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. 2026 Feb 8;15(4):530. doi: 10.3390/plants15040530

Comprehensive Genome-Wide Analysis of the AP2/ERF Transcription Factor Family and Examination of Potential Regulators CpERF140 and CpERF138 in the Biosynthesis of Lobetyolin in Codonopsis pilosula

Xiaobi Zhang 1, Jiaxin Zhang 1, Zhengqin Pan 2, Ping Wang 1, Linqiang Zhang 1, Jiayi Xu 1, Junmei Lian 1, Yihan Wang 1, Xinyue Zhang 1, Yan Sui 1, Yan Yan 1,*, Jian Zhang 1,3,4,*, Peng Di 1,*
Editor: Hisato Kunitake
PMCID: PMC12944265  PMID: 41754237

Abstract

Lobetyolin is a key bioactive compound in Codonopsis pilosula, but the existing literature has not well explored the regulatory mechanisms governing its biosynthesis. This study performed a genome-wide identification of 175 AP2/ERF transcription factors (TFs), categorizing them into the AP2, ERF, and RAV subfamilies. Promoter analysis revealed numerous hormone- and stress-responsive cis-elements, with a notable prevalence of methyl jasmonate (MeJA)-related motifs. In MeJA-treated hairy root cultures, we observed a significant increase in lobetyolin accumulation. Integrated transcriptomic and metabolomic analyses demonstrated the activation of jasmonate signaling and secondary metabolism pathways. Two MeJA-induced TFs, CpERF140 and CpERF138, were identified as key regulatory candidates. Subsequent experiments confirmed their nuclear localization and specific binding affinity for GCC-box motifs. This study constitutes the inaugural genome-wide characterization of the AP2/ERF family in C. pilosula, employing hairy root culture as an effective platform for functional investigation. Our findings offer novel perspectives on the transcriptional regulation of lobetyolin biosynthesis, providing a foundation for the future metabolic engineering of this medicinal plant.

Keywords: C. pilosula, CpAP2/ERF, lobetyolin, methyl jasmonate, secondary metabolism

1. Introduction

Codonopsis pilosula is a perennial herbaceous vine within the Campanulaceae family, with roots utilized as the primary medicinal organ due to their rich phytochemical profile. These encompass diverse bioactive compounds, including alkaloids, terpenoids, flavonoids, lignans, steroids, glycosides [1], and polyacetylenes [2]. The pharmacological activities of these components are multifaceted, including neuroprotection, metabolic regulation of glucose and lipid levels, immune modulation, digestive support, and improvement of microcirculation, as well as antibacterial and antiviral effects, thereby supporting its traditional uses in promoting overall health [3]. Using transcriptomic profiling, Gao et al. identified high expression of genes driving the synthesis of UDP-L-arabinose and UDP-xylose, both critical for polysaccharide biosynthesis in C. pilosula [4]. Furthermore, multi-omics investigations by Li et al. suggested that bHLH2, bHLH3, and AP2/ERF2 may significantly influence biosynthesis of triterpenoids and sesquiterpenoids by directly regulating CpLUP4-14, CpSQLE-2, and CpFDFT1-8 transcription [5]. Lobetyolin, an important polyacetylene glycoside in C. pilosula, consists of the aglycone lobetyol linked to a glucose moiety [6]. Primarily extracted from C. pilosula roots [6], lobetyolin reportedly enhances immune function, reduces blood pressure, stimulates appetite, and alleviates gastric ulcers [7]. However, its biosynthetic pathway remains poorly defined.

Plants contain a substantial superfamily of transcription factors referred to as APETALA2/ethylene-responsive factors (AP2/ERFs). These factors are distinguished by a highly conserved DNA-binding AP2 domain that spans approximately 57–66 amino acids [8]. This superfamily has been identified across diverse species, including Arabidopsis thaliana, Oryza sativa, and Sorghum bicolor [9]. Based on the criteria of AP2 domain copy number and overall sequence similarity, the AP2/ERF superfamily is categorized into three principal families: AP2, ERF (further divided into the ERF and DREB subgroups), and RAV [10,11,12]. Structurally, the AP2 family is defined by two tandem AP2 domains, whereas ERF members contain a single domain; the RAV family is distinct for harboring both AP2 and B3 DNA-binding domains [10,13]. Based on the amino acid sequence of the double AP2 domain and the nuclear localization signal, the AP2 family is further categorized into the euAP2, euAINTEGUMENTA (euANT), and baseant groups [14,15,16]. The AP2/ERF gene family critically dominating plant defense and hormone responses. For instance, AtERF2 plays a positive role in plant defense mechanisms and responses [17,18]. Moreover, this family significantly modulates secondary metabolite production. For example, expression of AaERF1, AaERF2, and AaORA in Artemisia annua upregulates AaADS and AaCYP71AV1, increasing artemisinin accumulation [19,20]. Similarly, in Salvia miltiorrhiza, SmERF1L1 upregulates SmDXR expression, promoting tanshinone and phenolic acid biosynthesis [21].

Natural products in medicinal plants include primary and secondary metabolites. Typically, secondary metabolites such as saponins, terpenoids, alkaloids, flavonoids, and volatile oils have therapeutic value for human diseases [22]. However, the content of secondary metabolites is generally low, often less than 1% of the plant’s dry weight, because it occurs during specific physiological and developmental stages and is influenced by external factors [23,24]. To overcome this issue and investigate biosynthetic pathways, in vitro elicitation techniques are frequently employed. The application of biotic or abiotic elicitors to stimulate the biosynthesis of bioactive secondary metabolites [25,26], followed by comprehensive transcriptomic and metabolomic analyses to examine associated genes and elucidate molecular mechanisms, is a common practice. Nirin Udomsom et al. elucidated the efficacy of AP2/ERF transcription factors in regulating metabolism in Ophiorrhiza pumila through an integrated transcriptomic and metabolomic analysis [27]. Chenghao Zhu et al. uncovered distinct regulatory mechanisms for underlying ear underling bolting and flowering across varieties of the Chinese herb Angelica sinensis through transcriptomic and metabolomic analyses, providing new insights into the mechanisms in Aspergillus and other Apiaceae medicinal plants [28]. Jiaojiao Ji et al. induced adventitious roots of C. pilosula with MeJA and, through combined metabolite and biosynthetic gene correlation analysis, revealed that MeJA promotes bioactive metabolite accumulation by regulating the expression of CpUGPase and CpPMK [29].

This study is the first one to comprehensively analyze the AP2/ERF gene family in C. pilosula. Therefore, we first conducted a genome-wide identification and bioinformatic analysis of this family to establish a candidate pool. Subsequently, we utilized MeJA-treated hairy roots as a platform and integrated targeted metabolomic and transcriptomic analyses to screen for key AP2/ERF transcription factors potentially involved in lobetyolin biosynthesis. A total of 175 members of the AP2/ERF gene family were identified and subjected to bioinformatics analysis. Simultaneously, MeJA was applied to elicit responses in C. pilosula hairy roots. By integrating metabolomic and transcriptomic analyses, the primary secondary metabolites and differentially expressed genes (DEGs) were identified in hairy roots treated with MeJA. Two AP2/ERF transcription factors potentially involved in lobetyolin biosynthesis were screened. Subcellular localization confirmed that both transcription factors are nuclear, and yeast one-hybrid assays verified their specific binding to the GCC-box. The findings provide a molecular framework for regulating the biosynthetic pathway of lobetyolin in C. pilosula.

2. Results

2.1. Identification of the CpERF Gene Family

A comprehensive genome-wide analysis of the AP2/ERF transcription factor family was performed in C. pilosula to ascertain phylogenetic relationships and assess their regulatory mechanisms in biosynthesis. This well assists in investigating metabolic mechanisms and molecular breeding strategies.

A total of 175 genes belonging to the AP2/ERF transcription factor family were identified (Supplementary Table S1). Of these, 169 TFs were distributed across eight chromosomes (Figure 1) and designated CpERF1 to CpERF169. Chromosomal distribution was uneven; for example, on chromosomes 1 and 8, genes were densely clustered in the middle and lower regions. Genes on chromosomes 3 and 4 were concentrated at both ends, with no distribution in the middle areas. Chromosome 2 contained the highest gene density (39 genes), while chromosomes 3, 5, and 8 harbored the fewest (12 genes).

Figure 1.

Figure 1

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Genomic localization of CpAP2/ERF transcription factors in C. pilosula. The eight chromosomes are numbered in yellow. A color scale extending from dark blue to red represents gene density across specific chromosomal regions.

Phylogenetic analysis facilitates the understanding of evolutionary lineages and reveals sequence parallels and potential functional homologies. Employing 140 transcription factors from the A. thaliana AP2/ERF gene family as a reference, a phylogenetic tree was built with the 175 CpAP2/ERFs (Figure 2, Supplementary Table S3). The results indicated that the C. pilosula AP2/ERF gene family comprises three subfamilies: ERF, AP2, and RAV. The ERF subfamily is further categorized into the ERF and DREB subgroups. These two groups are distinguished by the amino acid residue at the fourteenth position; specifically, an alanine (Ala) or aspartic acid (Asp) residue is conserved in the ERF subfamily, whereas a valine (Val) or glutamic acid (Glu) residue characterizes the DREB subfamily [13]. The ERF family contained the highest number of members, the RAV family the lowest (merely 8 members), the AP2 family comprised 20 members, the DREB family comprised 51 members, and the remainder fell into the ERF gene family. The ERF subfamily collectively accounts for 84% of all CpAP2/ERF gene family members.

Figure 2.

Figure 2

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Phylogenetic analysis of CpAP2/ERF genes, including 140 AP2/ERF reference sequences from A. thaliana. Color-coded subfamilies are as follows: Green: AP2 gene family; red: RAV gene family; yellow: ERF gene family; blue: DREB gene family.

Multiple sequence alignment was performed on 175 members of the CpAP2/ERF transcription factor family to elucidate structural and functional conservation and similarity. The analysis (Figure S1) revealed that the C. pilosula AP2/ERF domain comprises 71 amino acid residues. Among these, 35 residues exhibited > 50% conservation, and 15 residues exhibited > 95% conservation (e.g., positions 11, 20, 45, and 46). Although the AP2/ERF domain is relatively conserved, variations were noted in select members. For instance, CpERF36/57/102/107/108/109/110/123 exhibited partial domain deletions, while CpERF153 showed a large fragment deletion. Additionally, CpERF22/78/95/100/112 harbored partial domain mutations.

Gene duplication crucially drives gene family evolution. Examining both segmental and tandem duplication events sheds light on the underlying mechanisms that promote the expansion of these families. To elucidate the expansion history of the CpAP2/ERF family, duplication patterns were analyzed and found to be widespread across all C. pilosula chromosomes. Our analysis identified 28 duplication events: 27 segmental duplications and one tandem duplication (Figure 3).

Figure 3.

Figure 3

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Collinearity analysis of the AP2/ERF gene family in C. pilosula. Red lines within the circle denote segmental duplication events. The yellow numbers indicate chromosome numbers, tracks (a) and (b) denote GC content and gene density r, respectively.

2.2. Bioinformatics Analysis of CpAP2/ERF

To clarify the evolutionary development of the CpAP2/ERF family, the gene structures of all 175 members were analyzed and predicted. Among the 175 CpAP2/ERF genes, ten conserved motifs were identified (Figure S2). Except for CpERF39, which lacked motif 1, all other genes harbored motif 1—every CpAP2/ERF transcription factor possessed at least one motif 2 or motif 4. Consequently, motifs 1, 2, and 4 jointly comprise the conserved domain of the AP2/ERF gene family, with motif 1 designated as the binding motif. Examination of the CpAP2/ERF gene structures revealed that exon count varied from 1 to 17, with CpERF101 possessing the maximum number of exons. Most family members harbored only one exon. Intron numbers ranged from 0 to 16, with most family members lacking introns.

An analysis of cis-acting elements was performed on the 2000 bp upstream sequences of all 175 CpAP2/ERFs to elucidate transcriptional regulatory mechanisms (Figure 4 and Figure S3, Supplementary Table S4). The elements were categorized into five functional groups: basic elements; light-responsive elements (GT1, Gbox, GATA, TCT, Box4, etc.); and plant hormone-responsive elements (TGACG, CGTCA, ABRE, TGAelement, TCAelement, etc.). growth and development-responsive elements (O2-site, CAT-box, CCAAT-box, GCN4, circadian, etc.); and stress-related cis-acting elements (ARE, LTR, MBS, TC-rich repeats, GC, etc.). Scrutiny of the four element types (excluding basic elements) indicated that light-responsive elements were the most plentiful (2273, 48%), whereas growth and development elements were the scarcest (374, 7.9%). Plant hormone-responsive elements and stress-responsive elements constituted 1354 (28.6%) and 733 (15.5%), respectively. Box4, G-box, ABRE, ARE, and GT1-motifs ranked as the top five cis-acting elements, with tallies of 502, 478, 419, 344, and 296, respectively, constituting 42.4% of all elements. Within the C. pilosula AP2/ERF gene family, 123 members possess methyl jasmonate (MeJA)-responsive elements, 128 contain abscisic acid-responsive elements (ABRE), 86 have auxin-responsive elements (TGA element), 75 include salicylic acid-responsive elements (TCA element or SARE), 84 exhibit drought stress-responsive elements (CAACTG), and 129 members feature low-temperature-responsive elements (LTR).

Figure 4.

Figure 4

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Proportional distribution of cis-acting elements. (a) Proportion of the four major cis-element categories. (b) The top five cis-acting elements within the four major categories. (c) Proportion of the top 10 light-responsive elements. (d) Proportion of plant hormone-responsive elements. (e) Proportion of stress-responsive elements. (f) Proportion of plant growth and development-responsive elements.

2.3. Induction of C. pilosula Hairy Roots and the Effect of MeJA Treatment

To elucidate the role of the AP2/ERF family in secondary metabolite biosynthesis, a complete genetic transformation system for C. pilosula hairy roots was established and subjected to MeJA treatment. Roots and stems were infected with Agrobacterium rhizogenes C58C1 at OD600 0.4–0.6. To eradicate residual A. rhizogenes, the explants were transferred to a solid 1/2 MS medium supplemented with 500 mg/L cefotaxime sodium (Cef) following a two-day co-cultivation period. Subcultures were performed weekly with progressively lower Cef concentrations. Suspension cultures were initiated by transferring hairy roots (2–3 cm in length) into liquid 1/2 MS medium (Figure 5a). Three randomly selected hairy root lines showed growth rates ranging from 1.261 to 1.786 g/day. The growth of hairy roots was constrained by flask size; rates declined rapidly once roots contacted the flask walls (Figure 5b). The hairy root line with the highest growth rate was chosen for subsequent expansion. MeJA-treated hairy roots (Figure S4) exhibited a pale yellow phenotype in both the root system and the culture medium following induction.

Figure 5.

Figure 5

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Establishment of the genetic transformation system for C. pilosula hairy roots and treatment with different elicitors. (a) Schematic diagram of C. pilosula hairy root induction. (b) Changes in the growth rate of C. pilosula hairy roots in fixed containers with the same volume of medium.

2.4. Targeted Metabolomic Analysis of C. pilosula

The secondary metabolites lobetyolin, lobetyol, and lobetyolinin in MeJA-treated C. pilosula hairy roots were subjected to targeted analysis. Employing HPLC-MS/MS, these compounds were further profiled to examine the effects of MeJA on the accumulation of active components. Differences in their peak areas were analyzed for significance (Figure 6, Supplementary Table S5). Under MeJA treatment, the peak area of lobetyolin increased significantly relative to the control, whereas that of lobetyol decreased. The peak area of lobetyolinin showed no significant difference between MeJA treatment and the control.

Figure 6.

Figure 6

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Differential analysis of peak areas for three main secondary metabolites in C. pilosula (n = 3).

2.5. Transcriptomic Analysis

To elucidate the biosynthetic pathway of lobetyolin, transcriptomic sequencing was performed on MeJA-treated and control C. pilosula hairy roots. In total, 16.9 GB of clean data were produced, as detailed in Supplementary Table S6, with alignment rates of 97.24–98.93%. DEGs referred to those meeting thresholds of |log2 FC| ≥ 2 and p < 0.05 (Figure 7a)—comparison between control and retreatment groups, 1020 DEGs, comprising 852 upregulated and 168 downregulated genes.

Figure 7.

Figure 7

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Analysis of differential transcripts in C. pilosula hairy roots after treatment with different elicitors. (a) Volcano plot analysis of DEGs in C. pilosula hairy roots after MeJA treatment. (b) Functional enrichment analysis of DEGs in C. pilosula hairy roots after MeJA treatment (top 70 terms ranked by p-value). (c) KEGG functional enrichment analysis of DEGs in C. pilosula hairy roots after MeJA treatment. The top 20 pathways ranked by p-value were selected for data visualization.

2.6. Functional Enrichment of DEGs

Gene Ontology (GO) enrichment analysis is a standard bioinformatic approach for interpreting high-throughput transcriptomic and proteomic data. Based on transcriptomic comparisons of C. pilosula hairy roots under MeJA treatment and control (CK) conditions, GO functional enrichment analysis was conducted to elucidate MeJA-regulated biological processes and molecular functions (Figure 7b). DEGs were significantly enriched in several categories related to secondary metabolism, stress response, and transcriptional regulation, demonstrating significant biological relevance. At the molecular function level, DEGs were notably enriched in glycosyltransferase activities, including enzymes such as UDP-glucosyltransferase and β-glucosyltransferase. Furthermore, these genes showed significant enrichment for oxidoreductase and transcription factor activities, particularly those involving DNA-binding transcription factors. Simultaneously, the enrichment of transcription factor activity strengthens the hypothesis that MeJA regulates metabolic pathways through transcriptional reprogramming.

  • Regarding biological processes, DEGs were significantly enriched in the following categories: Jasmonic acid signaling pathway. Specifically, responses to jasmonic acid stimuli and regulation of mediated signaling pathways, confirming effective activation of the JA signaling pathway.

  • Secondary metabolic and terpenoid metabolic processes: Including sesquiterpenoid and apocarotenoid metabolic processes, which are highly relevant to the biosynthesis of lobetyolin, a polyacetylene terpenoid derivative [30].

  • Stress and stimulus response: Encompassing abiotic stimuli such as salt stress and wounding, indicating that MeJA treatment triggered broad defense and adaptation responses.

  • Cell wall organization and biogenesis: Notably, primary cell wall biogenesis may be related to hairy root growth and the compartmentalized storage of metabolites.

Notably, the hormone-mediated signaling pathway and abscisic acid metabolism were also enriched, suggesting potential crosstalk between MeJA and other hormone signals in regulating secondary metabolism.

KEGG pathway analysis revealed a close relationship among DEGs enriched in C. pilosula metabolic pathways under MeJA treatment (Figure 7c). The results demonstrated that DEGs under MeJA treatment were highly enriched in phenylpropanoid biosynthesis, indicating enhanced plant defense mechanisms. Additionally, lipid metabolism signaling and antioxidant/detoxification systems were widely enriched in the treatments, reflecting basic adaptive mechanisms under elicitor treatment. KEGG enrichment analysis identified a significant enrichment in flavonoid biosynthesis, as well as in the biosynthesis pathways of stilbenoids, diarylheptanoids, and gingerols, monoterpenoid biosynthesis, and carotenoid biosynthesis.

Transcriptomic analysis identified 86 transcription factors that were significantly different across 20 transcription factor families in the CK vs. MeJA comparison. The AP2/ERF gene family was the most represented among these transcription factors (Figure S5)

2.7. Annotation of Pathway Genes and Transcription Factors

Lobetyolin is composed of lobetyol and a UDP-glucoside, though its biosynthetic pathway is not fully elucidated. Current models suggest that fatty acids undergo sequential reactions to form unsaturated fatty acids, which are subsequently processed by acetylenase into lobetyol; finally, UGT73 family glycosyltransferases catalyze the conjugation of lobetyol with UDP-glucose to yield lobetyolin [31]. Accordingly, this study characterized a putative lobetyolin biosynthetic pathway and identified candidate enzymatic genes. In the proposed pathway, acetyl-CoA is converted to malonyl-CoA by ACC, which ACP/FAS converts into saturated fatty acids. FAD subsequently desaturates these into unsaturated fatty acids, which are processed into lobetyol via CYP-mediated acetylenase reactions. Finally, lobetyol reacts with UDP-glucose via UGT73 family enzymes to generate lobetyolin.

A total of 33 genes potentially involved in lobetyolin biosynthesis were annotated, 4 of which contain GCC-boxes (Supplementary Table S7). Their expression levels across treatments were visualized in Figure 8. The MeJA treatment group showed significant upregulation of early biosynthetic genes (e.g., FAS2, FAS3, ACP2, ACP4) and desaturase genes (FAD3, FAD4) versus the control (CK) group. Notably, this treatment exerted the most potent induction effect on post-modification genes: the expression of multiple CYP genes (e.g., CYP86A1, CYP86B1, CYP92C6) and UGT73 family genes (e.g., UGT73B4, UGT73C4, UGT73C11) increased dramatically.

Figure 8.

Figure 8

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Proposed biosynthetic pathway of lobetyolin. The square heatmap shows the expressions of key enzyme genes in C. pilosula hairy roots under MeJA elicitor treatments.

2.8. Screening of Key AP2 Transcription Factors

To explore transcription factors involved in lobetyolin biosynthesis in C. pilosula, a total of 24 AP2/ERF transcription factors were identified as significantly differentially expressed (Supplementary Table S8). Of these candidates, only CpERF140 and CpERF138 were successfully cloned. These two genes exhibited high homology (Supplementary Table S9). Motif analysis revealed that they possess identical motifs and are both characterized by a single exon. Promoter cis-element analysis showed that both genes harbor regulatory motifs associated with MeJA response, abscisic acid signaling, and stress responses. Based on the presence of these functionally relevant elements, CpERF140 and CpERF138 were prioritized for subsequent functional characterization.

2.9. Analysis of Subcellular Localization of CpERF140 and CpERF138

Determining a protein’s subcellular localization is fundamental to elucidating its functional mechanism. To achieve this aim, we engineered C-terminal YFP fusion vectors for CpERF140 and CpERF138, which were then transiently expressed in the leaves of Nicotiana benthamiana (Figure 9a, Supplementary Table S9). In contrast to the fluorescence signal of the free PHB-YFP control, which was distributed across both the cytoplasm and nucleus, the signals from the CpERF140-YFP and CpERF138-YFP fusions were localized solely within the nucleus. This observation confirms that CpERF140 and CpERF138 are nuclear-localized proteins.

Figure 9.

Figure 9

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(a) Subcellular localization of CpERF138 and CpERF140 in N. benthamiana leaves. Confocal microscopy was used to visualize fluorescence signals. Panels display the YFP signal, the bright-field image, and the merged view. Scale bar = 25 µm. (b) Yeast one-hybrid (Y1H) assay showing the interaction of CpERF138 and CpERF140 with the GCC-box motif. (α) Yeast strains harboring empty vectors (pHis2-GCC-box + AD), a positive control (pHis2-P53 + AD-rec-p53), or the candidate gene constructs were grown on medium lacking 3-AT. (β) Growth of the same strains on selective medium containing 10 mM 3AT.

2.10. Yeast One-Hybrid Assay of CpERF140 and CpERF138

The DNA-binding specificity of CpERF140 and CpERF138 was assessed via a yeast one-hybrid assay. Fusion constructs were engineered and co-transformed with a pHIS2 reporter carrying the GCC-box into Y187 yeast. While all strains grew on non-selective medium, only those co-expressing either CpERF140 or CpERF138 in conjunction with the GCC-box reporter exhibited growth on stringent selective medium (SD/-Trp/-Leu/-His + 10 mM 3-AT) (Figure 9b, Supplementary Table S9). This specific growth phenotype provides direct evidence that both proteins bind specifically to GCC-box motifs, implicating them in the transcriptional regulation of downstream genes. harboring this cis-element.

3. Discussion

3.1. The AP2/ERF Transcription Factor Family: Constructing a Regulatory Network for Lobetyolin Biosynthesis

The AP2/ERF superfamily represents a significant transcription factor family in plants, serving as a pivotal integrative node that connects environmental signals with the regulation of secondary metabolic gene expression [32]. Comprehensive analysis of the C. pilosula genome revealed 175 AP2/ERF transcription factors, with 169 (CpERF1–CpERF169) localized to the chromosomes. Phylogenetic reconstruction grouped them into the AP2, ERF (subdivided into ERF and DREB), and RAV subfamilies. The conserved domain architecture, defined by motifs 1, 2, and 4 was identified, with motif 1 annotated as the diagnostic DNA-binding motif. Cis-regulatory element analysis demonstrated that these genes are enriched for hormone-responsive motifs, including 123 and 128 members that harbor motifs associated with MeJA and abscisic acid response, respectively. Furthermore, auxin-responsive elements were identified in 86 members, salicylic acid-responsive elements in 75, drought stress-responsive elements in 84, and low-temperature-responsive elements (LTR) in 129. This indicates that most C. pilosula AP2/ERF gene family members are regulated by hormones and respond to various environmental stressors. Gene duplication serves as a primary driver of the expansion and functional diversification of TF families [33]; in this study, 28 duplication events were identified within the CpAP2/ERF family, comprising 27 segmental duplications and a single tandem duplication. This pattern suggests that segmental duplication events have played a more significant role than tandem amplifications in expanding this family [34].

Extensive studies have established that ERF subfamily members serve as key mediators linking stress and hormone signaling to secondary metabolism. This regulatory function is primarily mediated by their interactions with specific cis-elements, such as the GCC-box or DRE/CRT motifs, embedded within the promoters of their target genes [10]. For example, in Zanthoxylum bungeanum, MeJA-induced upregulation of ZaERF166 expression coincided with a marked increase in leaf terpenoids, suggesting a regulatory role in terpenoid metabolism [35]. Similarly, in Panax notoginseng, PnERF2 and PnERF3 exhibited significant correlation with key genes in the ginsenoside biosynthetic pathway, specifically dammarenediol-II synthase (DS) and squalene epoxidase (SE), in MeJA-induced roots [36]. In the present study, the finding that numerous CpAP2/ERF promoters are enriched with MeJA- and stress-responsive elements suggests an inherent capacity to integrate multiple signaling pathways.

3.2. Differential Mechanisms of MeJA Treatment in Regulating Lobetyolin Synthesis

The pronounced inductive effect of MeJA on lobetyolin is consistent with its recognized function as a principal regulator of plant defense mechanisms and the reprogramming of secondary metabolism, effectively activating the biosynthesis of various defense compounds such as terpenoids, alkaloids, and phenylpropanoids [37]. The transcriptomic data obtained in this study revealed significant enrichment of the jasmonic acid (JA) response pathway within secondary metabolic processes, providing robust evidence for the extensive involvement of MeJA in secondary metabolite synthesis. This regulatory pattern suggests that JA signaling, acting as a central defense modulator, can coordinately enhance the entire biosynthetic pipeline—from precursor supply to final product modification—thereby driving the rapid accumulation of defense compounds such as lobetyo. Furthermore, the enrichment of glycosyltransferase activity identified in the GO analysis suggests that MeJA may modulate the biosynthesis and modification of glycosidic compounds, such as lobetyolin, by regulating glycosylation processes that facilitate the rapid conversion of lobetyol into its glycosylated form. Given that glycosyltransferases are key enzymes involved in the glycosylation modification of natural products, this observation is consistent with patterns reported in other medicinal plant species. In Glycyrrhiza uralensis, MeJA induced the MYB transcription factors GlMYB4 and GlMYB88, which positively regulate flavonoid synthesis in licorice cells [38]. Similarly, in Panax ginseng, MeJA treatment strongly induced PgNAC72 expression. This induction consequently led to a marked increase in total saponin content, especially dammarane-type ginsenosides, primarily through the upregulation of dammarenediol synthase (PgDDS), a pivotal enzyme in the ginsenoside biosynthetic pathway [39]. These findings collectively indicate that MeJA treatment significantly enhances the synthesis of plant secondary metabolites across varying medicinal species, including C. pilosula. Here, MeJA treatment not only upregulated early precursor supply genes (e.g., FAS, ACP) in the proposed lobetyolin pathway but also more significantly upregulated key modification genes (e.g., CYP86A1, UGT73B4). These results suggest that MeJA does not merely enhance basal metabolism; instead, it likely utilizes its core transcriptional module—involving JAZ repressor degradation and downstream TF activation—to systematically coordinate the entire metabolic chain from initial precursors to final glycosylated products, thereby driving the efficient production of defensive polyacetylenes such as lobetyolin [40,41].

3.3. Potential Role of CpERF140 and CpERF138 in Lobetyolin Biosynthesis

Cross-omics screening identified CpERF140 and CpERF138 as core AP2/ERF genes differentially expressed and simultaneously upregulated by MeJA. Their nuclear localization and GCC-box binding affinities were experimentally validated in this study. Based on these findings, we propose a working model in which cellular perception of MeJA stress signals triggers signal transduction cascades—potentially via the COI1-JAZ module—that relieve the inhibition of downstream transcription factors, thereby activating the expression or activity of CpERF140/138 [42].

Once activated, these ERF proteins specifically bind to GCC-box motifs within the promoters of key lobetyolin biosynthetic genes, such as CpFAS1, CpFAD2, and CpCYP86B1, thereby driving their transcription and ultimately promoting lobetyolin accumulation. The coordinated upregulation of CpERF140/138 alongside multiple putative biosynthetic genes (e.g., CpCYP86B1, CpUGT73B4) following MeJA treatment provides robust co-expression evidence supporting this regulatory model (Figure 8). This pattern is consistent with their potential role as transcriptional regulators within a coordinated biosynthetic module.

The current findings provide an initial framework for what is likely a sophisticated regulatory network, given that functional redundancy, synergy, and antagonism are common among AP2/ERF family members. For example, different ERF members exhibit functional redundancy in Arabidopsis defense responses [43]. In future experiments, to further clarify the key role of these factors in the lobetyolin biosynthetic pathway, chromatin immunoprecipitation sequencing (ChIP-seq) could be employed to identify genome-wide direct target genes of CpERF140/138. Yeast two-hybrid or co-immunoprecipitation experiments could be used to investigate protein–protein interactions and identify upstream regulators and cofactors. CRISPR-Cas9 or RNAi technology represents a viable approach for generating knockout or knockdown mutants in C. pilosula hairy roots, enabling in planta validation of their requirement for lobetyolin biosynthesis [44]. Additionally, investigating potential hierarchical or synergistic relationships with other co-expressed transcription factor families (e.g., MYB, WRKY) may facilitate the construction of a comprehensive transcriptional regulatory network governing lobetyolin biosynthesis.

4. Materials and Methods

4.1. Retrieval and Identification of CpAP2/ERF Sequences

The study obtained the C. pilosula genome assembly from a public repository (http://medicinalplants.mau.edu.cn/unpublished_data/Genome/Assembly/Codonopsis%20pilosula-genome.fasta, Date of visit: 6 January 2024) [45]. To identify candidate AP2/ERF members, a Hidden Markov Model (HMM) search was conducted against the proteome using the AP2 domain profile (PF00847) from the Pfam database, with HMMER v3.2.1 under an E-value threshold of <1 × 10−2 [46]. The resulting genes were systematically designated CpERF1 through CpERF169 based on their genomic identifiers. Quantification of the expression levels of the CpAP2/ERF genes across various samples was achieved by virtue of the FPKM method. Subsequently, a heatmap visualizing these expression profiles was constructed using TBtools 1.120.

4.2. Phylogenetic Analysis and Synteny Analysis

For phylogenetic reconstruction, the AP2 domain sequences from C. pilosula and A. thaliana were aligned with MAFFT7.526. A maximum-likelihood tree was then generated via IQ-TREE, employing the JTTDCMult + F + R4 substitution model and 1000 bootstrap replicates to test branch support [47]. The resulting tree, comprising 175 CpAP2/ERF members and reference Arabidopsis sequences, was visualized and annotated in Evolview. Moreover, the intraspecific synteny relationships within the CpAP2/ERF family were examined and depicted usingTBtools.v1.120 [48].

4.3. Analysis of Gene Structure, Conserved Motifs, and Promoter Cis-Elements

Gene structure architectures, including exon–intron configurations, were visualized using the genome annotation (GFF) file. Conserved protein motifs were identified via the MEME Suite online platform (https://meme-suite.org). To explore potential transcriptional regulatory mechanisms, putative promoter regions—comprising the 2.0 kb genomic sequences upstream of translation start sites—were defined. Following extraction, these promoter sequences were analyzed for cis-acting regulatory elements using the PlantCARE database [49]. The distribution of stress-responsive cis-elements within individual promoters was subsequently mapped with TBtools v1.120 [48].

4.4. Induction of C. pilosula Hairy Roots

Seeds of C. pilosula (College of Chinese Medicinal Materials, Jilin Agricultural University) were surface-sterilized using a multi-step protocol: 70% ethanol for 20 s, followed by five to six rinses with sterile water; disinfection in 2% sodium hypochlorite for 20 min; and a final series of five to six sterile water washes (3–5 min each). Following sterilization, the seeds were germinated on Murashige and Skoog (MS) medium (5% sucrose, 1% agar; pH 5.8–6.0) and maintained at 25 ± 1 °C with a 16 h photoperiod for 4 weeks to obtain aseptic seedlings. Stems and roots from four-week-old aseptic seedlings were wounded and pre-cultured on 1/2 MS medium in the dark for two days. Explants were then inoculated with A. rhizogenes strain PHB-C58C1 (OD600 = 0.6–0.8) for 15 min, followed by 2 days a co-cultivation on solid 1/2 MS medium. Following inoculation, the explants were placed onto solid 1/2 MS medium supplemented with 500 mg/L cefotaxime sodium (Cef) to remove bacteria; the Cef concentration was reduced incrementally every 7 days until reaching 0 mg/L to facilitate hairy root induction. All steps were carried out in the dark and maintained at a temperature of 25 ± 1 °C.

4.5. Treatment of C. pilosula Hairy Roots with Methyl Jasmonate (MeJA) and Sample Preparation

Consistent with previously established methodologies, hairy root cultures were elicited with 100 μM methyl jasmonate (MeJA) [29] for a duration of six days, employing three biological replicates per treatment group. After the treatment phase, all samples underwent liquid nitrogen freezing, followed by being preserved at −80 °C. Tissues were then lyophilized and homogenized into a fine powder using a mixer mill equipped with zirconia beads; then, 50 mg of the resultant dry powder was extracted with 1 mL of 100% methanol. The resulting suspension was sonicated and centrifuged. The supernatant was then collected, diluted with methanol, and passed through a 0.22 μm PTFE membrane filter (JINTENG, Tianjin, China) before analysis via UPLC-MS/MS.

4.6. UPLC-MS/MS Conditions

Liquid chromatographic separation was conducted with a mobile phase comprising 0.1% methanol (A) and water (B) at 0.2 mL/min. The gradient elution profile was programmed as follows: 0–2 min, 5–20% A; 2–10 min, 20–100% A; 10–13 min, 100% A; and 13–15 min, 100–10% A. The column temperature was 35 °C, and the sample injection volume was 2 μL. Heated electrospray ionization (HESI) served for mass spectrometric detection in negative ion mode. Operating parameters were optimized as follows: spray voltage, 3.00 kV; scan range, 100–1500 m/z; full MS resolution, 70,000; and MS/MS resolution, 17,500. Additional settings included a sheath gas flow of 35 arbitrary units (arb), an auxiliary gas flow of 10 arb, an ion transfer tube temperature of 320 °C, an auxiliary gas heater temperature of 310 °C, and a collision energy of 35 eV.

4.7. Transcriptomic Analysis(Gene Denovo, Guangzhou, China)

Total RNA was extracted from MeJA-elicited and control C. pilosula hairy roots using a specialized plant RNA isolation kit. Biological replicates were designated as CK1–3 (controls) and MeJA1–3 (treated samples). RNA concentration and purity assessment relied on a Nanophotometer N50 (Implen, Munich, Germany); only high-integrity samples meeting strict quality thresholds were utilized for downstream library construction. Following extraction, ribosomal RNA (rRNA) was depleted, and the remaining mRNA was enriched using capture beads, thermally fragmented, and reverse-transcribed into first-strand cDNA. Then, after second-strand cDNA synthesis, end repair, poly-A tailing, and adapter ligation were conducted in sequence. Target fragments were size-selected and purified with Hieff NGS® DNA Selection Beads (Yeasen Biotechnology, Shanghai, China) before PCR-based library amplification and high-throughput sequencing.

To identify DEGs, an analysis was conducted on the Express Analyst platform using thresholds of |log2 (fold change)| ≥ 1 and a p-value < 0.05. Visualization of the results, including volcano plots and KEGG enrichment analysis, was performed using integrated bioinformatic web tools. Further functional assessments—namely GO enrichment and KEGG pathway analysis—were performed via TBtools-II v2.142. The same software was employed to generate heatmaps visualizing DEG expression profiles. Throughout the study, the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) method was adopted for gene expression quantification.

4.8. Subcellular Localization and the Transcriptional Autoregulation Assay

The coding sequences of CpERF140 and CpERF138 were amplified via PCR with primers flanked by HindIII and SacI restriction sites (Table S9). To generate fusion constructs, the amplified fragments were cloned into the PHB-YFP vector, yielding PHB-YFP-CpERF plasmids, which, after verification, were transformed into A. tumefaciens strain GV3101. For infiltration, a single colony was cultured, and the cells were collected and resuspended in an induction buffer (2 mM MgCl2, 2 mM MES, 150 μM acetosyringone) to a final OD600 of 0.4–0.6. This suspension was used to infiltrate leaves of 30-day-old N. benthamiana plants. The bacterial suspension was injected into the abaxial leaf surface using a sterile needleless syringe until a visible semi-transparent infiltration zone (1–2 cm diameter) formed. Excess suspension was blotted away. Following a 12 h dark period, infiltrated plants were returned to standard growth conditions (16 h light/8 h dark cycle). After 48–72 h, fluorescence signals were visualized via a Stellaris 5 confocal laser scanning microscope. YFP signals were captured in the green using 405 nm excitation and 420–480 nm emission detection.

4.9. Yeast One-Hybrid Assay

After the amplification of coding sequences of CpERF140 and CpERF138 with primers containing EcoRI and BamHI restriction sites, the resulting fragments were subsequently cloned into the pGADT7 vector to form prey constructs. Concurrently, a double-stranded DNA fragment harboring the GCC-box motif was designed with SacI/EcoRI overhangs, annealed, and ligated into the pHIS2 vector to produce the pHIS2-GCC-box reporter. The bait (pHIS2-GCC-box) and prey (pGADT7-CpERF) constructs, alongside empty vector controls (pHIS2 + pGADT7) and a positive control (pHIS2-P53 + pGADT7-p53), were co-transformed into Y187 yeast competent cells. Transformants were selected on SD/-Leu/-Trp medium at 30 °C in the dark for three days. The optimal concentration of 3-amino-1,2,4-triazole (3-AT) required to suppress background growth was subsequently determined using strains containing the pHIS2-GCC-box reporter. Finally, yeast strains carrying the target gene constructs were tested for growth on selective SD/-Leu/-Trp/-His medium supplemented with the predetermined concentration of 3-AT.

4.10. Statistical Analysis

Statistical analyses relied on IBM SPSS 27.0 Statistics (Armonk, NY, USA). For group comparisons, either the two-tailed Student t-test or Duncan’s multiple range test (following one-way ANOVA) was employed. p ≤ 0.05 indicated statistical significance (p * ≤ 0.05; p ** ≤ 0.01; p *** ≤ 0.001). All data represent findings from three independent biological replicates and are expressed as mean ± SD.

5. Conclusions

In the study, the AP2/ERF transcription factor family in Codonopsis pilosula was characterized on a genome-wide scale for the first time. This comprehensive analysis identified 175 family members and provided a systematic evaluation of their genomic features, including chromosomal distribution, phylogenetic classification, conserved domains, and cis-element characteristics. Using a hairy root genetic transformation system combined with methyl jasmonate (MeJA) treatment, we confirmed that MeJA significantly enhances lobetyolin accumulation. Integrated transcriptomic and targeted metabolomic analyses further revealed that MeJA activates the JA signaling pathway and related secondary metabolic pathways, regulating the expression of key biosynthetic genes, including glycosyltransferases and cytochrome P450 enzymes. The study successfully screened two AP2/ERF transcription factors, CpERF140 and CpERF138, which were strongly induced by MeJA elicitation. Experiments confirmed their nuclear localization and specific binding to the GCC-box regulatory element, suggesting their potential involvement in lobetyolin biosynthesis by regulating downstream target genes. These findings not only provide preliminary comprehension into the regulatory role of the AP2/ERF family in C. pilosula secondary metabolism but also establish a theoretical basis for further elucidation of the lobetyolin biosynthetic pathway, discovery of key regulatory genes, and molecular breeding in C. pilosula. Future work will utilize functional validation, interacting protein screening, and genetic modification to fully characterize the specific functions and mechanisms of CpERF140/138 within the metabolic regulatory network.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants15040530/s1.

plants-15-00530-s001.zip (7.1MB, zip)

Author Contributions

Conceptualization, X.Z. (Xiaobi Zhang), P.W., L.Z., J.X. and J.L.; methodology, X.Z. (Xiaobi Zhang), P.W., J.Z. (Jiaxin Zhang) and J.L.; software, X.Z. (Xiaobi Zhang), X.Z. (Xinyue Zhang) and Y.W.; formal analysis, X.Z. (Xiaobi Zhang), P.W., L.Z., J.X. and J.L.; investigation, Z.P.; resources, P.D.; data curation, X.Z. (Xiaobi Zhang); writing—original draft preparation, X.Z. (Xiaobi Zhang); writing—review and editing, Y.Y., J.Z. (Jian Zhang) and P.D.; visualization, Y.S., Y.W.; project administration, Y.Y., J.Z. (Jian Zhang) and P.D.; funding acquisition, J.Z. (Jian Zhang) and P.D. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data are contained within the article and Supplementary Material.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

The authors declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Key Research and Development Program of China (2022YFC3501703), the Jilin Agricultural University High-Level Researcher Grant (JLAUHLRG20102006), the Jilin Provincial Department of Human Resources and Social Security grant (No. 201020012), and The 111 Project, Northeast Advantageous Characteristic Resources and Health Food Discipline Innovation Introduction Base Grant (No: D23007).

Footnotes

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Supplementary Materials

plants-15-00530-s001.zip (7.1MB, zip)

Data Availability Statement

Data are contained within the article and Supplementary Material.

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