated metabolites (Table S3). Furthermore, a single metabolitecan be regulated by multiple genes, while conversely, a single gene can be regulated bymultiple metabolites.Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 6 of 19 Figure 3. qRT-PCR Validation of DEGs randomly selected from three tissues of P. quinquefolius. 2.4. Correlation Analysis of Transcriptomic and Metabolomic Data Correlation analysis was conducted to explore potential regulatory networks be-tween genes and metabolites. Transcriptomic and metabolomic data revealed significant differenc s in metabolite accumulation and gene expression between the aerial tissues (leaves and flowers) and the underground tissue (roots) of P. quinquefolius. Therefore, tran-scriptomic data from the leaves, representing the aerial parts, and the roots, representing the underground parts, were selected for comparison, with 18,946 highly expressed genes (FPKM > 5) and 141 metabolites analyzed for correlations. As shown in the nine-quadrant plot (Figure 4A), genes and metabolites in the fifth quadrant, representing non-differentially expressed elements, were relatively few. Anal-ysis of the third and seventh quadrants identified 13 DEGs significantly correlated with 27 differen ially accumulated met bol t s (Table S3). Furthermore, a sing e metabolite can be regulated by multip e genes, while conversely, a single gene can be regulated by mul-tiple metabolites. Figure 4. Integrated metabolomic and transcriptomic analysis across three tissues of P. quinquefolius, (A) Nine-quadrant plot of genes and metabolites. (B) Correlation analysis of DEGs and DAMs se-lected from the third and seventh quadrants. Based on the results from the third and seventh quadrants, a correlation network analysis was conducted between the identified DEGs and DAMs (Figure 4B), which re-Figure 4. Integrated metabolomic and transcriptomic analysis across three tissues of P. quinquefolius,(A) Nine-quadrant plot of genes and metabolites. (B) Correlation analysis of DEGs and DAMsselected from the third and seventh quadrants.Based on the results from the third and seventh quadrants, a correlation network analy-sis was conducted between the identified DEGs and DAMs (Figure 4B), which revealed thatthe UDP-glycosyltransferases (UGTs) gene positively regulates the biosynthesis of luteolin3′,7-di-O-glucoside. Among the three tissues of P. quinquefolius, chrysoeriol 7-O-acetyl-hexoside is exclusively present in roots, where it is associated with the regulatory activityof cytochrome CYP (Cytochrome P450) and the transferase HCT (Hydroxycinnamate: CoAligase). The metabolites isoschaftoside, Limocitrin O-hexoside, Apigenin O-hexosyl-O-pentoside, Apigenin O-malonylhexoside, Tricin di-O-hexoside, Tricin 5-O-feruloylhexoside,Luteolin O-hexosyl-O-hexosyl-O-hexoside, Chrysoeriol O-hexosyl-O-pentoside, Chryso-eriol, Apigenin, Apigenin 4-O-rhamnoside, Luteolin 7-O-glucoside, Tricetin and Butinaccumulated in leaves and flowers but were absent in roots. Other flavonoid metaboliteswere detected in roots, leaves, and flowers, although their accumulation in roots wasconsistently lower than in the aerial parts. This suggests that the high expression levels ofthese genes in leaves and flowers may contribute to the increased metabolite accumulationin these tissues, resulting in a greater diversity and concentration of flavonoids in the aerialparts compared to the roots. These findings provide new insights into the biosyntheticmechanisms of flavonoid compounds in different tissues of P. quinquefolius.Int. J. Mol. Sci. 2025, 26, 2669 7 of 182.5. Changes in Genes and Metabolites in the Flavonoid Biosynthesis Regulatory NetworkTo analyze the differences in flavonoid compounds across different tissues of P. quinque-folius, metabolomic and transcriptomic data were integrated. A pathway map for flavonoidbiosynthesis was constructed based on the detected metabolites (Figure 5), and DEGsinvolved in flavonoid biosynthesis, including Cinnamate-4-hydroxylase (C4H), ChalconeSynthase (CHS), Chalcone Isomerase (CHI), Flavanone 3-Hydroxylase (F3H), CYP, UGT,and Flavonol Synthase (FLS), were identified (Table S4). Apigenin, luteolin, and isovitexinaccumulated at higher levels in flowers and leaves than in roots. Their corresponding gly-cone also showed lower accumulation in roots compared to leaves and flowers, correlatingwith the higher expression levels of UGTs in the aerial tissues. Among the 11 flavonoidcompounds derived from tricin through UGT activity, only tricin 7-O-acetylglucosideaccumulated at higher levels in roots than in leaves and flowers, and its correspondingUGT (Pq0G410310.1) showed the highest expression in roots. The remaining flavonoidcompounds exhibited higher accumulation in leaves and flowers, which may explainthe overall higher flavonoid content in these tissues. CHIs (Pq0G103550.1, Pq0G177470.1,Pq0G640290.1) and its downstream genes F3H (Pq0G325260.1) and FLS (Pq0G614560.1)had the highest expression in flowers. In contrast, C4Hs (Pq0G238520.1, Pq0G244320.1,Pq0G244740.1) and all annotated pathway genes of the CYPs family exhibited distinctexpression patterns from other genes in this pathway, with the highest expression levelsobserved in the roots. The distribution patterns of differentially accumulated flavonoidcompounds were consistent with the expression patterns of the related genes. For example,FLS (Pq0G614560.1) exhibited higher expression levels in the aerial parts than in the un-derground parts, and its downstream metabolites, such as quercetin and kaempferol, alsoaccumulated at higher levels in the aerial parts than in the underground parts.Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 8 of 19 Figure 5. Flavonoid biosynthesis pathway in P. quinquefolius. Square heatmaps represent the expres-sion levels of differentially expressed key enzyme genes across different tissues, while circular heatmaps represent the accumulation levels of differentially accumulated flavonoid metabolites in various tissues. These results indicate the existence of a complex regulatory network linking the ac-cumulation of secondary metabolites and gene expression levels. Notably, key flavonoid biosynthetic genes, including PAL, CHS, and FLS, exhibited significantly higher expres-sion levels in flowers and leaves compared to roots, aligning with the metabolic differ-ences observed among these three tissues. Furthermore, comparative analysis with other Panax species, such as Panax ginseng and Panax notoginseng, revealed that while P. quin-quefolius shares conserved regulatory mechanisms in flavonoid biosynthesis, it also exhib-its species-specific regulatory characteristics. This finding underscores the necessity for further investigation. Therefore, future studies should focus on the functional validation of these key genes to elucidate their precise roles in flavonoid biosynthesis in P. quinque-folius. 2.6. Functional Validation of Flavonoid-Related TFs and UGTs in P. quinquefolius A correlation analysis was conducted using 26 highly expressed PqUGTs and 13 PqMYBs identified in the flavonoid biosynthetic pathway (Table S5, Figure 6A). To further select candidate UGTs, a phylogenetic tree was constructed based on previously reported flavonoid UGTs and PqUGTs with high degree values from the correlation analysis (Fig-ure S9). This analysis revealed clustering of certain glycosyltransferases from P. quinque-folius with those of other species (Figure 6B). Figure 5. Flavonoid biosynthesis pathway in P. quinquefolius. Square heatmaps represent the ex-pression levels of differentially expressed k y enzym genes across diff rent tissues, while circularheatmaps represent the a cumulation levels of differentially a cumulated flavonoid metabolites invarious tissues.Int. J. Mol. Sci. 2025, 26, 2669 8 of 18These results indicate the existence of a complex regulatory network linking the ac-cumulation of secondary metabolites and gene expression levels. Notably, key flavonoidbiosynthetic genes, including PAL, CHS, and FLS, exhibited significantly higher expressionlevels in flowers and leaves compared to roots, aligning with the metabolic differencesobserved among these three tissues. Furthermore, comparative analysis with other Panaxspecies, such as Panax ginseng and Panax notoginseng, revealed that while P. quinquefoliusshares conserved regulatory mechanisms in flavonoid biosynthesis, it also exhibits species-specific regulatory characteristics. This finding underscores the necessity for further inves-tigation. Therefore, future studies should focus on the functional validation of these keygenes to elucidate their precise roles in flavonoid biosynthesis in P. quinquefolius.2.6. Functional Validation of Flavonoid-Related TFs and UGTs in P. quinquefoliusA correlation analysis was conducted using 26 highly expressed PqUGTs and13 PqMYBs identified in the flavonoid biosynthetic pathway (Table S5, Figure 6A). Tofurther select candidate UGTs, a phylogenetic tree was constructed based on previouslyreported flavonoid UGTs and PqUGTs with high degree values from the correlation anal-ysis (Figure S9). This analysis revealed clustering of certain glycosyltransferases fromP. quinquefolius with those of other species (Figure 6B).To confirm the functional role of these glycosyltransferases in flavonoid glycosylation,five candidate genes were selected for in vitro validation (Table S6). The gene sequences ofthe five PqUGTs are shown in Table S7. Prokaryotic expression vectors were constructed,and recombinant proteins were expressed and purified. The concentrated crude proteinswere subsequently tested for in vitro enzymatic activity using flavonoid substrates. Theresults (Figure 6C,D) indicated that only PqUGT19 could catalyze apigenin and naringeninto form apigenin-7-O-glucoside and naringenin-7-O-glucoside, confirming that PqUGT19functions as a flavonoid glycosyltransferase.To investigate the subcellular localization of PqMYB7, PqMYB9, and PqMYB13, theirencoding genes were fused with the PHB-YFP vector and subsequently transformed into to-bacco leaves. We utilized the Leica STELLARIS 5 laser confocal microscope. In the softwaresettings, we assigned the YFP marker to the green channel to display green fluorescenceduring observation. The merged images display overlay fields: YFP fluorescence imagesunder 514 nm excitation indicate green fluorescent protein expression, while bright-fieldimages reveal cellular structures. The results indicated that, in contrast to the control vector(YFP), which was distributed throughout both the nucleus and cytoplasm, PqMYB7-YFPand PqMYB13-YFP exhibited fluorescence exclusively in the nucleus. In addition, PqMYB9-YFP displayed fluorescence in both the nucleus and cytoplasm. These findings indicatethat PqMYB7 and PqMYB13 are localized in the nucleus, supporting their predicted role asTFs (Figure 6E).The promoter of PqUGT19 was analyzed using the PlantCARE database, which iden-tified cis-elements for MYB TFs, suggesting that PqUGT19 may be regulated by MYBTFs. Promoter-binding elements for MYBs were also identified in the promoter regionof UGT19 (Figure S8). The yeast one-hybrid assay results further indicated that PqMYB7and PqMYB13 can bind to the promoter elements of PqUGT19 (Figure 6F). This observa-tion is consistent with related studies on Citrus aurantium L. (sour orange), where MYBtranscription factors regulate the expression of UGT genes, participate in the biosynthesispathway of flavonoid compounds, and play a key role in the developmental process of sourorange. Taken together, these findings suggest that PqMYB7 and PqMYB13 regulate theexpression of PqUGT19 in P. quinquefolius by binding to its promoter, thereby influencingthe biosynthesis of apigenin-7-O-glucoside and naringenin-7-O-glucoside.Int. J. Mol. Sci. 2025, 26, 2669 9 of 18Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 9 of 19 Figure 6. Functional validation of PqUGTs and PqMYBs in P. quinquefolius. (A) Correlation analysis of 27 highly expressed PqUGTs and 13 PqMYBs. (B) Phylogenetic analysis of the amino acid se-quences of selected PqUGTs and UGT genes from various plant species, with the selected candidate genes highlighted in the blue box. (C) UPLC-MS chromatogram showing the catalytic product of PqUGT19 with apigenin as the substrate. (D) UPLC-MS chromatogram showing the catalytic prod-uct of PqUGT19 with naringenin as the substrate. (E) Subcellular localization of PqMYB7, PqMYB9, and PqMYB13 in vivo. (F) Yeast one-hybrid assay demonstrating the interaction of PqMYB7 and PqMYB13 with the PqUGT19 promoter. To confirm the functional role of these glycosyltransferases in flavonoid glycosyla-tion, five candidate genes were selected for in vitro validation (Table S6). The gene se-quences of the five PqUGTs are shown in Table S7. Prokaryotic expression vectors were constructed, and recombinant proteins were expressed and purified. The concentrated crude proteins were subsequently tested for in vitro enzymatic activity using flavonoid substrates. The results (Figure 6C,D) indicated that only PqUGT19 could catalyze apigenin and naringenin to form apigenin-7-O-glucoside and naringenin-7-O-glucoside, confirm-ing that PqUGT19 functions as a flavonoid glycosyltransferase. To investigate the subcellular localization of PqMYB7, PqMYB9, and PqMYB13, their encoding genes were fused with the PHB-YFP vector and subsequently transformed into tobacco leaves. We utilized the Leica STELLARIS 5 laser confocal microscope. In the soft-ware settings, we assigned the YFP marker to the green channel to display green fluores-cence during observation. The merged images display overlay fields: YFP fluorescence Figure 6. Functional validation of PqUGTs and PqMYBs in P. quinquefolius. (A) Correlation analysis of27 highly expressed PqUGTs and 13 PqMYBs. (B) Phylogenetic analysis of the amino acid sequencesof s lected PqUGTs and UGT genes fr m various pl nt species, with the selected candidate geneshighlighted in the blue box. (C) UPLC-MS chromatogram showing the catalytic product of PqUGT19with apigenin as the substrate. (D) UPLC-MS chromatogram showing the catalytic product ofPqUGT19 with naringenin as the substrate. (E) Subcellular localization of PqMYB7, PqMYB9, andPqMYB13 in vivo. (F) Yeast one-hybrid assay demonstrating the interaction of PqMYB7 and PqMYB13with the PqUGT19 promoter.3. DiscussionP. quinquefolius harbors a diverse repertoire of metabolites and an intricate gene ex-pression regula ory network, with a distinct regulatory emphasis on flavonoid biosynthe-sis. To elucidate the factors contributing to variations in gene expression and metaboliteaccumulation within the flavonoid biosynthetic pathway, we analyzed gene-metabolitecorrelations in distinct tissues of P. quinquefolius. Given that gene expression is influencedInt. J. Mol. Sci. 2025, 26, 2669 10 of 18by various internal and external factors [34], transcriptome analysis alone is insufficientto fully elucidate the patterns of metabolite accumulation [35–37]. As the end products ofbiosynthetic processes, metabolites are regulated by numerous endogenous and exogenousfactors [38]. Therefore, integrating transcriptomic and metabolomic approaches can aidin identifying key functional genes involved in critical metabolic pathways or molecularmechanisms [39,40]. For example, combined transcriptomic and metabolomic analyseshave successfully identified key genes in flavonoid biosynthesis in blackberry [41] andcandidate genes involved in phenolic acid biosynthesis in Cyclocarya paliurus leaves [42].3.1. Transcriptome Analysis and qRT-PCR ValidationIn this study, transcriptome analysis was initially conducted to identify DEGs indifferent tissues of P. quinquefolius. The reliability of the transcriptomic data was verifiedthrough qRT-PCR. A total of 15,551 DEGs were identified in the root vs. leaf comparison,while 16,604 DEGs were detected in the root vs. flower comparison. P. quinquefoliusexhibited a significantly higher number of DEGs compared to other plant species, such asTartary buckwheat [43] and Camellia lanceoleosa [44]. This difference may be attributed toits polyploid genome, complex secondary metabolic pathways, and distinct physiologicaladaptations between underground (roots) and aerial (leaves and flowers) tissues [45–47].3.2. Metabolite ProfilingFurthermore, metabolomic analysis quantified metabolites in roots, flowers, and leavesof P. quinquefolius [48,49]. Metabolite profiling demonstrated tissue-specific accumulationpatterns, with most flavonoid compounds exhibiting higher concentrations in leaves andflowers than in roots. A limited number of flavonoids, however, accumulated at higherlevels in roots.3.3. Flavonoid Biosynthesis Pathway and Tissue-Specific AccumulationBy integrating metabolomic and transcriptomic data, this study examined the dif-ferences in flavonoid content and gene expression patterns across various tissues ofP. quinquefolius. Based on the detected differentially accumulated metabolites and DEGs, aflavonoid biosynthesis pathway map was constructed, identifying 13 types key DEGs inthis pathway, including OMTs, CHIs, HCTs, HIDHs, UGTs, CHRs, CHSs, and CYPs. Amongthese pathway genes, UGT (Pq0G410310.1) expression was significantly higher in leavesand flowers, which may explain the elevated accumulation of flavonoid glycosides inthese tissues. The distribution pattern of flavonoid compounds across different tissues wasconsistent with the expression patterns of the related genes. This result aligns with previousfindings [50], which demonstrated that flavonoid compounds are widely distributed invarious plant tissues, with significantly higher accumulation in the aerial parts (particu-larly leaves and flowers) compared to roots in most species. Based on these findings, itis hypothesized that the differential expression of flavonoid-modifying genes may play acrucial role in the tissue-specific accumulation patterns of these metabolites. Flavonoidcompounds have been shown to effectively prevent and reduce potential root damage inplants caused by various diseases and pests [51,52]. Studies indicate that isoflavonoids inpea roots, such as biochanin A, maackiain, and genistein, exhibit antifungal activity againstsoil fungi in the pea rhizosphere and possess antioxidant properties [53]. This may explainthe high concentrations of certain flavonoid compounds in roots.3.4. Regulation of Flavonoid Biosynthesis by TFsAdditionally, these tissue-specific metabolite variations may be driven by the reg-ulation of TFs and structural genes. The biosynthesis of flavonoids involves a complextranscriptional regulatory network composed of multiple TFs, which can bind to genesInt. J. Mol. Sci. 2025, 26, 2669 11 of 18encoding key enzymes in the flavonoid biosynthetic pathway, thereby influencing theentire pathway. The MYB TFs family plays a crucial regulatory role in the biosynthesis offlavonoid compounds, primarily controlling the biosynthesis of flavonoids, cellulose, andlignin, as well as the formation of secondary cell walls in plants [54]. MYB TFs can directlyinfluence the expression of key flavonoid genes or modulate the process by interactingwith other TFs. For example, in A. thaliana, MYB111 positively regulates the expressionof CHS, F3H, and FLS1 genes, enhancing the plant’s salt stress tolerance [55]. In Panaxginseng, the R2R3-MYB transcription factor PgTT2 regulates proanthocyanidin biosynthesisthrough interaction with the PgTTG1 protein [56]. In Scutellaria baicalensis, SbMYB3 regu-lates flavone synthase II-2 (SbFNSII-2), thereby increasing the levels of baicalin, baicalein,and wogonin [57]. MYB TFs modulate the accumulation of flavonoids in Panax notoginsengby regulating the expression of genes involved in flavonoid biosynthesis [58].3.5. Glycosylation of Flavonoids by UGTsThe glycosylation of flavonoids is primarily catalyzed by UGTs, with flavonoids inplants typically existing in glycoside forms. Previous studies have demonstrated thatcelery UGT94AX1 can catalyze the biosynthesis of the flavone glycoside apiin [59]. In thisstudy, the PqUGTs identified through screening were found, by in vitro assays, to catalyzethe glycosylation of quercetin and luteolin at the 7-OH position. BLAST, version 2.12.0.https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 25 February 2025) analysis revealedthat PqUGT19 exhibits high sequence similarity with members of the UGT71A family. Thisfamily is known to participate in the glycosylation of flavonoid compounds. For example,a novel 7-O-glycosyltransferase CmGT from Cucurbita moschata was cloned, which couldefficiently convert luteolin into luteolin-7-O-glucoside under optimal conditions [60]. Thisenzyme family is involved in the glycosylation of flavonoid compounds, playing a criticalrole in plant defense, hormone regulation, and the biosynthesis of secondary metabolites,which enables plants to adapt to fluctuating environmental conditions [61]. It has beenextensively characterized in plants such as A. thaliana [62] and Panax notoginseng [63].Consequently, PqUGT19 was classified as PqUGT71A1.3.6. Regulatory Network of UGTs and MYB Transcription Factors in Flavonoid AccumulationUGTs and MYB transcription factors not only function independently but may alsowork together through a complex regulatory network to regulate the accumulation offlavonoid compounds in different plant tissues. The role of the MYB-UGT regulatoryaxis in the biosynthesis of flavonoid compounds in P. quinquefolius is shown in Figure 7.Additionally, in blueberries (Vaccinium spp.), VcMYB forms a regulatory network withabscisic acid response element binding factors, bHLH, and flavonoid biosynthetic genes tocontrol the accumulation of flavonoid metabolites in drought-induced blueberry leaves [64].Therefore, the flavonoid accumulation patterns in the roots and leaves of P. quinquefoliusmay be coordinately regulated by the expression patterns of MYB transcription factorsand UGT genes. MYB TFs regulate the expression of UGT genes, thereby affecting thedistribution of flavonoid compounds in different plant tissues. Furthermore, in our pre-vious study [65], some R2R3-MYB TFs were hypothesized to be involved in flavonoidbiosynthesis in P. quinquefolius. In this study, PqMYB7 and PqMYB13 from the C17 subfam-ily of the previously identified MYB family were further studied. Validation experimentsrevealed that these TFs can regulate the expression of PqUGT71A1, which is associatedwith corresponding changes in flavonoid content across the roots, leaves, and flowers ofP. quinquefolius. The functional characterization of UGTs and TFs in this study provides afoundation for further research into the regulatory mechanisms of flavonoid biosynthesisin P. quinquefolius.Int. J. Mol. Sci. 2025, 26, 2669 12 of 18Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 12 of 19 which enables plants to adapt to fluctuating environmental conditions [61]. It has been extensively characterized in plants such as A. thaliana [62] and Panax notoginseng [63]. Con-sequently, PqUGT19 was classified as PqUGT71A1. 3.6. Regulatory Network of UGTs and MYB Transcription Factors in Flavonoid Accumulation UGTs and MYB transcription factors not only function independently but may also work together through a complex regulatory network to regulate the accumulation of fla-vonoid compounds in different plant tissues. The role of the MYB-UGT regulatory axis in the biosynthesis of flavonoid compounds in P. quinquefolius is shown in Figure 7. Addi-tionally, in blueberries (Vaccinium spp.), VcMYB forms a regulatory network with abscisic acid response element binding factors, bHLH, and flavonoid biosynthetic genes to control the accumulation of flavonoid metabolites in drought-induced blueberry leaves [64]. Therefore, the flavonoid accumulation patterns in the roots and leaves of P. quinquefolius may be coordinately regulated by the expression patterns of MYB transcription factors and UGT genes. MYB TFs regulate the expression of UGT genes, thereby affecting the distribution of flavonoid compounds in different plant tissues. Furthermore, in our previ-ous study [65], some R2R3-MYB TFs were hypothesized to be involved in flavonoid bio-synthesis in P. quinquefolius. In this study, PqMYB7 and PqMYB13 from the C17 subfamily of the previously identified MYB family were further studied. Validation experiments re-vealed that these TFs can regulate the expression of PqUGT71A1, which is associated with corresponding changes in flavonoid content across the roots, leaves, and flowers of P. quinquefolius. The functional characterization of UGTs and TFs in this study provides a foundation for further research into the regulatory mechanisms of flavonoid biosynthesis in P. quinquefolius. Figure 7. Schematic representation of the MYB-UGT regulatory axis in flavonoid biosynthesis inP. quinquefolius.4. Materials and Methods4.1. Plant Materials and Sample CollectionTo perform transcriptomic and metabolomic analyses of P. quinquefolius roots, leaves,and flowers. The P. quinquefolius plant material used in this study was collected fromWanliang Township, Fusong County (42◦26′9.22′′ N, 127◦17′21.00′′ E) on 9 June 2018, aperiod characterized by stable plant growth. The specimens were identified as 4-year-oldP. quinquefolius by Professor Tian Yixin from Jilin Agricultural University. Three plants ofsimilar size and growth status were selected for sampling. From each plant, the main roots(2 cm below the rhizome), leaves, and flower buds were thoroughly washed, cut into smallpieces, flash-frozen in liquid nitrogen, and divided into two aliquots for storage at −80 ◦Cuntil further analysis.4.2. Sample Preparation and ExtractionTo extract flavonoid compounds for UPLC-MS/MS analysis. Samples (100 mg each)were freeze-dried using a vacuum freeze dryer. The dried samples were then groundusing a bead mill (MM400, RETSCH) at 30 Hz for 1.5 min. The powdered samples weresuspended in 70% methanol (1000 µL) and stored at 4 ◦C overnight. During extraction, thesamples were vortexed three times and centrifuged at 4 ◦C at 10,000× g for 15 min. Thesupernatant was then filtered through a 0.22 µm pore size filter membrane (SCAA-104,ANPEL, Shanghai, China) and used for UPLC-MS/MS analysis.Int. J. Mol. Sci. 2025, 26, 2669 13 of 184.3. UPLC-MS/MS Determination of Flavonoid CompoundsTo detect flavonoid compounds in P. quinquefolius, the filtered supernatant from thesample extraction was analyzed using a UPLC-MS/MS system (HPLC: Shim-pack UFLCSHIMADZU CBM30A; MS: Applied Biosystems 6500 QTRAP). The liquid chromatographyconditions were as follows: (i) Column: Waters ACQUITY UPLC HSS T3 C18, 1.8 µm,2.1 mm × 100 mm; (ii) Mobile phases: ultrapure water with 0.04% acetic acid as the aque-ous phase, and acetonitrile with 0.04% acetic acid as the organic phase; (iii) Elution gradient:0 min, water/acetonitrile (95:5, v/v); 11.0 min, 5:95 (v/v); 12.0 min, 5:95 (v/v); 12.1 min,95:5 (v/v); 15.0 min, 95:5 (v/v); (iv) Flow rate: 0.4 mL/min; column temperature: 40 ◦C;injection volume: 5 µL.4.4. Identification and Quantification of Flavonoid MetabolitesTo analyze the accumulation differences of flavonoid metabolites between differenttissues of P. quinquefolius, the acquired mass spectrometry data were processed usingAnalyst software for quality control of pooled samples. Qualitative analysis of the widelytargeted metabolomics results was performed based on public databases, a custom-builtdatabase (MWDB, Metware Database), and commercially purchased standards. Peakdetection and annotation were conducted using in-house R scripts and databases. Variableimportance in projection (VIP) values of the first principal component in OPLS-DA analysiswere obtained to visualize group separation and identify metabolites with significantchanges. The VIP values summarize each variable’s contribution to the model, withVIP > 1, fold change ≥ 2 or ≤0.5, and p < 0.05 indicating DAMs.4.5. RNA Extraction and Transcriptome SequencingTo analyze the transcriptome data differences between different tissues of P. quin-quefolius. Total RNA was extracted from the nine above mentioned samples using theRNeasy Plant Mini Kit (Qiagen, Germany), following the manufacturer’s instructions.RNA quality was assessed prior to library construction, which was performed usingsynthesis-based techniques to create complementary DNA (cDNA) libraries. Sequenc-ing was conducted on the PacBio Sequel and Illumina HiSeq 2000 high-throughputplatforms. The clean reads we reported previously [66] were aligned to the P. quin-quefolius genome [67] using the workflow as reported [68]. Gene expression quantifi-cation was based on FPKM values (Fragments Per Kilobase of transcript per Millionmapped reads). Differential expression analysis was conducted using the DESeq R, version1.30.1 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html accessed on25 February 2025) package. Genes with adjusted p < 0.05 were considered significantlydifferentially expressed. Hierarchical clustering analysis was performed on DEGs to assessgene expression patterns. GO enrichment and KEGG pathway enrichment analyses ofdifferentially expressed genes were conducted using R with a hypergeometric test.4.6. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) AnalysisTo validate the reliability of the transcriptomic data, twelve flavonoid-related DEGswere selected for qRT-PCR. The primers used were synthesized by Sangon Biotech(Shanghai, China) and their sequences are listed in Table S1. Total RNA was extracted fromthe samples using Esippur Plana Kit (Transon Bioteh, Beijing, China) and Renas-Friednasyi(Transon Bioteh) to eliminate DNA contamination. The concentration and quality ofthe RNA samples were assessed using a NanoPhotometer N50 (Implen, GER, München,Germany). Subsequently, the Perfect-Start Uni qRT-PCR Kit (TransGen Biotech) was usedto reverse transcribe RNA into cDNA. Two-step qRT-PCR kit was used for analysis, and thereaction procedure is as follows: predenaturation 94 ◦C, 30 s; denature 94 ◦C, 5 s; annealingInt. J. Mol. Sci. 2025, 26, 2669 14 of 18temperature 60 ◦C, 30 s; 45 cycles. The reaction system is 20 µL: 2 × PerfectStart® GreenqPCR SuperMix 10 µL, F, R primers all 1 µL, cDNA 1 µL, filled to 20 µL with ddH2O. TheP. quinquefolius GAPDH gene was used as the internal reference. The expression level of thecorresponding genes was calculated using the 2−∆∆CT method.4.7. Correlation Analysis of Transcriptome and MetabolomeThrough the integrated analysis of transcriptomic and metabolomic data, key PqMYBsand PqUGTs involved in the regulation of flavonoid biosynthesis were identified. Functionalvalidation experiments, such as subcellular localization and yeast one-hybrid assays, weresubsequently performed to further investigate the interaction between PqMYBs and PqUGTsand their roles in the regulation of flavonoid biosynthesis in P. quinquefolius.The Pearson correlation coefficient test was used to calculate the correlation betweenDEGs and DAMs related to the flavonoid biosynthesis pathway in P. quinquefolius. APearson correlation coefficient (PCC) ≥ 0.9 and p-value ≤ 0.05 were selected as thresholds,and results were visualized using nine-quadrant plots. Key genes and metabolites wereidentified based on PCC > 0.9 and |log2(FC)| > 2 through correlation network analysis.The subcellular localization method was as follows: The selected PqMYBs were in-serted into the PHB-YFP vector. The correctly sequenced PqMYB-YFP recombinant plasmidwas then transferred into the Agrobacterium tumefaciens strain GV 3101. The GV 3101strain containing the recombinant plasmid was used to infect tobacco leaves, with theempty PHB-YFP vector serving as the control. The samples were observed using the LeicaSTELLARIS 5 laser confocal microscope.The yeast one-hybrid assay was conducted to verify the DNA binding of PqMYBs tothe promoter regions of PqUGTs by constructing the PqMYB-pGADT7 and MYB-bindingsite-p53-His2 vectors. First, the pGADT7 vector was digested with EcoR I and BamH I, andthe PqMYBs fragment was recombined with the vector and transformed into Escherichia coli.Correct recombinant plasmids were selected and verified. Next, a DNA fragment containingMYB-binding sites was synthesized and ligated into the p53-His2 vector, followed bysequencing verification. Finally, the constructed plasmids were co-transformed into yeastcompetent cells. After culturing and colorimetric reaction, the interaction between PqMYBsand the promoter regions of PqUGTs was validated.5. ConclusionsBy integrating transcriptomic and metabolomic analyses, this study systematicallyelucidates the tissue-specific accumulation patterns of flavonoids in P. quinquefolius andtheir underlying molecular regulatory networks. A total of 141 flavonoid metabolites wereidentified in P. quinquefolius for the first time, revealing significant enrichment of flavones,flavonols, and C-glycosylflavones in aerial tissues (leaves and flowers). Additionally, adistinct tissue-specific accumulation pattern was observed for certain glycosylated com-pounds, such as tricin 7-O-acetylglucoside, which was predominantly accumulated inroots. Transcriptomic analysis identified 15,551–18,946 DEGs, highlighting the phenyl-propanoid biosynthetic pathway as a key regulatory hub and uncovering a coordinatedinteraction between UGTs and MYB TFs in flavonoid glycosylation through co-expressionnetwork analysis.Functional validation experiments demonstrated, for the first time, that PqUGT71A1catalyzes the glycosylation of apigenin and naringenin at the 7-OH position. Furthermore,PqMYB7 and PqMYB13 were found to directly bind to the promoter region, activatingPqUGT71A1 expression and thereby establishing the MYB-UGT regulatory axis. Thesefindings not only address a critical gap in understanding the spatial dynamics of flavonoidInt. J. Mol. Sci. 2025, 26, 2669 15 of 18metabolism in P. quinquefolius but also provide a novel framework for elucidating thetissue-specific regulation of secondary metabolite biosynthesis in medicinal plants.This study lays a theoretical foundation for metabolic engineering and the optimizationof bioactive compounds in medicinal plants. Targeted regulation of the MYB-UGT axisenables precise modulation of flavonoid biosynthesis in specific tissues of P. quinquefolius.For instance, gene editing or overexpression strategies could be employed to enhance theproduction of high-value flavonoids in aerial tissues or to regulate the accumulation ofroot-specific glycosides for the development of novel medicinal compounds.Moreover, the integrative analytical approach established in this study is broadlyapplicable to other medicinal plants, facilitating the elucidation and utilization of natu-ral product biosynthetic pathways. Future research could further explore the ecologicalfunctions of flavonoids in P. quinquefolius, such as their roles in stress resistance and symbi-otic signaling, through multi-omics approaches to gain deeper insights into the dynamicinterplay between metabolites and gene regulation.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26062669/s1.Author Contributions: Conceptualization, P.D. and Y.W. (Yumeng Wang); methodology, Y.W. (Yu-meng Wang); software, P.D.; validation, Y.W. (Yumeng Wang), Y.L. and J.C. (Jiankang Chen); formalanalysis, Y.W. (Yumeng Wang); investigation, P.W., J.L., J.C. (Junwen Chi) and J.Z. (Jiaxin Zhang); re-sources, Y.W. (Yihan Wang); data curation, Y.W. (Yumeng Wang); writing—original draft preparation,Y.W. (Yumeng Wang); writing—review and editing, J.M. and M.K.D.; visualization, X.P., X.L. andY.X.; supervision, Y.Y.; project administration, J.Z. (Jian Zhang); funding acquisition, P.D. and Y.W.(Yingping Wang). All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the Natural Science Foundation of China (U21A20405)and the National Key Research and Development Program of China (2021YFD1600900), and alsosupported by the 111 Project, Northeast Advantageous Characteristic Resources and Health FoodDiscipline Innovation Introduction Base, Grant No: D23007, the Jilin College Students Innovationand Entrepreneurship Training Program (S202310193106), and the China National University StudentInnovation & EntrepreneurshipDevelopment Program (202410193085).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: Data are contained within the article.Conflicts of Interest: This research was conducted in the absence of any commercial or financialrelationships that could be construed as a potential conflict of interest.References1. Kim, D.H. 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flavonoid biosynthesis
American ginseng
Panax quinquefolius
transcriptomic and metabolomic integration study
Integrated Transcriptomic and Metabolomic Analysis Reveals Tissue-Specific Flavonoid Biosynthesis and MYB-Mediated Regulation of UGT71A1 in Panax quinquefolius
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