Next Article in Journal
Distinct Morphokinetic Signature of Human Embryos with Chromosomal Mosaicism
Previous Article in Journal
The Role of VEGFA in T2DM-Nephropathy: A Genetic Association Study and Meta-Analysis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 16
Issue 11
10.3390/genes16111387
genes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

MeJA Elicitation on Flavonoid Biosynthesis and Gene Expression in the Hairy Roots of Glycyrrhiza glabra L.

by
Yutao Zhu
1,*,
Bohan Wang
1,
Bingyi Xue
1,
Runqian Wang
1,
Ganlin Tang
1,
Tao Zhu
1,
Mei Zhao
1,
Taotao Li
1,
Chunli Liao
1,
Huamin Zhang
1,
Dongxiao Liu
1,
Jianhua Chen
2 and
Lianzhe Wang
1
1
College of Life Science and Engineering, Henan University of Urban Construction, Pingdingshan 467036, China
2
Pingdingshan Academy of Agricultural Sciences, Pingdingshan 467003, China
*
Author to whom correspondence should be addressed.
Genes 2025, 16(11), 1387; https://doi.org/10.3390/genes16111387
Submission received: 23 October 2025 / Revised: 8 November 2025 / Accepted: 14 November 2025 / Published: 18 November 2025
(This article belongs to the Section Plant Genetics and Genomics)
Browse Figures
Review Reports Versions Notes

Abstract

Background/Objectives: Licorice (Glycyrrhiza glabra L.) is a highly important medicinal plant that is widely used in China owing to its active ingredients. Its main active components are flavonoids, including liquiritigenin, liquiritin and licochalcone A. The hairy roots (HRs) induced by Agrobacterium rhizogenes are a commonly used chassis in synthetic biology to enhance the production of active compounds in medicinal plants. Methods: A biosynthesis system to acquire the active ingredients of G. glabra was established using an HR culture system. It employed a transcriptome analysis to identify the change in gene expression following treatment with methyl jasmonate (MeJA). Results: After 28 days of suspension culture, the biomass of HRs increased by approximately 34.5-fold and reached 1.83 g/100 mL flask. Treatment with MeJA significantly increased the contents of liquiritigenin, liquiritin, and glabridin in the HRs. The transcriptome data indicated that MeJA activated the flavonoid biosynthetic pathway genes in the HRs, which was largely consistent with the qRT-PCR results. Furthermore, the overexpression of the GgCHS6 gene substantially increased the content of flavonoids in HRs. Conclusions: Collectively, this study established an HR system to biosynthesize the active ingredients of G. glabra using metabolic engineering and genetic engineering techniques and provides several valuable candidate genes for further functional study.

1. Introduction

Licorice is the dried root and rhizome of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L., which are plants in the legume family; it has been used medicinally for thousands of years in China [1]. The flavonoids in licorice are more diverse, and the primary pharmacologically active compounds include liquiritigenin, isoliquiritigenin, liquiritin, licochalcone A, and glabridin [2]. However, the reduction in wild licorice resources and the decline in the quality of cultivated varieties have severely constrained the acquisition of medicinal components derived from this plant. Given the critical importance of the active ingredients of licorice in medical and food applications, their rapid and efficient biosynthesis through synthetic biology methods is highly significant and of practical value.
HRs are types of root-like tissues that are produced at a wound site after plants have been infected by Agrobacterium rhizogenes [3]. Owing to their advantages, such as a short growth cycle, high degree of differentiation, hormone-free culture, genetic stability, and ease of manipulation, HRs have become a commonly used plant chassis for the biosynthesis of plant secondary metabolites [4,5,6]. There are more than 200 medicinal plants that have established HR culture systems for the biosynthesis of saponins, terpenoids, phenylpropanoids, alkaloids, flavonoids, anthraquinones, and polysaccharides [7]. There are typically economically valuable plant secondary metabolites at low concentrations in both plant tissues and HRs under natural growth conditions. Altering the culture environment, such as the type of light and its intensity, temperature, and rotation speed, can enhance the active components in HRs. For example, Afsharzadeh et al. demonstrated that the use of red-blue composite light significantly increased the content of total phenolic in HRs of G. glabra [8]. In addition, the incorporation of exogenous plant hormones, chitin, chitosan, and metal particles as elicitors in the HR cultivation system can also significantly enhance the content of plant secondary metabolites in HRs [7]. Among the commonly used in vitro elicitors, the plant hormone methyl jasmonate (MeJA) has been widely employed to dramatically increase the production of plant secondary metabolites [9,10].
Numerous studies have shown that the exogenous application of MeJA can enhance the content of secondary metabolites in HRs. Wongwicha et al. (2011) demonstrated that the addition of MeJA to the HR culture system of G. inflata significantly increased the content of glycyrrhizin [11]. MeJA can significantly promote the gene expression of genes for the tanshinone and phenolic acid biosynthetic pathways in the HRs of Salvia przewalskii Maxim., thus leading to a substantial increase in the contents of caffeic acid, salvianolic acid B, and tanshinone IIA [12]. Similarly, the content of total phenolics and flavonoids in Ficus carica. Cv. Siah HRs were increased by MeJA elicitation, which may be the result of a significant enhancement in the expression of chalcone synthase (CHS) and flavonoid 3-hydroxylase (F3H) genes [13]. In addition, Bao et al. (2022) found that MeJA treatment of broccoli (Brassica oleracea var. italica) HRs activated the expression of the key genes that regulate the biosynthesis of sulforaphane, which significantly enhances its production [14].
Flavonoids are the primary active components in licorice, and they possess a variety of significant pharmacological activities. In this study, the addition of MeJA can promote an increase in the contents of isoliquiritigenin, liquiritigenin, liquiritin, licochalcone A, and glabridin in the HRs, while salicylic acid (SA) was less effective at promoting such effects. A transcriptome analysis revealed that treatment with MeJA activated the flavonoid biosynthetic pathway in the HRs, and quantitative reverse-transcription PCR (qRT-PCR) data confirmed the significant upregulation of key flavonoid biosynthetic genes. Furthermore, the overexpression of GgCHS6 substantially enhanced the levels of flavonoids in the HRs. Collectively, this study provides a new perspective to explore the complex molecular mechanism of the accumulation of flavonoids mediated by MeJA and provides several valuable candidate genes for further functional study.

2. Materials and Methods

2.1. Plant Materials

G. glabra seeds were collected from Altay, Xinjiang, China. Plump seeds were soaked in concentrated sulfuric acid for 60 min, thoroughly rinsed with tap water, and then transferred to sterile centrifuge tubes. Subsequent sterile washes included the following: (1) three rinses with sterile water, (2) 10 min of disinfection in 75% ethanol, (3) 10 min treatment with 10% sodium hypochlorite, and (4) three final sterile water rinses. Finally, the sterilized seeds were then cultured on 1/2 MS solid medium in a growth chamber maintained at 25 °C with a 16/8 h (light/dark) photoperiod.

2.2. Inoculation of A. rhizogenes K599

This study used the A. rhizogenes K599 strain, which is resistant to streptomycin and harbors the Ri plasmid pRi2659, to induce the HRs [15]. A volume of 1 mL of activated K599 bacteria was added to 100 mL of LB liquid medium that contained 50 μg/mL streptomycin. The culture was then incubated at 28 °C and 200 rpm in the dark in a shaker for 24–48 h. The activated culture was then centrifuged and resuspended in a 1/2 MS liquid medium that contained 100 μmol/L acetosyringone (AS), with an OD600 that ranged from 0.4 to 1.0.

2.3. Induction and Parameter Optimization of the HRs

The roots of 5-day-old G. glabra seedlings were excised to leave the hypocotyls with cotyledons as explants. The explants were immersed in a suspension of K599 for 10 min and then placed on 1/2 MS solid medium that contained 100 μM AS for culture in the dark for 2 d. After 2 d, the explants were rinsed with 1/2 MS liquid medium that contained 500 mg/L Timentin and transferred to 1/2 MS solid medium that contained 500 mg/L Timentin for induction culture. The induction rate of HRs was counted after 7 d [16]. Various parameters, such as the density of bacterial suspension (OD600 of 0.4, 0.6, 0.8, and 1.0), infection time (5, 10, 15, and 20 min), types of culture media (MS, 1/2 MS, 6,7-V, and B5), and concentration of AS (50, 100, 150, and 200 μmol/L), were studied to determine the optimal conditions to induce the HRs in G. glabra.

2.4. PCR Analysis to Screen the Transgenic HRs

The T-DNA that contained the rolB and rolC was responsible for the induction of HRs in plants. The successfully transformed HRs were screened by extracting the genomic DNA of the HRs using the CTAB method, followed by PCR analysis to detect the presence of rolB and rolC genes in the T-DNA (GenBank: Z29365.1) [17,18,19]. Additionally, the VirG gene was also detected to confirm the absence of A. rhizogenes in the HRs [20]. The primers are listed in Table S1. The PCR was conducted by amplification with an initial denaturation at 95 °C for 3 min, followed by 32 cycles of denaturation at 94 °C for 25 s, annealing at 57 °C for 25 s (rolB at 57 °C, rolC at 55 °C, and VirG at 56 °C), and extension at 72 °C for 15 s with a final extension of 72 °C for 5 min using a PCR instrument (Bio-Rad, Hercules, CA, USA).

2.5. Analysis of the Growth Kinetics of HRs

A total of 2–3 cm (approximately 50 mg fresh weight) of successfully transformed and young HR shoots were used to inoculate them into 100 mL of 1/2 MS liquid medium that contained 3% sucrose and 200 mg/L Timentin (using a 250 mL conical flask). All the cultures were incubated at 150 rpm on a rotary shaker at 28 ± 1 °C. The growth curve was analyzed by harvesting three independent fresh HRs at 7, 14, 21, 28, 35, and 42 days to determine their fresh weight (FW).

2.6. HRs Treatments by Elicitors

For the induction experiment, the HRs were inoculated into 100 mL of 1/2 MS liquid medium that contained 200 mg/L Timentin in 250 mL conical flasks for subculture to facilitate the subsequent induction treatments. The flasks were shaken continuously at 150 rpm on a rotary shaker and cultured in the dark at 28 ± 1 °C for 28 days. MeJA (Sigma-Aldrich, St. Louis, MO, USA) and salicylic acid (SA) (Sigma-Aldrich) were dissolved separately in absolute ethanol and 20% ethanol, respectively, to prepare 100 mM stock solutions, which were filter-sterilized and added to the medium to achieve a final concentration of 100 μM [21]. Samples were collected at 0, 3, and 5 days after the elicitor treatment, and there were three replicates for each treatment group. The harvested HRs were weighed to determine their FW, rapidly frozen in liquid nitrogen, and stored at −80 °C until further analysis.

2.7. Use of HPLC-MS to Quantify the Contents of Flavonoids in the HRs

The active compounds in HRs were detected as previously described [22,23] with modifications. In brief, 100 mg of freeze-dried HRs were vortexed to mix with 1 mL of extraction solvent (methanol/water = 4:1 [v/v]) for 5 min for extraction. Following 10 min of ultrasonication and centrifugation at 1000 rpm, the supernatant (1 mL) was purified using 100 mg of PSA and filtered through a 0.22 μm membrane into an autosampler vial. The contents of isoliquiritigenin, liquiritigenin, liquiritin, licochalcone A, and glabridin in the HRs were determined using an Agilent 6495D triple quadrupole liquid chromatography-mass spectrometry (LC-MS) system (Agilent 1290 Infinity II, Agilent Technologies, Santa Clara, CA, USA), which was equipped with a C18 column (2.1 × 50 mm, 1.8 µm). Mobile phases A and B were acetonitrile and 0.1% formic acid in water, respectively. A linear gradient elution program was employed as follows: 0 min: 5% A; 4 min: 95% A; 5 min: 95% A; 5.1 min: 5% A; and 6.1 min: 95% A with a flow rate of 0.2 mL/min. The injection volume was 2 μL. The mass spectrometry parameters of the flavonoid standards are shown in Table S2.

2.8. Transcriptome Analysis and Gene Annotation

HRs treated with 100 μM MeJA were chosen for further transcriptome analysis. The HRs that had grown to day 28 were treated with 100 μM MeJA and harvested at 0, 3, and 5 d. There were three biological replicates for each treatment. The HRs were quickly frozen in liquid nitrogen and then stored at −80 °C until the transcriptome analysis. A total of 1 μg total RNA was obtained from the HRs and used to construct RNA-seq libraries. An Illumina HiSeq/Illumina Novaseq/MGI2000 instrument (Illumina, San Diego, CA, USA) with a 2 × 150 paired-end (PE) configuration was used for the high-throughput sequencing. The raw sequencing reads in the FASTQ format were processed using Cutadapt (v. 1.9.1) to remove the technical sequences and low-quality bases, which can introduce bias and reduce the reliability of analysis [24]. Clean reads were aligned to the G. uralensis Fisch. Reference genome [25] using HISAT2 (v. 2.2.1) [26]. A transcriptome assembly was performed using StringTie (v. 1.3.3b) [27], and the novel transcripts were identified by a comparison of the assembled transcripts to the reference annotation using cuffcompare (v. 2.2.1) [28]. Differentially expressed genes (DEGs) were identified using DESeq2 (v. 1.34.0) (FDR < 0.05 and |log2FC| ≥ 1) [29]. A functional enrichment analysis interprets the biological significance of DEGs by mapping them to the Gene Ontology (GO) terms [30] and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways [31]. The DEGs were clustered using K-means clustering of the DEGs based on their Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values using the Mfuzz package in R [32].

2.9. Validation of Gene Expression by Quantitative Real-Time PCR

To verify the reliability of the RNA-seq data, an RT-qPCR expression analysis was used to identify candidate genes on the KEGG synthesis pathway, including GgPAL3, GgCHS6, GgCHS7, GgCHI1, GgUGT, GgHI4OMT, GgCYP81E1, GgCYP81E4, and GgVR1. An RNA extraction kit (Biomed Gene Technology, Co., Ltd., Beijing, China) was used to extract the total RNA from HRs. The cDNA was synthesized from 1000 ng total RNA using an M5 First Strand cDNA Synthesis Kit (Mei5 Biotechnology, Co., Ltd., Beijing, China). A qRT-PCR was conducted using 2 × M5 HiPer Realtime PCR Super mix with Low (SYBRgreen, with anti-Taq) (Mei5 Biotechnology, Co., Ltd.) on a QuantStudio 6 Pro system (Applied Biosystems, Waltham, MA, USA). Gene expression was normalized using actin (EU190972.1) as a reference gene to normalize the cDNA [33]. The relative gene expression was calculated using the 2−ΔΔCt method [34]. All the qRT-PCR experiments were performed in three biological replicates. All the primers are listed in Table S1.

2.10. Construction of GgCHS6 That Overexpresses the HRs

The coding sequence of the chalcone synthase gene GgCHS6 with Nco I and Spe I was inserted into the pCAMBIA 1302 vector that harbored hygromycin phosphotransferase (hptII) and green fluorescent protein (GFP) under the control of the CaMV 35S promoter (Figure S1). The construct was then introduced into A. rhizogenes K599 by the freeze–thaw method [35], and the transgenic HRs were induced as described in Section 2.3. The HRs transformed with the 1302 empty vector were used as the control. A total of 2–3 cm HRs were inoculated into 100 mL of 1/2 MS liquid medium that contained 3% sucrose and 200 mg/L Timentin (using a 250 mL conical flask). After 28 d of cultivation, the HRs were harvested and analyzed by high-pressure liquid chromatography-mass spectrometry (HPLC-MS) to determine the contents of isoliquiritigenin, liquiritigenin, liquiritin, licochalcone A and glabridin.

2.11. Data Analysis

Data are obtained from three independent replicates per treatment and presented as mean ± standard deviation. Significant differences between groups were compared with a confidence level of p < 0.05 by Duncan’s multiple range test using statistical software IBM SPSS Statistics 20.

3. Results

3.1. Optimization of the Induction and Condition of the HRs

The hypocotyls (with cotyledons) of 5-day-old G. glabra were soaked in A. rhizogenes strain K599. The HRs could be observed at the wounded site as early as 2 d after the end of co-cultivation and elongated further at 5 d (Figure 1A,B). The rol genes, such as rolB and rolC, in the T-DNA region of the Ri plasmid, play a significant role in inducing the formation of HRs and are ultimately integrated into the plant cell genome [7]. Therefore, they are commonly used as reference genes to confirm the formation of HR using PCR. The presence of PCR amplification products of rolB (381 bp) and rolC (408 bp) was observed in randomly selected HRs, while the untransformed normal roots did not show any amplification (Figure 1C,D). Furthermore, the absence of VirG genes in the HRs confirmed that the use of antibiotics successfully eradicated A. rhizogenes from the culture (Figure 1E). At 14 days, the HRs further elongated and developed dense branches (Figure 1F). Subsequently, 2–3 cm of HRs identified by PCR were excised and cultured in 100 mL of 1/2 MS liquid medium, and the HRs fully occupied the culture vessel by 28 d (Figure 1G).
The effects of density of bacterial suspension, infection time, co-culture medium, and AS on the rate of induction of the HRs were studied to increase the rate of induction of the HRs. As shown in Figure 2A, the induction rate is higher when a density of 0.6, 0.8, or 1.0 OD600 is used. This led to an average induction rate that exceeded 40%. When the infection time was 10 and 15 min (Figure 2B), the rates of induction of the HRs were 50.62 ± 2.70% and 43.41 ± 8.95%, respectively, while the rate of induction significantly decreased when the infection time was 5 or 20 min. Figure 2C indicates that the rate of induction of the HRs reaches a higher level of 52.58 ± 7.07% when the co-culture medium is 1/2 MS, while there is no significant difference in the effect of MS, 6,7-V, and B5 on the induction rate of G. glabra HRs. The addition of AS, a phenolic compound, to the co-culture medium facilitated the induction of HRs in the plant. Our findings suggested that the addition of 100 μmol/L AS effectively enhanced the efficiency of inducing HRs (Figure 2D). Various factors indicated that the most effective conditions to induce the subsequent HR included bacteria at a density of 0.6 and an infection time of 10 min, followed by transfer to ½ MS medium that contained 100 μmol/L AS for co-cultivation.

3.2. Growth Kinetics of G. glabra HRs

The growth kinetics of HRs were studied in 1/2 MS medium over a period of 42 d to identify the proper harvest time. There were few short HR branches during the initial incubation period (7 d). However, the number of HR branches increased significantly at day 14. At 28–35 d, the growth of HR branches reached its peak. By day 42, the growth of HR branches instead stagnated and declined (Figure 3A). We hypothesized that the stagnation of the growth of HRs at 42 d may be owing to the depletion of nutrients in the culture medium and the limited growth space. We measured the biomass of HR throughout the cultivation process, and the results were largely consistent with those of Figure 3A. As the cultivation time increased, the biomass in the shake flasks gradually increased. The biomass increased significantly on day 14. The biomass of the HRs peaked on day 28 and increased from 0.05 g/flask to 1.83 g/flask (Figure 3B). Consistent with the observed results, the FW of HRs in the liquid suspension culture medium increased by 24-fold on day 21 and approached 34.5-fold on day 28 (Figure 3C). The HRs subsequently entered a stable growth phase. The FW remained stable during this phase and did not increase significantly.

3.3. Effects of MeJA and SA on the Content of Flavonoids in G. glabra HRs

The ability of MeJA and SA to elicit the biosynthesis of flavonoids was studied by treating 28-day-old G. glabra HRs with 100 μM MeJA and 100 μM SA followed by continuous cultivation for 3 d and 5 d. The contents of isoliquiritigenin, liquiritigenin, liquiritin, licochalcone A, and glabridin were analyzed. The levels of isoliquiritigenin and licochalcone A in the HRs increased by 30.25% and 51.08%, respectively, on day 5 of the MeJA treatment (Figure 4A,D). Compared to the control group, the contents of liquiritigenin, liquiritin, and glabridin in HRs significantly increased at all time points following the treatment with MeJA. The content of liquiritigenin in the HRs increased by 59.09% and 100.03% after the MeJA treatment for 3 d and 5 d, respectively (Figure 4B). The content of liquiritin in the HRs increased by 28.23% and 53.55% after treatment with MeJA for 3 d and 5 d, respectively (Figure 4C). In addition, the content of glabridin in the HRs also increased by 23.70% and 36.83% (Figure 4E). After the SA treatment, the content of liquiritin in the HRs increased significantly on both the 3 and 5 days, while the contents of isoliquiritigenin, liquiritigenin, licochalcone A, and glabridin only showed significant increases on day 5 (Figure 4F–J). These results indicated that MeJA was a suitable stimulant to induce the biosynthesis of flavonoids in the G. glabra HRs.

3.4. GO Association and KEGG Enrichment Analysis in Two Comparisons

The gene regulatory mechanism of MeJA on the biosynthetic pathway of flavonoids in the G. glabra HRs was studied using GO classification analyses on all the selected up-regulated and downregulated DEGs in the two groups (0 vs. 3 d, 0 vs. 5 d) (Figure S2). The GO classification analysis categorized the annotated genes into the following three major functional classes: molecular functions, cellular components, and biological processes; the annotated genes encompassed 30 functional groups (Figure S2). The dominant groups between “0 vs. 3 d” within the molecular function, cellular component, and biological process categories were “sequence-specific DNA binding transcription factor activity” (216 members), plasma membrane (448 members), and protein phosphorylation (170 members) (Figure S2A). The dominant groups in the categories of molecular function, cellular component, and biological process in the comparison between “0 vs. 5 d” were “sequence-specific DNA binding transcription factor activity” (95 members), extracellular region (96 members), and defense response (64 members), respectively (Figure S2B). On days 3 and 5 of the MeJA treatment, 25 and 13 annotated genes were classified into the “flavonoid biosynthetic process”, respectively.
The biological pathways involved in the secondary metabolites were explored further by performing a KEGG pathway enrichment analysis using the upregulated and down-regulated DEGs (0 vs. 3 d, 0 vs. 5 d) to reveal the significantly enriched pathways. After 3 d and 5 d of MeJA treatment, the following KEGG pathways were mostly enriched in “metabolic pathways (ko01100)” (516 DEGs at 3 d and 251 DEGs at 5 d, respectively) and “biosynthesis of secondary metabolites (ko01110)” (343 DEGs at 3 d and 148 DEGs at 5 d, respectively) (Figure S3). Notably, the phenylpropanoid biosynthesis (ko00940), flavonoid biosynthesis (ko00941), isoflavonoid biosynthesis (ko00943), and flavone and flavanol biosynthesis (ko00944) were also significantly enriched in the HRs treated with MeJA, thus indicating their pivotal role in mediating the biosynthesis of flavonoids in the HRs (Figure S4). Additionally, on days 3 and 5, 130 and 41 DEGs potentially related to flavonoid synthesis were identified, respectively (Figure S5 and Table S3). Among them, 65% of the DEGs were upregulated on d 3, while 56% of the genes were up-regulated on d 5.

3.5. Expression Clustering Revealed Patterns of Flavonoids Associated with Genes

The dynamics of gene expression in the HRs under different durations of MeJA treatments were studied by performing a K-means clustering analysis on the transcriptomic data collected at various time points. The genes were divided into six clusters (Figure S6 and Table S4). In Cluster 1, the gene expression increased at d 3 and decreased at d 5. In Cluster 2, the gene expression significantly decreased at 3 d and slightly rebounded at 5 d. In Cluster 3, the gene showed no significant change at 3 d but increased significantly at 5 d. In cluster 4, the gene expression exhibited a sustained increase at 3 d and 5 d. The gene expression in cluster 5 significantly decreased on both days 3 and 5, while Cluster 6 showed a significant increase at 3 d and a slight increase at 5 d (Figure S6). A KEGG analysis was employed to focus on the DEGs that were upregulated at both 3 and 5 d, such as the genes in Cluster 4 and Cluster 6 (Figure S7). The genes in Cluster 4 were primarily enriched in the pathways of “Glycolysis/Gluconeogenesis (ko00010)”, “Ga-lactose metabolism (ko00052)”, and “Plant hormone signal transduction (ko04075)”. The genes in Cluster 6 were primarily enriched in the pathways of “Glycolysis/Gluconeogenesis (ko00010)”, “Phenylpropanoid biosynthesis (ko00940)”, and “Flavonoid biosynthesis (ko00941).” In addition, the genes involved in “Phenylalanine (ko00400)” and “Isoflavonoid biosynthesis (ko00943)” were also significantly enriched. These results indicate that the Cluster 6 genes may be involved in the biosynthesis of flavonoids in the HRs after MeJA treatment.

3.6. MeJA Effects the Levels of Transcription of the Genes Related to the Biosynthesis of Flavonoids in the G. glabra HRs

The schematic biosynthetic pathway of flavonoids/isoflavonoids and the involved genes are mapped in Figure 5. Numerous studies have shown that MeJA affects the biosynthesis of flavonoids in plants [13,36]. A KEGG enrichment analysis and gene functional annotation revealed that a total of 33 genes and 10 corresponding enzymes were recognized in phenylpropanoid biosynthesis, flavonoid biosynthesis, and isoflavonoid biosynthesis pathways. The sequences and FPKM values are listed in Table S5. As shown in Figure 6, there were eight significantly upregulated DEGs (1 PAL, 3 CHS, 1 UGT, 1 HI4OMT, 1 CYP81E, and 1 VR) and four downregulated DEGs (1 CHS, 1 CHI, and 2 CYP81E) in both the 3 d and 5 d. The biosynthesis of flavonoids occurs through the phenylpropanoid metabolic pathway. As the first enzyme in this pathway, phenylalanine ammonia-lyase (PAL) plays a pivotal role in linking the primary metabolism with the phenylpropanoid metabolic pathway [37]. We found that the expression of GgPAL3 was significantly upregulated on both days 3 and 5 after the MeJA treatment in HRs. 4: Coumarate-CoA ligase (4CL) is a key branch point enzyme in the phenylpropanoid metabolic pathway, and the gene Gg4CL1 was significantly upregulated on day 3 after the MeJA treatment. CHS is the first key enzyme and rate-limiting enzyme in the flavonoid biosynthetic pathway. It catalyzes the formation of chalcone (precursor of isoliquiritigenin) from coumaroyl-CoA. Here, we found that five GgCHSs genes, including GgCHS1, GgCHS3, GgCHS6, and GgCHS9, inducing at all time points of the treatment, while GgCHS7 was significantly downregulated. As previously reported in the literature, many genes in the flavonoid pathway of Glycyrrhiza species encode the same enzymes but exhibit different orientations of expression [38,39,40]. This indicates the complexity of the expression of flavonoid biosynthetic genes, and the direction and magnitude of the transcriptional responses of these genes were not conservative. In the biosynthesis of liquiritin, GgUGT, which was strongly induced by MeJA, may play a significant role. In the biosynthesis of isoflavones, GgHI4OMT, GgCYP81E4, and GgVR1 were significantly upregulated by the MeJA treatment, while GgCYP81E1 and GgCYP81E2 were significantly downregulated.

3.7. qRT-PCR Validation of Key Genes in the Biosynthesis of Flavonoids

qRT-PCR was used to detect the expression of key genes involved in the biosynthesis of flavonoids in the G. glabra HRs after 0, 3, and 5 d of MeJA induction. As shown in Figure 7, the results of qRT-PCR of the selected genes related to the biosynthesis of flavonoids matched well with the transcriptome results. The results showed that the level of expression of GgPAL3 exhibited an initial increase followed by a decrease (Figure 7A), indicating that it may play a role during the precursor stage of biosynthesis. As a ligase that connects phenylpropanoid metabolism and flavonoid metabolism, the expression level of expression of GgCHS6 increased significantly (Figure 7B). In addition, several other genes, including GgUGT, GgHI4OMT, GgCYP81E4, and GgVR1, that are involved in the synthesis of flavonoids/isoflavones also exhibited varying degrees of upregulation (Figure 7C–F). The level of expression of GgCHS7, GgCHI1, and GgCYP81E2 was largely consistent with the transcriptome data, and it also showed a downward trend under the MeJA treatment (Figure 7G–I).

3.8. Determination of the Contents of Flavonoid in GgCHS6 That Overexpressed HRs

Enhancing the content of plant secondary metabolites through genetic engineering is a significant approach in synthetic biology. To further increase the contents of flavonoids in the G. glabra HRs, we transformed and obtained HRs that overexpressed GgCHS6 (Figure S8). As shown in Figure 8A, there was no significant morphological difference between the 1302 (empty vector) HRs and GgCHS6-overexpressing HRs. Additionally, there was no notable difference in the FW and growth rate between them (Figure S9). These results indicate that the overexpression of GgCHS6 does not affect the growth of HRs. The HPLC-MS analysis results indicated that the contents of five flavonoids all increased in HRs overexpressing the GgCHS6 gene. Compared to the 1302, the contents of isoliquiritigenin, liquiritigenin, liquiritin, and licochalcone A in HRs that overexpressed GgCHS6 increased by 75.81%, 133.03%, 84.81%, and 67.76%, respectively, while the content of glabridin only increased by 39.74% (Figure 8B–F).

4. Discussion

Plant secondary metabolites have garnered attention due to their extensive applications in the fields of healthcare and food processing [41,42]. Over recent decades, plant HRs have emerged as an ideal biological system for both the large-scale biosynthesis of valuable secondary metabolites and the exploration of bioactive compound pathways [7,43]. This study systematically investigated the key parameters that affected the efficiency of inducing HRs (Figure 2). The concentration of A. rhizogenes critically influences the induction of HR. The results of this study demonstrated optimal G. glabra HR induction at bacterial suspension densities of 0.6–1.0 OD600 (Figure 2A). Within this range, the bacterial density correlates proportionally with the induction efficiency, although excessive densities cause browning and necrosis of the explants [7]. Currently, the concentration of A. rhizogenes used to induce the formation of roots is typically adjusted to an OD600 of 0.6–1.0 [7,17,44]. Similarly to the effect of bacterial suspension density, within a certain range, a longer infection time of the bacterial suspension results in a higher rate of the induction of HRs [45]. Our optimization identified 10 min as the optimal infection time for strain K599, which caused the maximal induction of HRs (Figure 2B). Appropriate co-culture media maintain the viability and cellular activity of the explants, which facilitate the induction of HRs [7]. We confirmed that the 1/2 MS medium was superior at inducing the HRs (Figure 2C). This is consistent with the findings of research on Astragalus membranaceus [46]. As a signaling molecule that activates the rol genes of A. rhizogenes, the phenolic compound AS can also enhance the induction rate of HRs at plant wound sites [7,47]. As expected, the supplementation with AS significantly increased the induction rates, and 100 μM was determined as the optimal concentration (Figure 2D).
The scaled-up cultivation of plant HRs in bioreactors is an essential pathway for the scaling and commercialization of the biosynthesis of secondary metabolites [4,48]. The rapidly growing HRs are crucial for the accumulation of secondary metabolites. Thus, it is imperative to observe the growth kinetic curve of HRs. In this study, selected HR lines showed a 34.5-fold increase in biomass and reached 1.83 g/flask compared to its initial FW (0.05 g/flask) after 28 d (Figure 3). The time course showed that the growth of G. glabra HRs followed a 42-day cycle in 100 mL liquid MS medium, including four distinct phases. These included a lag phase (0–14 d), a log phase (14–28 d), a stationary phase (28–35 d), and a phase of decline (after 35–42 d). This phased growth pattern aligns with previous research on Rubia yunnanensis Diels [49], Cannabis sativa L. [50], and Polygala tenuifolia Willd. [51]. These results indicate that under the current shake flask conditions, 28 d is the optimal period to harvest G. glabra HRs. Consistent with the observations in C. sativa L. [50], A. membranaceus [46], and Dalea purpurea [52], the growth of HRs plateaus at 28–35 d, and the biomass declines after day 35. In fact, an extension of the growth period depletes the nutrients and limits the growth space within the shake flask or bioreactors; these are the key factors that lead to the stagnation and retardation of plant HRs [53,54].
In plant HR cultures, elicitors are a successful and widely used method to induce the biosynthesis of secondary metabolites. Previous studies have shown that the cellulase from Aspergillus niger, MeJA, and Rhizobium leguminosarum can significantly increase the content of glycyrrhizin in HRs of G. glabra [55,56]. This study demonstrated that treatment with MeJA significantly increased the contents of isoliquiritigenin, liquiritin, and glabridin at all the time points (Figure 4). Thus, the elicitation of MeJA is a promising method to enhance the biosynthesis of flavonoid in G. glabra HRs. The transcription analysis enabled observations of the biosynthesis of flavonoids in G. glabra HRs under the regulation of MeJA. The GO classification analyses showed that the upregulated and downregulated DEGs were enriched in the following three functional groups: biological process, cellular component, and molecular function (Figure S2). These results indicated that the treatment with MeJA profoundly reprogrammed the biochemistry in G. glabra HRs. The KEGG enrichment of upregulated and downregulated DEGs (0 d vs. 3 d and 0 d vs. 5 d) identified metabolic pathways (ko01100) and secondary metabolite biosynthesis (ko01110) as significantly altered pathways (Figure S4). Importantly, the trends of expression of genes involved in phenylpropanoid biosynthesis (ko00940), flavonoid biosynthesis (ko00941), and isoflavonoid biosynthesis (ko00943) were upregulated by MeJA induction (Figures S6 and 7), which confirms its pivotal role in mediating the biosynthesis of flavonoids. As a signaling molecule, MeJA has been reported to promote the biosynthesis and accumulation of flavonoids in Ficus pandurate [57], G. inflata [58], Dioscorea composita [59], and Hedera helix [60].
Numerous rate-limiting biosynthetic enzymes encoded by multigene families, including PALs, 4CLs, CHSs, CHIs, and GTs, play a key role in the synthesis of flavonoids (Figure 5). This study identified 33 genes and 10 corresponding enzymes within the phenylpropanoid, flavonoid, and isoflavonoid biosynthetic pathways (Figure 6). More importantly, eight genes were significantly upregulated at all the time points following treatment with MeJA. The data of qRT-PCR further confirmed the trend of upregulation of these genes (Figure 7). Consistent with previous studies, treatment with MeJA can promote the upregulation of genes involved in flavonoid biosynthesis in licorice [39,61]. Phenylpropanoid biosynthesis is a major pathway for the biosynthesis of various specialized metabolites to defend against environmental stresses and changes [37]. Treatment with MeJA significantly induced the upregulation of GgPAL3, thus suggesting an enhancement of phenylpropanoid metabolism in the HRs, which can provide the precursor chemicals for the subsequent biosynthesis of flavonoids. CHS catalyzes the formation of chalcone from p-coumaroyl-CoA and is a key enzyme that directs the phenylpropanoid biosynthesis towards the biosynthesis of flavonoid [62]. MeJA induces the significant upregulation of multiple CHS genes, which indicates that MeJA can substantially promote the biosynthesis of flavonoids. More importantly, the overexpression of the GgCHS6 gene in the HRs of G. glabra can also increase the content of flavonoids (Figure 8). Furthermore, the genes involved in the isoflavone biosynthetic pathway, such as GgHI4OMT, GgCYP81E4, and GgVR1, were also significantly upregulated. This indicates that treatment with MeJA promotes the biosynthesis of more downstream metabolites in the HRs. In fact, subsequent research should employ multiple approaches, such as gene editing, gene silencing, or gene overexpression, to further validate the roles of these genes in HRs. Overall, MeJA may induce a regulatory chain that involves PAL/CHS genes in the biosynthesis of flavonoids in HRs, but its complete and precise regulatory network still merits further study.

5. Conclusions

In summary, we established an optimized HR culture system of G. glabra for the biosynthesis of various medicinally active ingredients. We identified MeJA as an effective elicitor that can significantly stimulate the accumulation of flavonoids, such as isoliquiritigenin, liquiritigenin, and liquiritin in HRs. The results of a transcriptome analysis revealed that the treatment with MeJA substantially upregulated the flavonoid pathway genes, such as PAL and CHS in the HRs, which effectively activated the biosynthesis of flavonoids. These findings were further validated by the qRT-PCR data. Additionally, the overexpression of GgCHS6 in the HRs by genetic engineering significantly increased the content of flavonoids. Collectively, these results elucidate the molecular mechanisms that underlie the flavonoid biosynthesis elicited by MeJA and establish an experimental framework to enhance the production of flavonoids by G. glabra through metabolic and genetic engineering. Further research on the genes identified is merited to unravel the complex network of flavonoid biosynthesis in licorice.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes16111387/s1, Figure S1: Construction of GgCHS6 overexpression vector; Figure S2: Gene Ontology (GO) functional classification analysis of DEGs; Figure S3: The bar chart of KEGG analysis showing the main enriched metabolic pathways; Figure S4: Bubble plot of KEGG analysis showing significantly enriched metabolic pathways; Figure S5: After 3 and 5 days of treatment with MeJA, the metabolic pathways were enriched by up-regulated and down-regulated genes; Figure S6: K-means clustering of gene expression profiles in all samples; Figure S7: KEGG enrichment analysis of differentially expressed genes in cluster 4 and 6; Figure S8: Identification of GgCHS6-overexpressing hairy roots by PCR; Figure S9: (A) The fresh weight of 1302 (empty vector) hairy roots and GgCHS6-overexpressing hairy roots at 28 days. (B) The fold increase in fresh weight of wild-type hairy roots and GgCHS6-overexpressing hairy roots at 28 days. Different small letters in the figure showed a significant difference (p < 0.05). Table S1: Primers used in this study; Table S2: Mass spectrometry parameters of isoliquiritigenin, liquiritigenin, liquiritin, licochalcone A, and glabridin; Table S3: The metabolic pathways were enriched by up-regulated and down-regulated genes; Table S4: K-means clustering analysis of genes contained in different clusters; Table S5: Sequences, chromosomal locations, and expression levels of significantly up-regulated or down-regulated genes. The Supplementary Material presented in this study can be found in online repositories. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics and Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA029106) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa (accessed on 15 October 2025).

Author Contributions

Conceptualization, Y.Z., B.W. and B.X.; methodology, Y.Z. and R.W.; software, Y.Z., G.T., T.Z. and M.Z.; validation, T.L. and C.L.; formal analysis, H.Z.; investigation, D.L.; writing—original draft preparation, Y.Z.; writing—review and editing, Y.Z.; visualization, J.C.; supervision, L.W.; project administration, Y.Z.; funding acquisition, Y.Z., M.Z., T.L. and L.W. All authors have read and agreed to the published version of the manuscript.

Funding

The authors declare that financial support was received for the research, authorship, and/or publication of this article. Henan Province Science and Technology Research Project (Grant No. 242102311202, 252102110247, and 252102111101), the Natural Science Foundation of Henan (Grant No. 252300420651); the Key General Project of Henan Province (Grant No. 252300421409) and the Henan University of Urban Construction Doctoral Research Start-up Fund Funding Project (Grant No. K-Q2023018).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and Supplementary Materials.

Acknowledgments

We thank the six fund projects for supporting this research. We also appreciate the editors and reviewers for their constructive comments.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Wang, C.; Chen, L.; Xu, C.; Shi, J.; Chen, S.; Tan, M.; Chen, J.; Zou, L.; Chen, C.; Liu, Z.; et al. A comprehensive review for phytochemical, pharmacological, and biosynthesis studies on Glycyrrhiza spp. Am. J. Chin. Med. 2020, 48, 17–45. [Google Scholar] [CrossRef]
  2. Wahab, S.; Annadurai, S.; Abullais, S.S.; Das, G.; Ahmad, W.; Ahmad, M.F.; Kandasamy, G.; Vasudevan, R.; Ali, M.S.; Amir, M. Glycyrrhiza glabra (Licorice): A comprehensive review on its phytochemistry, biological activities, clinical evidence and toxicology. Plants 2021, 10, 2751–2787. [Google Scholar] [CrossRef]
  3. Zheng, Y.P. Visual analysis of research progress and trend on hairy roots. Front. Plant Sci. 2025, 16, 1580007–1580023. [Google Scholar] [CrossRef]
  4. Gerszberg, A.; Wiktorek-Smagur, A. Hairy root cultures as a multitask platform for green biotechnology. Plant Cell Tissue Organ. Cult. 2022, 150, 493–509. [Google Scholar] [CrossRef]
  5. Gantait, S.; Mukherjee, E. Hairy root culture technology: Applications, constraints and prospect. Appl. Microbiol. Biotechnol. 2021, 105, 35–53. [Google Scholar] [CrossRef] [PubMed]
  6. Singh, R.S.; Singh, P.; Kumari, R.; Kumar, S. Hairy root cultures for secondary metabolite production. In Genetic Manipulation of Secondary Metabolites in Medicinal Plant; Singh, R., Kumar, N., Eds.; Springer Nature Singapore: Singapore, 2023; pp. 205–223. [Google Scholar]
  7. Zhu, Y.; Zhu, X.; Wen, Y.; Wang, L.; Wang, Y.; Liao, C.; Zhao, M.; Li, T.; Liu, D.; Li, B.; et al. Plant hairy roots: Induction, applications, limitations and prospects. Ind. Crops Prod. 2024, 219, 119104–119123. [Google Scholar] [CrossRef]
  8. Afsharzadeh, N.; Paltram, R.; Jungwirth, A.; Tabrizi, L.; Nazeri, V.; Kalantari, H.; Halbwirth, H.; Samiei, L.; Sheehan, H.; Shokrpour, M. Advancing Glycyrrhiza glabra L. cultivation and hairy root transformation and elicitation for future metabolite overexpression. Horticulturae 2025, 11, 62–79. [Google Scholar] [CrossRef]
  9. Venkatasai, N.N.; Shetty, D.N.; Vinay, C.M.; Sekar, M.; Muthusamy, A.; Rai, P.S. A comprehensive review of factors affecting growth and secondary metabolites in hydroponically grown medicinal plants. Planta 2025, 261, 48–66. [Google Scholar] [CrossRef]
  10. Liu, S.; Zhang, Q.; Kollie, L.; Dong, J.; Liang, Z. Molecular networks of secondary metabolism accumulation in plants: Current understanding and future challenges. Ind. Crops Prod. 2023, 201, 116901–116917. [Google Scholar] [CrossRef]
  11. Wongwicha, W.; Tanaka, H.; Shoyama, Y.; Putalun, W. Methyl jasmonate elicitation enhances glycyrrhizin production in Glycyrrhiza inflata hairy roots cultures. Z. Für Naturforschung C 2011, 66, 423–428. [Google Scholar] [CrossRef]
  12. Li, J.; Li, B.; Luo, L.; Cao, F.; Yang, B.; Gao, J.; Yan, Y.; Zhang, G.; Peng, L.; Hu, B. Increased phenolic acid and tanshinone production and transcriptional responses of biosynthetic genes in hairy root cultures of Salvia przewalskii Maxim. treated with methyl jasmonate and salicylic acid. Mol. Biol. Rep. 2020, 47, 8565–8578. [Google Scholar] [CrossRef]
  13. Amani, S.; Mohebodini, M.; Khademvatan, S.; Jafari, M.; Kumar, V. Modifications in gene expression and phenolic compounds content by methyl jasmonate and fungal elicitors in Ficus carica. Cv. Siah hairy root cultures. BMC Plant Biol. 2024, 24, 520–534. [Google Scholar] [CrossRef]
  14. Bao, J.; Lu, X.; Ma, L.; Zhang, X.; Tian, P.; Zhang, X.; Li, S.; Ma, S.; Yang, J.; Lu, Y.; et al. Transcriptome analysis of genes related to glucoraphanin and sulforaphane synthesis in methyl jasmonate treated broccoli (Brassica oleracea var. italica) hairy roots. J. Plant Res. 2022, 135, 757–770. [Google Scholar] [CrossRef] [PubMed]
  15. Veremeichik, G.N.; Bulgakov, D.V.; Solomatina, T.O.; Makhazen, D.S. In the interkingdom horizontal gene transfer, the small rolA gene is a big mystery. Appl. Microbiol. Biotechnol. 2023, 107, 2097–2109. [Google Scholar] [CrossRef] [PubMed]
  16. Chen, X.; Wang, Y.; Sun, J.; Wang, H. Induction and cultivation of hairy roots of Zanthoxylum dissitum. Plant Physiol. J. 2023, 59, 2117–2125. [Google Scholar] [CrossRef]
  17. Foti, C.; Pavli, O.I. High-efficiency Agrobacterium rhizogenes-mediated transgenic hairy root induction of Lens culinaris. Agronomy 2020, 10, 1170–1181. [Google Scholar] [CrossRef]
  18. Medina, F.; Condori, J.; Rimando, A.M.; Hubstenberger, J.; Shelton, K.; O’Keefe, S.F.; Bennett, S.; Dolan, M.C. Production and secretion of resveratrol in hairy root cultures of peanut. Phytochemistry 2007, 68, 1992–2003. [Google Scholar] [CrossRef]
  19. Serino, G.; Clerot, D.; Brevet, J.; Costantino, P.; Cardarelli, M. rol genes of Agrobacterium rhizogenes cucumopine strain: Sequence, effects and pattern of expression. Plant Mol. Biol. 1994, 26, 415–422. [Google Scholar] [CrossRef]
  20. Grąbkowska, R.; Krzemińska, M.; Gaweda-Walerych, K.; Kiss, A.K.; Pluta, K.; Grzegorczyk-Karolak, I. Enhancement of rosmarinic acid production in hairy root cultures of Perovskia atriplicifolia Benth. Int. J. Mol. Sci. 2025, 26, 3187–3208. [Google Scholar] [CrossRef]
  21. Su, L.; Li, S.; Qiu, H.; Wang, H.; Wang, C.; He, C.; Xu, M.; Zhang, Z. Full-length transcriptome analyses of genes involved in triterpenoid saponin biosynthesis of Psammosilene tunicoides hairy root cultures with exogenous salicylic acid. Front. Genet. 2021, 12, 657060–657076. [Google Scholar] [CrossRef]
  22. Shang, Z.; Tian, Y.; Yi, Y.; Li, K.; Qiao, X.; Ye, M. Comparative bioactivity evaluation and chemical profiling of different parts of the medicinal plant Glycyrrhiza uralensis. J. Pharm. Biomed. Anal. 2022, 215, 114793–114803. [Google Scholar] [CrossRef]
  23. Zhu, L.; Li, M.; Yang, W.; Zhang, J.; Yang, X.; Zhang, Q.; Wang, H. Effects of different drying methods on drying characteristics and quality of Glycyrrhiza uralensis (Licorice). Foods 2023, 12, 1652–1665. [Google Scholar] [CrossRef] [PubMed]
  24. Martin, M. CUTADAPT removes adapter sequences from high-throughput sequencing reads. EMBnet.J. 2011, 17, 10–12. [Google Scholar] [CrossRef]
  25. Mochida, K.; Sakurai, T.; Seki, H.; Yoshida, T.; Takahagi, K.; Sawai, S.; Uchiyama, H.; Muranaka, T.; Saito, K. Draft genome assembly and annotation of Glycyrrhiza uralensis, a medicinal legume. Plant J. 2017, 89, 181–194. [Google Scholar] [CrossRef] [PubMed]
  26. Kim, D.; Langmead, B.; Salzberg, S.L. HISAT: A fast spliced aligner with low memory requirements. Nat. Methods 2015, 12, 357–360. [Google Scholar] [CrossRef]
  27. Pertea, M.; Pertea, G.M.; Antonescu, C.M.; Chang, T.C.; Mendell, J.T.; Salzberg, S.L. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 2015, 33, 290–295. [Google Scholar] [CrossRef]
  28. Trapnell, C.; Williams, B.A.; Pertea, G.; Mortazavi, A.; Kwan, G.; van Baren, M.J.; Salzberg, S.L.; Wold, B.J.; Pachter, L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 2010, 28, 511–515. [Google Scholar] [CrossRef]
  29. Love, M.I.; Anders, S.; Huber, W. Differential analysis of count data-the DESeq2 package. Genome Biol. 2015, 15, 10-1186. [Google Scholar]
  30. Harris, M.A.; Clark, J.; Ireland, A.; Lomax, J.; Ashburner, M.; Foulger, R.; Eilbeck, K.; Lewis, S.; Marshall, B.; Mungall, C.; et al. The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res. 2004, 32, D258–D261. [Google Scholar] [CrossRef]
  31. Kanehisa, M.; Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000, 28, 27–30. [Google Scholar] [CrossRef]
  32. Kumar, L.; Futschik, M.E. Mfuzz: A software package for soft clustering of microarray data. Bioinformation 2007, 2, 5–7. [Google Scholar] [CrossRef]
  33. Maroufi, A. Selection of reference genes for real-time quantitative PCR analysis of gene expression in Glycyrrhiza glabra under drought stress. Biol. Plant. 2016, 60, 645–654. [Google Scholar] [CrossRef]
  34. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  35. Jyothishwaran, G.; Kotresha, D.; Selvaraj, T.; Srideshikan, S.; Rajvanshi, M. A modified freeze-thaw method for efficient transformation of Agrobacterium tumefaciens. Curr. Sci. 2007, 93, 770–772. [Google Scholar]
  36. Kaushik, S.; Ranjan, A.; Singh, A.K.; Sirhindi, G. Methyl jasmonate reduces cadmium toxicity by enhancing phenol and flavonoid metabolism and activating the antioxidant defense system in pigeon pea (Cajanus cajan). Chemosphere 2024, 346, 140681–140694. [Google Scholar] [CrossRef]
  37. Ninkuu, V.; Aluko, O.O.; Yan, J.; Zeng, H.; Liu, G.; Zhao, J.; Li, H.; Chen, S.; Dakora, F.D. Phenylpropanoids metabolism: Recent insight into stress tolerance and plant development cues. Front. Plant Sci. 2025, 16, 1571825–1571846. [Google Scholar] [CrossRef]
  38. Li, X.; Xu, Y.; Zhang, J.; Xu, K.; Zheng, X.; Luo, J.; Lu, J. Integrative physiology and transcriptome reveal salt-tolerance differences between two licorice species: Ion transport, Casparian strip formation and flavonoids biosynthesis. BMC Plant Biol. 2024, 24, 272–298. [Google Scholar] [CrossRef]
  39. Yao, H.; Wang, F.; Bi, Q.; Liu, H.; Liu, L.; Xiao, G.; Zhu, J.; Shen, H.; Li, H. Combined analysis of pharmaceutical active ingredients and transcriptomes of Glycyrrhiza uralensis under PEG6000-induced drought stress revealed glycyrrhizic acid and flavonoids accumulation via JA-mediated signaling. Front. Plant Sci. 2022, 13, 920172–920186. [Google Scholar] [CrossRef]
  40. Zhong, C.; Chen, C.; Gao, X.; Tan, C.; Bai, H.; Ning, K. Multi-omics profiling reveals comprehensive microbe-plant-metabolite regulation patterns for medicinal plant Glycyrrhiza uralensis Fisch. Plant Biotechnol. J. 2022, 20, 1874–1887. [Google Scholar] [CrossRef]
  41. Ozyigit, I.I.; Dogan, I.; Hocaoglu-Ozyigit, A.; Yalcin, B.; Erdogan, A.; Yalcin, I.E.; Cabi, E.; Kaya, Y. Production of secondary metabolites using tissue culture-based biotechnological applications. Front. Plant Sci. 2023, 14, 1132555–1132583. [Google Scholar] [CrossRef]
  42. Mipeshwaree Devi, A.; Khedashwori Devi, K.; Premi Devi, P.; Lakshmipriyari Devi, M.; Das, S. Metabolic engineering of plant secondary metabolites: Prospects and its technological challenges. Front. Plant Sci. 2023, 14, 1171154–1171170. [Google Scholar] [CrossRef]
  43. Mirmazloum, I.; Slavov, A.K.; Marchev, A.S. The untapped potential of hairy root cultures and their multiple applications. Int. J. Mol. Sci. 2024, 25, 12682–12718. [Google Scholar] [CrossRef]
  44. Ebrahimi, T.; Piri, K.; Abdoli, A.; Tohidfar, M. Developing an efficient protocol for hairy root induction in Lythrum salicaria L. Biologia 2023, 78, 2667–2677. Biologia 2023, 78, 2667–2677. [Google Scholar] [CrossRef]
  45. Zhao, S.; Zhao, K.; Yang, J.; Huasong, W. Research and application of synthesis of secondary metabolites in hairy roots induced by Agrobacterium rhizogenes. Mol. Plant Breed. 2023, 21, 7554–7565. [Google Scholar] [CrossRef]
  46. Hwang, C.; Yan, S.; Choe, Y.; Yun, C.; Xu, S.; Im, M.; Xue, Z. Efficient hairy root induction system of Astragalus membranaceus and significant enhancement of astragalosides via overexpressing AmUGT15. Plant Cell Rep. 2024, 43, 285–298. [Google Scholar] [CrossRef] [PubMed]
  47. Jiao, J.; Gai, Q.Y.; Wang, X.; Liu, J.; Lu, Y.; Wang, Z.Y.; Xu, X.J.; Fu, Y.J. Effective production of phenolic compounds with health benefits in pigeon pea [Cajanus cajan (L.) Millsp.] hairy root cultures. J. Agric. Food Chem. 2020, 68, 8350–8361. [Google Scholar] [CrossRef]
  48. Hussain, M.J.; Abbas, Y.; Nazli, N.; Fatima, S.; Drouet, S.; Hano, C.; Abbasi, B.H. Root cultures, a boon for the production of valuable compounds: A comparative review. Plants 2022, 11, 439–477. [Google Scholar] [CrossRef]
  49. Miao, Y.; Hu, Y.; Yi, S.; Zhang, X.; Tan, N. Establishment of hairy root culture of Rubia yunnanensis Diels: Production of rubiaceae-type cyclopeptides and quinones. J. Biotechnol. 2021, 341, 21–29. [Google Scholar] [CrossRef]
  50. Mahendran, G.; Vimolmangkang, S. Effect of carbon source and elicitors on biomass production and accumulation of friedelin and epifriedelanol in hairy roots of hemp (Cannabis sativa L.). Plant Cell Tissue Organ Cult. (PCTOC) 2024, 156, 61–77. [Google Scholar] [CrossRef]
  51. Ji, H.; Yang, B.; Jing, Y.; Luo, Y.; Li, B.; Yan, Y.; Zhang, G.; Zhao, F.; Wang, B.; Peng, L.; et al. Trehalose and brassinolide enhance the signature ingredient accumulation and anti-oxidant activity in the hairy root cultures of Polygala tenuifolia Willd. Ind. Crops Prod. 2023, 196, 116521–116536. [Google Scholar] [CrossRef]
  52. Sharma, A.R.; Gajurel, G.; Abdel-Karim, S.; Alam, M.A.; Shields, R.C.; Medina-Bolivar, F. Production of malheuran A, a geranylated flavonoid with antimicrobial and anti-inflammatory activities, in hairy root cultures of Dalea purpurea. Plants 2025, 14, 259–281. [Google Scholar] [CrossRef] [PubMed]
  53. Verma, P.; Khan, S.A.; Alhandhali, A.J.A.; Parasharami, V.A. Bioreactor upscaling of different tissue of medicinal herbs for extraction of active phytomolecules: A step towards industrialization and enhanced production of phytochemicals. In Plant Growth Regulators: Signalling under Stress Conditions; Aftab, T., Hakeem, K.R., Eds.; Springer International Publishing: Cham, Switzerland, 2021; pp. 455–481. [Google Scholar]
  54. Liu, C.; Ahmad, N.; Tao, Y.; Hussain, H.; Chang, Y.; Umar, A.W.; Liu, X. Reprogramming hairy root cultures: A synthetic biology framework for precision metabolite biosynthesis. Plants 2025, 14, 1928–1945. [Google Scholar] [CrossRef] [PubMed]
  55. Allahdou, M.; Mehravaran, L.; Khajeh, H. Improving the production of glycyrrhizin and glycyrrhetinic acid by eliciting hairy root of Glycyrrhiza glabra. Russ. J. Plant Physiol. 2025, 72, 3. [Google Scholar] [CrossRef]
  56. Behdad, A.; Ganjeali, A. Phytohormones and microbial elicitation on glycyrrhizin production and gene expression in the hairy root of Glycyrrhiza glabra L. Plant Cell Tissue Organ Cult. (PCTOC) 2024, 160, 4–22. [Google Scholar] [CrossRef]
  57. Yang, B.; Pan, F.; Yasmeen, F.; Shan, L.; Pan, J.; Zhang, M.; Weng, X.; Wang, M.; Li, M.; Wang, Q.; et al. Integrated multi-omic analysis reveals the cytokinin and sucrose metabolism-mediated regulation of flavone glycoside biosynthesis by MeJA exposure in Ficus pandurata Hance. Food Res. Int. 2023, 174, 113680–113695. [Google Scholar] [CrossRef]
  58. Zeng, J.; Ma, X.; Li, Y.; Zhou, L.; Fu, J.; Wang, H.; Liu, Y.; Yuan, L.; Wang, Y.; Li, Y. Histone deacetylases repress the accumulation of licochalcone A by inhibiting the expression of flavonoid biosynthetic pathway-related genes in licorice (Glycyrrhiza inflata). Mol. Hortic. 2025, 5, 32–49. [Google Scholar] [CrossRef]
  59. Yu, S.; Zhang, J.; Cao, Y.; Xie, J.; Zhong, C. Combined transcriptome and metabolome analysis reveals that DcWRKY11 promotes methyl jasmonate-induced proanthocyanidin biosynthesis in Dioscorea composita. Plant Cell Environ. 2025, 48, 6782–6795. [Google Scholar] [CrossRef]
  60. Zheng, F.; Xu, Z.; Deng, X.; Wang, X.; Sun, Y.; Shen, X.; Yu, Z. Genome-wide transcriptome analysis reveals GRF transcription factors involved in methyl jasmonate-induced flavonoid biosynthesis in Hedera helix. Plants 2025, 14, 2094–2103. [Google Scholar] [CrossRef]
  61. Zhao, H.-H.; Du, R.; Han, Y.-L.; Yang, Z.-H.; Qiu, X.; Li, Y.-Q.; Zhang, J.-G.; Cheng, Z.-W. Effects of methyl jasmonate and salicylhydroxamic acid on the biosynthesis of flavonoids in Glycyrrhiza glabra L. hairy roots. Plant Stress 2025, 15, 100774–100786. [Google Scholar] [CrossRef]
  62. Yonekura-Sakakibara, K.; Higashi, Y.; Nakabayashi, R. The origin and evolution of plant flavonoid metabolism. Front. Plant Sci. 2019, 10, 943–959. [Google Scholar] [CrossRef]
Genes 16 01387 g001
Figure 1. Induction and establishment of A. rhizogenes strain K599 mediated HR culture from G. glabra. (A) At 2 days of co-cultivation, HRs formed at the wound site. (B) On the 5th day, the continuously elongating HRs. (C) The size of the rolB gene is 381 bp. (D) The size of the rolC gene is 408 bp. (E) The size of the VirG gene is 319 bp. M: marker; NR: The normal root of licorice. 1–8: Different hairy root samples. (F) Highly branched HRs formed in two weeks. (G) HRs were grown in a flask containing 1/2 MS liquid medium for 28 d.
Figure 1. Induction and establishment of A. rhizogenes strain K599 mediated HR culture from G. glabra. (A) At 2 days of co-cultivation, HRs formed at the wound site. (B) On the 5th day, the continuously elongating HRs. (C) The size of the rolB gene is 381 bp. (D) The size of the rolC gene is 408 bp. (E) The size of the VirG gene is 319 bp. M: marker; NR: The normal root of licorice. 1–8: Different hairy root samples. (F) Highly branched HRs formed in two weeks. (G) HRs were grown in a flask containing 1/2 MS liquid medium for 28 d.
Genes 16 01387 g001
Genes 16 01387 g002
Figure 2. Establishment and optimization of the induction system for G. glabra HRs. Effects of the density of bacterial suspension on the rate of HR induction in G. glabra (A), effects of infection time (B), types of co-culture medium (C), and AS concentration (D) on HR induction. The data were shown as the mean ± standard deviation, and the different letters represent significant differences (p < 0.05).
Figure 2. Establishment and optimization of the induction system for G. glabra HRs. Effects of the density of bacterial suspension on the rate of HR induction in G. glabra (A), effects of infection time (B), types of co-culture medium (C), and AS concentration (D) on HR induction. The data were shown as the mean ± standard deviation, and the different letters represent significant differences (p < 0.05).
Genes 16 01387 g002
Genes 16 01387 g003
Figure 3. Growth kinetics of G. glabra HRs in phytohormone-free 1/2 strength liquid MS medium. (A) Change in the growth status of HRs cultured in medium for 42 days. (B) The growth curve of fresh weight of HRs at different time periods. (C) The curve of fresh weight shows multiple changes in HRs at different time periods. Data were obtained from three independent biological replicates and presented as the mean ± standard deviation.
Figure 3. Growth kinetics of G. glabra HRs in phytohormone-free 1/2 strength liquid MS medium. (A) Change in the growth status of HRs cultured in medium for 42 days. (B) The growth curve of fresh weight of HRs at different time periods. (C) The curve of fresh weight shows multiple changes in HRs at different time periods. Data were obtained from three independent biological replicates and presented as the mean ± standard deviation.
Genes 16 01387 g003
Genes 16 01387 g004
Figure 4. Effects of MeJA and SA on the content of flavonoids in G. glabra HRs. (A–E) Effects of MeJA on the content of licochalcone A, liquiritin, liquiritigenin, isoliquiritigenin, and glabridin in G. glabra HRs. (F–J) Effects of SA on the content of licochalcone A, liquiritin, liquiritigenin, isoliquiritigenin, and glabridin in G. glabra HRs. The data were shown as the mean ± standard deviation, and the different letters represent significant differences (p < 0.05).
Figure 4. Effects of MeJA and SA on the content of flavonoids in G. glabra HRs. (A–E) Effects of MeJA on the content of licochalcone A, liquiritin, liquiritigenin, isoliquiritigenin, and glabridin in G. glabra HRs. (F–J) Effects of SA on the content of licochalcone A, liquiritin, liquiritigenin, isoliquiritigenin, and glabridin in G. glabra HRs. The data were shown as the mean ± standard deviation, and the different letters represent significant differences (p < 0.05).
Genes 16 01387 g004
Genes 16 01387 g005
Figure 5. Expression profiles of enzyme-encoding genes involved in phenylpropanoid and flavonoid/isoflavone biosynthesis. Abbreviations: PAL (phenylalanine ammonialyase), C4H (cinnamate 4-hydroxylase), 4CL (4-coumaroyl CoA ligase), CHS (chalcone synthase), CHR (chalcone reductase), CHI (chalcone isomerase), UGT (UDP-glucosyltransferase), F2H (flavanone 2′-hydroxylase), LMT (licodione 2′-O-methyltransferase), IFS (2-hydroxyisoflavanone synthase), HID (2-hydroxyisoflavanone dehydratase), HI4OMT (isoflavone 4′-O-methyltransferase), CYP81E (isoflavone 2′-hydroxylase), IFR (isoflavone reductase), VR (vestitone reductase), PTS (pterocarpan synthase), PTR (pterocarpan reductase), PT (prenyltransferases), DMT (demethylases), and OC (Isopentenyl cyclase).
Figure 5. Expression profiles of enzyme-encoding genes involved in phenylpropanoid and flavonoid/isoflavone biosynthesis. Abbreviations: PAL (phenylalanine ammonialyase), C4H (cinnamate 4-hydroxylase), 4CL (4-coumaroyl CoA ligase), CHS (chalcone synthase), CHR (chalcone reductase), CHI (chalcone isomerase), UGT (UDP-glucosyltransferase), F2H (flavanone 2′-hydroxylase), LMT (licodione 2′-O-methyltransferase), IFS (2-hydroxyisoflavanone synthase), HID (2-hydroxyisoflavanone dehydratase), HI4OMT (isoflavone 4′-O-methyltransferase), CYP81E (isoflavone 2′-hydroxylase), IFR (isoflavone reductase), VR (vestitone reductase), PTS (pterocarpan synthase), PTR (pterocarpan reductase), PT (prenyltransferases), DMT (demethylases), and OC (Isopentenyl cyclase).
Genes 16 01387 g005
Genes 16 01387 g006
Figure 6. The heat graph shows the up-regulated and down-regulated genes involved in phenylpropanoid and flavonoid/isoflavone biosynthesis in G. glabra HRs under MeJA treatment. The heatmap values represent the Log2FPKM value of each sample.
Figure 6. The heat graph shows the up-regulated and down-regulated genes involved in phenylpropanoid and flavonoid/isoflavone biosynthesis in G. glabra HRs under MeJA treatment. The heatmap values represent the Log2FPKM value of each sample.
Genes 16 01387 g006
Genes 16 01387 g007
Figure 7. The qRT-PCR analysis of selected DEGs in G. glabra HRs under the MeJA treatment. GgPAL3 (A), GgCHS6 (B), GgUGT (C), GgHI4OMT (D), GgCYP81E4 (E), GgVR1 (F), GgCHS7 (G), GgCHI1 (H), GgCYP81E1 (I). Column diagram represents the relative expression level of qRT-PCR (left y-axis). The line chart represents the relative expression level of RNA-Seq (right y-axis). Different small letters in the figure showed a significant difference (p < 0.05).
Figure 7. The qRT-PCR analysis of selected DEGs in G. glabra HRs under the MeJA treatment. GgPAL3 (A), GgCHS6 (B), GgUGT (C), GgHI4OMT (D), GgCYP81E4 (E), GgVR1 (F), GgCHS7 (G), GgCHI1 (H), GgCYP81E1 (I). Column diagram represents the relative expression level of qRT-PCR (left y-axis). The line chart represents the relative expression level of RNA-Seq (right y-axis). Different small letters in the figure showed a significant difference (p < 0.05).
Genes 16 01387 g007
Genes 16 01387 g008
Figure 8. The impact of GgCHS6 overexpression on G. glabra HRs. (A) The impact of GgCHS6 gene overexpression on the morphology of HRs. (B–F) The overexpression of the GgCHS6 gene affects the content of isoliquiritigenin (B), liquiritigenin (C), liquiritin (D), licochalcone A (E), and glabridin (F). The data were shown as the mean ± standard deviation, and the different letters represent significant differences (p < 0.05).
Figure 8. The impact of GgCHS6 overexpression on G. glabra HRs. (A) The impact of GgCHS6 gene overexpression on the morphology of HRs. (B–F) The overexpression of the GgCHS6 gene affects the content of isoliquiritigenin (B), liquiritigenin (C), liquiritin (D), licochalcone A (E), and glabridin (F). The data were shown as the mean ± standard deviation, and the different letters represent significant differences (p < 0.05).
Genes 16 01387 g008
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zhu, Y.; Wang, B.; Xue, B.; Wang, R.; Tang, G.; Zhu, T.; Zhao, M.; Li, T.; Liao, C.; Zhang, H.; et al. MeJA Elicitation on Flavonoid Biosynthesis and Gene Expression in the Hairy Roots of Glycyrrhiza glabra L. Genes 2025, 16, 1387. https://doi.org/10.3390/genes16111387

AMA Style

Zhu Y, Wang B, Xue B, Wang R, Tang G, Zhu T, Zhao M, Li T, Liao C, Zhang H, et al. MeJA Elicitation on Flavonoid Biosynthesis and Gene Expression in the Hairy Roots of Glycyrrhiza glabra L. Genes. 2025; 16(11):1387. https://doi.org/10.3390/genes16111387

Chicago/Turabian Style

Zhu, Yutao, Bohan Wang, Bingyi Xue, Runqian Wang, Ganlin Tang, Tao Zhu, Mei Zhao, Taotao Li, Chunli Liao, Huamin Zhang, and et al. 2025. "MeJA Elicitation on Flavonoid Biosynthesis and Gene Expression in the Hairy Roots of Glycyrrhiza glabra L." Genes 16, no. 11: 1387. https://doi.org/10.3390/genes16111387

APA Style

Zhu, Y., Wang, B., Xue, B., Wang, R., Tang, G., Zhu, T., Zhao, M., Li, T., Liao, C., Zhang, H., Liu, D., Chen, J., & Wang, L. (2025). MeJA Elicitation on Flavonoid Biosynthesis and Gene Expression in the Hairy Roots of Glycyrrhiza glabra L. Genes, 16(11), 1387. https://doi.org/10.3390/genes16111387

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop