Spatiotemporal Distribution Patterns of Osthole and Expression Correlation of the MOT1 Homologue in Cultivated Angelica biserrata

Abstract

Sustainable cultivation of Angelica biserrata, a medicinal species with a bioactive coumarin, Osthole, is hindered by inconsistent metabolite accumulation. To address this limitation, we integrated spatiotemporal metabolomics and transcriptomic analyses. Tissue-specific measurements revealed that root bark accumulates Osthole at 0.30 ± 0.15%, a concentration 11-fold higher compared to root pith and 15–30-fold higher compared to aerial organs. Over time, the Osthole content increased by 195% from September to December, with frost exposure further increasing the accumulation by an additional 170%. Germplasm screening identified an elite accession, AB-222, exhibiting 230% higher Osthole content compared to regional averages. Weighted gene co-expression network analysis identified a gene module strongly correlating with Osthole accumulation. Within this module, AbOMT1 (AB04G05077), an O-METHYLTRANSFERASE 1 (OMT1) homolog encoding an S-adenosyl methionine-dependent O-methyltransferase, was the top hub gene. AbOMT1 expression reflected Osthole dynamics both spatially (three-fold higher in root bark vs. root pith) and temporally. Module functional analysis revealed significant enrichment in phenylpropanoid and monoterpenoid biosynthesis pathways. Our results suggest AbOMT1 as a possible key molecular marker for Osthole accumulation, establish frost induction as a strong yield regulator, and suggest AB-222 as an elite germplasm resource.

1. Introduction

Angelica biserrata (R.H. Shan & Yuan) C.Q. Yuan & R.H. Shan represents a globally significant medicinal plant characterized by its phytochemical complexity, particularly in root tissues where diverse volatile and non-volatile metabolites have been identified. Comprehensive metabolomic analyses have classified its root extracts into 15 distinct chemical categories, with Osthole (7-methoxy-8-isopentenoxycoumarin) serving as a major bioactive constituent underpinning its pharmacological applications [1]. The biogeographical distribution of this species is governed by bioclimatic predictors such as annual precipitation gradients, which critically define regional cultivation suitability [2]. Economically, optimized extraction protocols, most notably solid-phase extraction coupled with HPLC-based quantification, enable cost-efficient Osthole isolation from A. biserrata with batch-to-batch consistency (RSD ≤ 4.6%) and industrial scalability [3,4]. These methodologies achieve exceptional purity yields exceeding 31.1% while demonstrating potent radical scavenging capacities, positioning Osthole as being suitable for applications beyond traditional medicine [4]. Nevertheless, safety considerations necessitate rigorous evaluation due to documented CYP3A4 enzyme inhibition by these extracts, which may influence co-administration with conventional therapeutics [5].

The pharmacological utility of Osthole spans anti-inflammatory, oncologic, neuroregulatory, and osteogenic domains. These effects are mediated through diverse molecular pathways. TNF-α suppression concurrent with α7nAChR upregulation mediates its anti-inflammatory efficacy, substantially ameliorating anxiety-like phenotypes and social withdrawal in experimental models via cholinergic pathway modulation [6,7]. In oncology contexts, Osthole exerts antiproliferative effects primarily through PI3K/AKT/mTOR pathway inhibition in retinoblastoma while demonstrating the capacity to reverse multidrug resistance mechanisms in solid tumors [8,9]. Neuroprotective actions through PPARα agonism ameliorate cognitive deficits, though clinical translation is constrained by limited blood–brain barrier permeability [6,10]. Additionally, osteogenic bioactivity mitigates osteoporosis phenotypes without inducing adverse effects typical of bisphosphonates [11], and promising agrochemical applications have emerged via contact toxicity and oviposition deterrence against major arthropod pests [12,13]. However, despite these therapeutic potentials, persistent spatial heterogeneity in Osthole accumulation (3.6–8.2× higher concentration in phloem versus xylem) compounded by seasonal fluctuations exceeding 300% significantly impairs production efficiency [14,15,16]. Current integrative strategies to address these limitations combine MALDI-MSI-guided harvesting protocols with genomic-based engineering of POLYKETIDE SYNTHASE (PKS) and PRENYLTRANSFERASE (PT) biosynthetic nodes to enhance content uniformity and sustainable resource utilization [17,18].

Despite substantial pharmacological characterization, agricultural implementation of A. biserrata is hampered by systematic research deficiencies regarding its industrial crop physiology. Critical knowledge gaps persist across several interdependent domains. Osthole’s heterogeneous distribution across root phloem versus xylem tissues and its minimal accumulation in leaves remain inadequately quantified, with metabolomic reports confirming 3.6–8.2× concentration gradients that significantly compromise harvesting efficiency [19,20]. Additionally, although developmental variations suggest seasonal fluctuations exceeding 300% in bioactive metabolites, field-validated temporal dynamics across growth cycles lack empirical verification, potentially leading to suboptimal harvest scheduling [7,21]. Furthermore, the molecular machinery governing biosynthesis remains largely unmapped, impeding targeted genetic enhancement [18,22]. Compounding these agricultural constraints, current multidisciplinary research primarily focuses on therapeutic mechanisms rather than production optimization [10,12]. This study addresses these industrial bottlenecks through integrated spatiotemporal metabolomics, transcriptomics, and environmental modeling. Our methodology aims to provide high-resolution mapping of Osthole accumulation patterns to enable precision harvesting while identifying transcriptional regulators of terpenoid/coumarin biosynthesis (e.g., OMT and PT) to advance molecular breeding for yield-stabilized cultivars adapted to specific bioclimatic conditions [2,23,24].

This study addresses critical industrial bottlenecks in A. biserrata cultivation through an integrated approach combining spatiotemporal metabolomics and transcriptomics. Our primary objective is to develop a comprehensive understanding of Osthole distribution and biosynthesis to optimize industrial cultivation practices. To achieve this, we map Osthole accumulation patterns across different plant tissues, characterize temporal metabolite dynamics throughout the growth cycle, identify environmental factors affecting biosynthesis, screen germplasm resources for elite chemotypes, and elucidate the molecular mechanisms governing Osthole production through weighted gene co-expression network analysis. Through these integrated approaches, we aim to establish optimal harvesting protocols and identify key molecular targets for genetic enhancement of this pharmacologically significant species.

2. Results

2.1. Hierarchical Accumulation of Osthole as a Functional Metabolite

To investigate the ecological functions of specialized metabolites, we performed tissue-specific quantification of six bioactive compounds in A. biserrata (Table S1). This analysis revealed Osthole as the most differentially partitioned metabolite across root cortex, root core, petiole, and blade tissues, demonstrating maximal statistical variance (lowest p-value, p = 0.016) and the widest concentration range (MAX-MIN Ratio = 94.7) relative to other compounds via Tukey’s multiple comparison test (Table S2). Such significant tissue heterogeneity suggests specific mechanisms governing Osthole biosynthesis or storage. Spatial mapping further identified a steep concentration gradient: root bark accumulated 0.3 ± 0.15% Osthole, which is 11-fold higher than root pith (0.028 ± 0.007%; p = 0.002, Student’s t-test) and 15–30-fold greater than aerial parts (petioles: 0.02 ± 0.005 mg/g, blades: 0.010 ± 0.002 mg/g) (Figure 1a,b). This bark-centric accumulation localized 83.6% of the total Osthole in underground organs, suggesting that its primary biological roles may involve belowground ecological interactions.

2.2. Metabolic Variability and Core Germplasm Identification

To delineate metabolic variability implicated in our metabolite profiling, we performed multivariate analysis on 12 A. biserrata individuals derived from a half-sibling family (Table S3). Principal Component Analysis resolved 95.33% of compositional variance (PC1: 60.51%; PC2: 34.82%), isolating accessions AB-50, AB-222, and AB-SK2 as chemotypic outliers characterized by elevated PC1 scores (>1.5) and constrained PC2 values (0–0.5) (Figure 2a). These accessions consistently demonstrated the highest Osthole accumulation (Figure 2b). Coefficient of variation assessment further quantified heterogeneity: Osthole exhibited maximal variability (CV = 93.75%) compared to Osthenol (69.82%), Umbelliferone (64.7%), Columbianadin (57.8%), Isoimperatorin (33.56%), and Dihydrooroselol (27.11%) (Figure S1). This positions Osthole as the pivotal discriminator for germplasm screening, directly establishing AB-222 as the optimal core germplasm due to its exceptional chemometric stability.

2.3. Temporal Dynamics and Environmental Induction of Osthole Biosynthesis

Having established the distinct chemotypic variation among accessions (Figure 2), we further tracked temporal metabolite dynamics during rhizome maturation. Monthly monitoring of six bioactive markers in field-grown A. biserrata revealed a critical accumulation pattern for Osthole, which exhibited a content surge of 195% from September to November and continued rising, albeit at a reduced rate (17% increase), through December (Figure 3a; Table S4). These three sampling points were strategically selected to capture key developmental stages in A. biserrata’s biennial growth cycle: the early dormancy phase (September), when vegetative growth ceases and resources begin redirecting to rhizomes; mid-dormancy (November), when secondary metabolite accumulation typically intensifies; and deep dormancy (December), which is the stage following frost exposure. Earlier time points were not examined as preliminary screenings of summer samples showed negligible coumarin levels that fell below reliable quantification thresholds, consistent with the resource allocation to aerial growth observed in Apiaceae perennials during vegetative phases.

Contrastingly, Osthole’s direct precursor Osthenol remained statistically unchanged (p > 0.05), while the upstream precursor Umbelliferone underwent a sharp decline (−50%) by November followed by a December surge to 40.16%, going above September baseline levels. To dissect environmental triggers, we analyzed nine accessions pre- and post-frost in December. An unexpected early frost event presented a valuable research opportunity to capture immediate metabolic responses within a 24-h window. Frost exposure induced a rapid 1.73-fold escalation in Osthole concentration across all lines (p < 0.05) (Figure 3b; Table S4). The relative fold-change values displayed in Figure 3b (ranging from 1.63 to 1.95-fold across different accessions) highlight the remarkably consistent proportional increase in Osthole compared to the minimal relative changes in other metabolites, suggesting a specific frost-triggered biosynthetic shift favoring Osthole accumulation.

2.4. Transcriptomic Profiling and Co-Expression Network Analysis of A. biserrata

To identify molecular drivers of coumarin biosynthesis implicated by tissue-specific metabolite accumulation, we performed a transcriptome-wide investigation of the molecular mechanisms underlying coumarin biosynthesis. Total RNA sequencing of all corresponding metabolic samples generated comprehensive expression profiles (Table S5). Principal component analysis confirmed robust sample stratification, revealing eight distinct clusters corresponding to tissue types (leaf, petioles, root bark, and root pith) and temporal variations across developmental stages (September, November, and December roots), with genetic divergence among 12 half-sibling individuals forming a separate cohort (Figure S2).

To elucidate regulatory networks governing coumarin accumulation, we executed WGCNA using our 33-sample FPKM dataset. Convergent scale-free topology fit indices and mean connectivity analyses identified a network construction threshold of power = 4, generating 44 co-expression modules (Figure S3a–c). Integration of six key coumarin metabolite levels with expression clustering identified a gene module (colored purple in Figure S3d) that strongly correlated with Osthole accumulation. The subsequent module–trait association analysis demonstrated that this module exhibited exceptionally strong positive correlations with Osthole (r = 0.81, p = 6 × 10⁻⁴) and Umbelliprenone (r = 0.74, p = 1 × 10⁻⁶; Figure 4a). Hierarchical clustering confirmed that co-expression patterns were tightly linked to Osthole accumulation (Figure 4b). Gene significance (GS) and module membership (MM) analyses within the purple module revealed significant relationships between Osthole levels and hub gene expression (Table S6; Figure 4c), with topological visualization substantiating dense interconnection among key biosynthetic regulators (Figure S3e).

2.5. Identification of AbOMT1 as a Central Hub Gene in the Osthole-Associated Co-Expression Module

To elucidate the molecular drivers underlying Osthole accumulation, this study integrated co-expression networks with metabolite correlation screening. The gene module strongly correlating with Osthole accumulation (r = 0.81, p = 6 × 10⁻⁴, colored purple in our visualization) was subjected to hub gene identification. Within this module, gene AbOMT1 (AB04G05077), the homolog of OMT1 (METABOLIC O-METHYLTRANSFERASE 1), emerged as a possible significant hub gene, demonstrating a robust correlation with Osthole content traits (gene significance, GS = 0.9162; module membership, MM = 0.8434). Functional annotation revealed that this gene encodes an S-adenosyl methionine (SAM)-dependent methyltransferase harboring the conserved IPR029063 SAM_binding domain, which is essential for O-methylation during coumarin backbone modification. Key co-expressed genes within the module included prenyltransferases (AB10G00842, AB06G01999, AB07G04187, and AB11G02263); COUMARIN SYNTHASE (COSY) regulating umbelliferone biosynthesis (AB01G01375 and AB06G01114); and core phenylpropanoid biosynthetic genes (PAL, C4H, 4CL, and C2H). All these genes are key components in the pathway of Osthole biosynthesis from phenylalanine (Figure 5a).

KEGG enrichment analyses revealed significant enrichment of genes from the purple module in the “phenylpropanoid biosynthesis” (ko00940, p = 1.87 × 10⁻³), “Diterpenoid biosynthesis” (ko00904, p = 2.81 × 10⁻³), and “terpenoid backbone metabolism” (ko00900, p = 1.67 × 10⁻³) pathways (Figure S4a). GO enrichment analyses revealed significant enrichment of genes from the purple module in the “intramolecular oxidoreductase activity” (GO:0016860, p = 1.33 × 10⁻³), “lipid binding” (GO:0008289, p = 1.52 × 10⁻²) and “response to light stimulus” (GO:0009416, p = 4.21 × 10⁻³) pathways (Figure S4b), indicating coordinated metabolic flux across these pathways. In contrast, KEGG and GO enrichment analyses of the gray module, which showed very low correlation with Osthole biosynthesis (r = −0.0027), revealed no enrichment in any pathways related to Osthole biosynthesis (Figure S4c,d). This comparative analysis further supports the specificity of the purple module in Osthole biosynthesis regulation.

Validation via qRT-PCR using root samples representing different tissues, materials with varying Osthole contents, and different periods demonstrated that AbOMT1 expression patterns strongly co-varied with Osthole accumulation. Specifically, expression in the root bark was three-fold higher than in the root pith, and expression progressively increased from September to December.

Crucially, the reliability of the underlying transcriptomic data was confirmed by a strong correlation (n = 434, |R| = 0.8052) between gene expression levels and qRT-PCR quantification for 31 selected genes across these diverse samples (Figure S5a; Table S7). Furthermore, a highly significant direct correlation (n = 26, |R| = 0.8786; Figure S5b and Table S8) was observed between the expression level of the key enzyme gene AbOMT1 and Osthole accumulation intensity across these same sample sets (Figure 5b,c and Table S8). These collective results position AbOMT1 as a possible key regulatory factor in Osthole biosynthesis and as a prime candidate gene for metabolic engineering initiatives.

3. Discussion

3.1. Spatiotemporal Heterogeneity in Osthole Accumulation

The significant enrichment of Osthole in root bark (three-fold higher than in root pith) aligns with the established ecological defense roles of coumarins in subterranean tissues. This finding is supported by studies in the literature demonstrating that furocoumarins, including structurally related compounds, function as important natural biocides with specific “biodefense roles” in the roots of Angelica dahurica, where their accumulation is developmentally regulated [1]. Furthermore, complex coumarins in Apiaceae plants act as key regulators responding to environmental stimuli [25]. As derivatives of the phenylpropanoid pathway, this targeted enrichment represents a strategic defense mechanism against biotic stresses, providing a crucial physiological rationale for developing low-loss harvesting strategies focused on phloem-enriched tissues.

The pronounced increase in Osthole content (>150%) observed under seasonal changes and frost stress indicates a robust abiotic stress response mechanism. Cold stress activates the phenylpropanoid pathway, leading to enhanced accumulation of secondary metabolites like coumarins. This induction is mediated through signaling cascades involving endogenous hormones such as jasmonic acid and abscisic acid (ABA), which upregulate key biosynthetic and regulatory components [26]. Supporting this, cold stress has been shown to elevate phenylpropanoid derivatives, providing antioxidant protection against cold-induced oxidative damage [27]. Importantly, ABA signaling pathways are directly implicated in enhancing freezing tolerance [28], likely coordinating the observed metabolic shift towards coumarin accumulation. This suggests that controlled frost exposure could represent a viable agronomic strategy to boost Osthole yield in pharmaceutically relevant plants.

3.2. Expression of the AbOMT1 Correlates with Osthole Accumulation and Chemotype Stability

Elevated expression of AbOMT1 correlated significantly with increased Osthole accumulation. This indicates a potential functional involvement of this SAM-dependent AbOMT1 in Osthole biosynthesis. However, the specific catalytic role of AbOMT1 in mediating the critical methylation step from osthenol to Osthole requires direct biochemical validation. The enzymes catalyzing this critical step of the coumarins biosynthesis pathway remain unidentified in Angelica species [29]. While OMTs like AgOMT1 demonstrate functional methylation activity in related Apiaceae plants, such as Anethum graveolens for phenylpropene methylation [30], substrate specificity varies significantly across OMTs. Although we observed elevated expression of AbOMT1, its in vitro enzymatic characterization and confirmation of its ability to methylate specific intermediates like osthenol within A. biserrata are pending. Therefore, while our expression data strongly implicates AbOMT1, this evidence does not yet confirm it as the core catalytic enzyme for the final Osthole methylation, consistent with the knowledge gap highlighted in A. decursiva [29]. The observed expansion of PRENYLTRANSFERASE (PT) genes in Angelica sinensis for coumarin biosynthesis [15] further illustrates the complexity of the pathway and the need for functional validation of candidate genes like AbOMT1 in specific species and steps.

This robust expression–metabolite correlation suggests constitutively elevated AbOMT1 expression as an important factor underpinning the persistent, high-level Osthole production characteristic of the AB-222 chemotype. While we did not functionally assess specific regulatory polymorphisms as the causal mechanism for this differential expression, the correlative pattern itself aligns with established biological principles where stable metabolic phenotypes arise from genetically encoded differences in the regulation of key biosynthetic genes. For instance, stable variation driven by promoter polymorphisms controlling expression levels of key enzymes, such as OsTPS1 in rice disease resistance [31], exemplifies this well-documented link between regulatory mechanisms and consistent metabolic traits. OMT expression modulation, as seen in sesquiterpene production in agarwood [32], further supports this paradigm within specialized metabolism. Future studies should prioritize fine-mapping and functional validation of regulatory elements within the AbOMT1 locus in contrasting chemotypes, utilizing approaches successful in dissecting similar regulatory variation [31], to definitively elucidate the genetic basis for the persistent expression driving the economically valuable Osthole chemotype in AB-222.

3.3. Frost Stress Association with Osthole Levels and AbOMT1 Expression

Constitutively elevated AbOMT1 expression, correlating with a high Osthole content, suggests inherent genetic regulation underpinning this metabolic phenotype. While our findings demonstrate frost-induced increases in Osthole levels, the question regarding the mechanism of frost induction of AbOMT1 expression and subsequent ABA/ROS signaling as a contributor to Osthole accumulation remains open. No direct evidence has been established linking AbOMT1 induction to cold stress as OMT1 function is documented solely in the context of salt tolerance, where it enhances copper uptake to support ABA biosynthesis and compartmentalization of Na⁺ [33]. Consequently, a direct mechanistic link between frost stimuli and AbOMT1 expression/activity in modulating Osthole remains speculative based on current evidence.

It is well-established that cold stress, including frost, engages ABA and ROS signaling as core conserved pathways across plants [34,35]. Activation of OST1 kinase, induction of ABA biosynthetic genes like NCED1 [35], accumulation of ROS [36], and crosstalk with other hormones such as JA [37] are fundamental mechanisms for cold acclimation, aiming to balance stress protection with resources for growth [38]. Given our data implicating AbOMT1 in Osthole accumulation, it is plausible that perturbations in such core stress signaling networks could indirectly influence the expression or activity of biosynthetic genes or enzymes.

4. Materials and Methods

4.1. Plant Materials

Field specimens of A. biserrata were established on 20 March 2020 at an altitudinal range of 1200 m a.s.l. in Yesanguan Town, Badong County (30.632823° N, 110.336975° E), Hubei Province, a traditional cultivation region characterized by cambisol soil (pH 6.2; 0–20 cm depth: SOC: 22.7 g·kg⁻¹, total N 159.2 mg·kg⁻¹, Olsen-P 40.4 mg·kg⁻¹, exchangeable K 193.5 mg·kg⁻¹). Uniform one-year-old rootstocks showing vigorous growth (diameter ≥ 0.8 cm at root collar), absence of mechanical injury, and zero pathogenic symptoms were transplanted under non-fertilized cultivation at 75,000 plants·ha⁻¹.

4.2. Sample Collection for RNA Sequencing and Metabolomic Profiling

An integrative multi-omics sampling approach was stratified across tissue types, genetic lineages, and seasonal phases in biennial A. biserrata plants. Three focal accessions (P1, P2, and P3) contributed leaf blade, petiole, root stele, and root periderm samples for parallel RNA/metabolome extraction. Ten half-sib families (AB-1, AB-12, AB-131, AB-133, AB-222, AB-24, AB-5, AB-50, AB-62, and AB-SK2) provided root tissues for comparative transcriptome–metabolome analysis. Temporal dynamics were captured through AB-182 root samples collected in September, November, and December 2022. Frost response metabolomics targeted root tissues of nine distinct accessions (AB-3, AB-5, AB-25, AB-55, AB-60, AB-67, AB-69, AB-70, and AB-92) sampled during pre-frost and post-frost periods, exclusively for metabolomic analysis.

4.3. Metabolite Extraction and Quantification

Samples dissected into roots, petioles, blades, root cores, and root cortices underwent thermal enzyme inactivation (105 °C, 15 min) followed by drying at 70 °C for 6 h until a constant mass was achieved. After pulverization through a 60-mesh sieve, targeted constituents were extracted using 50–85% ethanol via percolation or reflux. Simultaneous quantification of Dihydrooroselol, Osthole, iso-Osthenol, and Columbianadin was performed using HPLC on an Agilent Extend C₁₈ column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of 0.1% aqueous formic acid (A) and acetonitrile (B) with a flow rate of 1.0 mL·min⁻¹. The gradient elution program was as follows: 0–5 min, 25–30% B; 5–15 min, 30–40% B; 15–25 min, 40–55% B; 25–35 min, 55–70% B; 35–40 min, 70–80% B; 40–45 min, 80–25% B; and 45–50 min, 25% B for column equilibration. The column temperature was maintained at 30 °C with detection at 330 nm and an injection volume of 10 μL. Method validation confirmed linear responses within different concentration ranges, namely Dihydrooroselol (1.27–11.43 μg·mL⁻¹, r ≥ 0.9996), Osthole (33.09–297.79 μg·mL⁻¹), iso-osthenol (1.00–8.96 μg·mL⁻¹), and Columbianadin (9.02–81.16 μg·mL⁻¹), with average recoveries of 98.4–100.4% (RSD 0.90–1.72%, n = 6). The optimal content of all analytes was achieved under the 70 °C/6-h drying protocol.

4.4. Transcriptome Profiling and Co-Expression Network Analysis

Transcriptome analysis commenced with RNA-seq library construction using the Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina, Inc., San Diego, CA, USA), followed by 150 bp paired-end sequencing on the NovaSeq 6000 platform (Illumina, Inc., San Diego, CA, USA).Raw reads underwent rigorous quality control through Trimmomatic v0.39 to eliminate adapter sequences and low-quality bases (Phred score cutoff: 20). High-quality reads were subsequently aligned to an unpublished chromosome-level A. biserrata reference genome using HISAT2 v2.2.1, with gene expression quantified as FPKM values via StringTie v2.2.0. Differential expression analysis was performed via DESeq2, applying thresholds of |log2(fold change)| > 1.5 and adjusted p < 0.05. Functional enrichment analysis of DEGs was conducted for Gene Ontology (GO) terms and KEGG pathways (clusterProfiler v4.6.2).

Weighted gene co-expression networks were constructed using the WGCNA R package (v1.72) based on FPKM matrices derived from all 33 samples. Following scale-free topology assessment (R² > 0.85), a soft-thresholding power of β = 4 was selected to build signed topological overlap matrices. Hierarchical clustering and dynamic tree-cutting identified co-expression modules (minimum size: 200 genes). Module–trait correlations were specifically calculated for Osthole content levels quantified via HPLC (significance threshold: |correlation| > 0.7, p < 0.05), alongside tissue types and seasonal timepoints. Hub genes were extracted by intersecting high module membership (MM > 0.9) and gene significance (GS > 0.8) metrics. Osthole-associated modules underwent functional annotation through integrated KEGG pathway and InterPro domain analysis, with putative molecular roles in coumarin biosynthesis assessed via qPCR on selected 30 hub genes.

4.5. Statistical Analysis of Metabolite Variation and Sample Classification

Statistical analyses were conducted to compare six metabolite levels across distinct plant parts using independent samples t-tests with significance defined at p < 0.05, while variation magnitude was quantified via the MAX-MIN Ratio. Principal Component Analysis (PCA) was applied to root samples from different raw material sources to resolve metabolic heterogeneity patterns. Discriminatory metabolites were screened through Coefficient of Variation (CV) assessment, where compounds exhibiting CV > 30% across samples were considered highly variable and thus potentially effective markers for material distinctness.

4.6. qRT-Pcr Validation

To validate transcriptome results, 30 key DEGs were selected for quantitative reverse transcription PCR (qRT-PCR). First-strand cDNA was synthesized from 1 μg of total RNA using the PrimeScript™ RT Reagent Kit (Takara, Kusatsu, Japan). Gene-specific primers (Table S9) were designed with Primer Premier 6.0 and synthesized by Sangon Biotech (Shanghai, China). Reactions were performed on a Bio-Rad CFX96 system with SYBR Green Master Mix (Roche, Basel, Switzerland) under the following conditions: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s, with a final extension at 72 °C for 5 min. Melt curve analysis confirmed amplification specificity. Relative expression levels were calculated using the 2^−ΔΔCt method with Rubisco as the internal control. Statistical significance was assessed via a one-way ANOVA (p < 0.05).

5. Conclusions

Collectively, this study bridges critical knowledge gaps in A. biserrata industrial cultivation through the following validated findings: We reveal an 11-fold enrichment of Osthole in root phloem relative to xylem tissues (p < 0.001), suggesting the possible industrial merit of epidermal skin harvesting protocols to maximize bioactive yield. Temporal quantification identified a 340% elevation in Osthole concentration during December versus September harvesting (p = 1.8 × 10⁻⁸), with a further 170% surge following frost events (p = 9 × 10⁻⁶), suggesting mid–late December post-frost as a potentially optimal agricultural window for maximal productivity. Germplasm screening identified elite accession AB-222 exhibiting 230% higher Osthole content versus regional population means, identifying this variant as a promising breeding material for yield stabilization. Weighted gene co-expression network analysis (WGCNA) delineated a gene module strongly correlating with biosynthesis (module-trait r = 0.81, p = 6 × 10⁻⁴) and identified a possible OMT1 homolog (AB04G05077) displaying robust correlation with Osthole accumulation (|R| = 0.88, p < 0.001), suggesting its potential utility as a molecular marker for precision breeding.

These integrated discoveries demonstrate a robust, constitutive association between sustained AbOMT1 gene expression patterns and elevated Osthole content across geographically distinct populations and developmental stages. Importantly, while environmental factors like frost exposure influence biosynthesis, our findings suggest that genetic regulation of AbOMT1 expression, rather than short-term abiotic triggers alone, is the primary driver defining the observed chemotype persistence in high-Osthole (H) and low-Osthole (L) variants.

Future studies should investigate the role of prolonged field-based frost exposure in modulating genetic pathways controlling AbOMT1 to account for its ecological significance in natural habitats. Thus, targeted chemotype selection based on stable AbOMT1 expression markers offers immediate potential for optimizing cultivation programs. Integrating H-chemotype germplasm into breeding strategies will accelerate the development of high-yielding, quality-controlled medicinal material for the reliable production of standardized, Osthole-rich botanical extracts.