Mingzhu Zhao

Panax ginseng C.A. Meyer is a valuable medicinal plant with considerable pharmaceutical and economic importance. However, long-term cultivation of ginseng faces several challenges, including replanting problems, yield decline, and unstable quality, which severely restrict the sustainable development of the ginseng industry. Plant endophytes are microorganisms that establish long-term associations with their hosts, and their bioactive metabolites have great potential for promoting host growth, enhancing stress tolerance, and modulating secondary metabolism. In this study, a total of 134 ginseng endophytes were isolated from 15 forest-grown ginseng samples. Among them, the pyrroloquinoline quinone (PQQ)-producing strain W7N-70 (PQQ yield: 41.99 mg/L), belonging to the genus Methylorubrum, was screened. The engineered strain W7N-70-PqqABCDE (PQQ yield: 92.55 mg/L) was constructed through overexpression of the PQQ biosynthetic gene cluster, and fermentation conditions were optimized using response surface methodology, resulting in a significantly enhanced PQQ yield of 149.29 mg/L. Furthermore, ginseng adventitious roots were treated with culture supernatants from strain W7N-70 and the engineered strain W7N-70-PqqABCDE, as well as exogenous PQQ. The results showed that the culture supernatant of the engineered strain significantly promoted adventitious root growth, increased indole-3-acetic acid (IAA) content, and enhanced antioxidant enzyme activities. Moreover, it significantly upregulated the expression of key enzyme genes involved in the protopanaxadiol (PPD)-type ginsenoside biosynthetic pathway and significantly increased the content of PPD-type ginsenosides. Overall, this study provides novel microbial resources for PQQ biosynthesis and preliminarily demonstrates the potential of PQQ-producing strains to promote ginseng growth, modulate physiological responses, and improve ginsenoside production.

Panax ginseng (Panax ginseng C.A. Mey.) produces pharmacologically valuable ginsenosides. WD40-repeat (WDR) proteins act as versatile regulators of plant specialized metabolism, yet their biological roles under methyl jasmonate (MeJA) elicitation remain largely uncharacterized in ginseng. In this study, we identified 29 PgWDR family members at the whole-genome level, and systematically analyzed their phylogeny, gene structure, cis-acting promoter elements, as well as organ- and development-dependent expression patterns. Six candidate genes potentially associated with ginsenoside biosynthesis were screened through integrating gene–metabolite correlation analysis and gene co-expression analysis. Under MeJA treatment, three of these candidates showed statistically significant expression responses, while the other three exhibited variable expression fluctuations with no statistical significance. PgWDR24 displayed a positive correlation with key ginsenoside biosynthetic enzyme genes, and a negative correlation with protopanaxadiol-type ginsenoside accumulation. Combined with its predicted nuclear localization, we hypothesize that PgWDR24 participates in the negative modulation of protopanaxadiol-type ginsenoside accumulation, although further genetic functional validation is still required. This work provides valuable candidate genes for deciphering ginsenoside regulatory networks and offers support for molecular-assisted breeding of high-quality ginseng.

Pathogenesis-related (PR) proteins are central to plant defense; however, their roles in regulating secondary metabolism remain largely unexplored. In Panax ginseng, the molecular characteristics and functional links of PR1 and PR10 gene families to ginsenoside biosynthesis remain unclear. Here, we conducted genome and transcriptome identification, evolutionary analysis, co-expression network construction, and methyl jasmonate (MeJA) induction assays to investigate their roles. We identified 34 PR1/PR10 family members (11 PgPR1s and 23 PgPR10s) unevenly distributed across 15 chromosomes, with gene duplication driving family expansion. Functional divergence was evident: PgPR1 members are evolutionarily conserved and root-preferential, associated with antibacterial defense, whereas PgPR10 underwent species-specific expansion, showed broad tissue expression, and possessed promoters enriched with hormone- and stress-responsive cis-elements. Co-expression analysis (|r| ≥ 0.3, p < 0.001) identified PgPR1-5 and PgPR10-8 as the only PR members significantly correlated with key ginsenoside biosynthetic enzymes. Under MeJA, the ginsenoside pathway displayed bidirectional temporal regulation, with core PR genes showing synchronized expression with the negatively regulated enzyme gene CAS_14. Our study characterizes the molecular and evolutionary features of PgPR1/PgPR10 families, identifies core PR genes associated with ginsenoside metabolism, and reveals their coordinated response to MeJA, providing key insights and candidate targets for exploring the potential link between PR proteins and ginsenoside biosynthesis and promising candidate genes for further research on P. ginseng.

Panax ginseng is an economically important medicinal plant, and the biosynthetic regulatory network of ginsenosides, its core bioactive constituents, is extremely complex. Fasciclin-like arabinogalactan proteins (FLAs) are a class of glycoproteins widely involved in plant growth and development that modulate secondary cell wall biosynthesis, phytohormone signal transduction, and stress responses in plants. In this study, we performed a genome-wide identification of the FLA gene family in P. ginseng and identified 21 PgFLA family members. Comprehensive analyses were subsequently conducted to characterize their gene structures, chromosomal distributions, collinearity relationships, phylogenetics, expression patterns, and cis-acting regulatory elements. Subsequently, combined with SNP/InDel-ginsenoside association analysis based on 344 ginseng accessions, correlation analysis between gene expression and ginsenoside content, as well as co-expression network analysis linking PgFLA genes with key enzyme genes of the ginsenoside biosynthetic pathway, eight candidate PgFLA genes associated with ginsenoside biosynthesis were identified. Further mediation effect analysis identified six PgFLA members that potentially affect ginsenoside biosynthesis by modulating the expression of key enzymes involved in the ginsenoside biosynthetic pathway. Quantitative real-time PCR (qRT-PCR) was used to validate the expression of these pivotal candidate genes in methyl jasmonate (MeJA)-treated ginseng adventitious roots. The results revealed that the transcript abundances of five genes (PgFLA02, PgFLA05-01, PgFLA05-02, PgFLA06, and PgFLA09-06) were significantly negatively correlated with the levels of protopanaxadiol-type ginsenosides (Rb1, Rb2, Rb3, Rc, and Rd). These findings provide a theoretical foundation for further dissecting the molecular regulatory network underlying ginsenoside biosynthesis and supply valuable candidate genes for marker-assisted breeding and quality improvement of cultivated P. ginseng.

Plant homeodomain (PHD) finger proteins are important histone modification readers that play broad roles in the epigenetic regulation of plant development and stress responses. However, the PHD finger gene family has not been systematically identified in Panax ginseng, and its association with ginsenoside biosynthesis remains unclear. In this study, we identified 63 PgPHD genes, corresponding to 163 transcripts, from the P. ginseng transcriptome using a combination of HMMER-based domain and BLAST-based homology searches (BLAST+ 2.16.0). Phylogenetic analysis classified these genes into 15 groups. Promoter cis-element prediction, co-expression network construction, and methyl jasmonate (MeJA) induction experiments were further performed to explore the possible regulatory associations of PgPHD genes. The PgPHD genes were distributed across all 24 chromosomes, and 18 segmentally duplicated gene pairs suggested that whole-genome duplication drove the expansion of this gene family. A total of 308 MeJA-responsive cis-elements were identified in the promoter regions, making them one of the most prevalent functional categories. Co-expression network analysis based on 42 landraces identified five candidate genes significantly associated with key enzyme genes involved in ginsenoside biosynthesis, among which PgPHD31-09 showed a significant expression correlation with nine key enzyme genes. qRT-PCR analysis showed that PgPHD31-09 was significantly upregulated in response to MeJA treatment, and its expression pattern was similar to that of several key enzyme-encoding genes related to ginsenoside biosynthesis. This study provides the first systematic analysis of the evolutionary and expression characteristics of the PHD gene family in P. ginseng, identifying PgPHD31-09 as a candidate gene potentially associated with MeJA responsiveness and ginsenoside biosynthesis.

Ginsenoside Rg1 has a broad spectrum of pharmacological activities, and its concentration is a key indicator of ginseng quality. Because ginsenoside levels are quantitative traits controlled by multiple genes and environmental factors, identifying genes involved in Rg1 biosynthesis is essential to improve its production in ginseng. Transcriptome analysis of 344 4-year-old ginseng roots identified 33 Candidate Genes implicated in Rg1 biosynthesis. These candidates were detected through differential expression analysis, SNP/InDel mutation screening, correlation analysis with Rg1 content variation, and co-expression network analysis. By integrating GO functional annotation, gene expression profiles, and Rg1 content correlations, a putative biosynthetic pathway for Rg1 was inferred and the roles of these genes were preliminarily clarified. Further study of MeJA regulation confirmed that PgRg1-021 is highly associated with ginsenoside Rg1 biosynthesis. Functional verification was performed via overexpression of PgRg1-021 and RNAi in ginseng hairy roots. RT-qPCR analysis showed that PgRg1-021 negatively regulated ginsenoside Rg1 biosynthesis: Rg1 saponin levels decreased after overexpression and increased after RNAi. This study was the first to verify PgRg1-021’s role in Rg1 biosynthesis and provided essential gene resources for pathway analysis. It established a foundation for targeted breeding and serves as a reference for identifying other genes associated with complex traits.

Panax ginseng is a medicinal plant with diverse pharmacological effects; its primary active components are ginsenosides. The biosynthesis of ginsenosides can be regulated by the plant hormone methyl jasmonate (MeJA) during in vitro culture of ginseng. The PYL proteins, which serve as abscisic acid (ABA) receptors, play a crucial role in plant hormone signaling transduction. However, there are no reports on the response of PYL genes to MeJA in ginseng. In this study, we identified 49 members of the PYL gene family in the ginseng genome and transcriptome from databases and conducted a systematic analysis. The results indicated that these PYL genes were unevenly distributed across the chromosomes and exhibited significant synteny. Gene Ontology (GO) functional annotation revealed considerable functional diversity in the PYL gene family in ginseng. Analysis of the promoter cis-acting elements showed that these genes are involved in various biological processes, including growth, development, and metabolic regulation. After treating ginseng adventitious roots with MeJA, we found that four PgPYL genes (PgPYL13, PgPYL17-01, PgPYL17-02, and PgPYL23) containing abundant MeJA-responsive elements exhibited distinct expression patterns and were negatively correlated with protopanaxadiol-type ginsenoside content. This study systematically elucidated the characteristics and functions of the ginseng PYL gene family, revealing its potential role in MeJA signal transduction and the regulation of ginsenoside biosynthesis. Our findings provide a theoretical basis for optimizing the in vitro culture of ginseng plant cells and enhancing ginsenoside production.

Panax ginseng is a traditional Chinese herbal medicine. Ginsenosides, the main bioactive components responsible for the medicinal value of ginseng, are regulated by transcription factors. Among these regulatory factors, basic leucine zipper (bZIP) transcription factors play crucial roles in plant development and secondary metabolism. To verify that members of the bZIP gene family are involved in regulating ginsenoside biosynthesis and explore their potential mechanisms of action, a correlation analysis was first conducted in this study between the expression levels of PgbZIP genes and ginsenoside content. Additionally, the effects of single-nucleotide polymorphisms (SNPs) and Insertions/Deletions (InDels) on ginsenoside content were analyzed in this study. Through these analyses, PgbZIP48-3, a gene highly associated with ginsenoside biosynthesis, was identified. Subsequently, we systematically analyzed PgbZIP48-3, including its gene structure, protein properties, and phylogenetic relationships. To further verify the function of PgbZIP48-3, an overexpression vector was constructed. Positive ginseng hairy roots were obtained via Agrobacterium-mediated transformation of explants, and the ginsenoside content in these positive hairy roots was determined. The results showed that in the PgbZIP48-3 overexpression positive lines, the contents of ginsenosides Re, Rb2, Rb3, Rc, and Rd were significantly higher than those in the control group, whereas the contents of ginsenosides Rg1, Rf, and Rb1 were lower than those in the control group. Finally, by investigating the SNP/InDel data of PgbZIP48-3 in 346 accessions of a natural P. ginseng population and constructing a predicted interaction map between PgbZIP48-3 and key enzyme genes involved in ginsenoside synthesis, this study preliminarily revealed the potential molecular mechanism by which PgbZIP48-3 regulates ginsenoside biosynthesis from two dimensions: gene mutation and gene expression correlation. Meanwhile, this study provides genetic resources for the breeding of ginseng cultivars with high ginsenoside content.

Ginseng (Panax ginseng) is a valuable medicinal plant whose primary active components, known as ginsenosides, play a significant role in anti-cancer, anti-inflammatory, and anti-diabetic effects. WRKY transcription factors represent a prominent class of transcription factors in higher plants, fulfilling essential functions in numerous processes such as plant growth and development, reactions to biotic and abiotic stresses, and the control of secondary metabolism. This study is based on the laboratory’s previous bioinformatics analysis of the WRKY gene family in ginseng. After in-depth analysis, the PgWRKY064-04 gene was identified, which is significantly associated with ginsenosides. The physicochemical properties and expression patterns of this gene were analyzed, indicating that its expression in ginseng is temporally and spatially specific. A subcellular localization vector for this gene was constructed, confirming that it functions in the cell nucleus. Subsequently, overexpression vectors and interference vectors for PgWRKY064-04 were constructed, and ginseng adventitious roots were transformed using Agrobacterium-mediated transformation, successfully yielding positive materials. Gene expression levels and saponin content in the positive materials were detected, preliminary findings indicate that the expression of the PgWRKY064-04 gene is negatively correlated with the biosynthesis of ginsenosides. This study complements research on the functional roles of WRKY transcription factor family genes in ginseng, paving the way for future efforts to enhance ginsenoside production using modern biotechnological approaches.

Ginseng (Panax ginseng) is a globally renowned medicinal plant. The primary active compounds in ginseng are ginsenosides, which have been shown to possess preventive and therapeutic properties against a range of ailments. WNK (with no lysine) kinases is a subfamily of serine/threonine protein kinases. Members of the WNK gene family play vital roles in the regulation of plant growth, development, and biological processes. Several studies have shown that jasmine methyl ester treatment can increase the content of protopanaxatriol-type ginsenosides in adventitious ginseng roots. However, there are no reports on the effects of WNK in ginseng under methyl jasmonate (MeJA) treatment. In this study, we conducted a detailed screening, identification, and systematic research on the WNK gene family in ginseng, including phylogenetic relationships, gene structure, chromosomal distribution, GO functional classification, cis-regulatory elements, co-expression network analysis, and gene expression pattern analysis. Furthermore, we identified candidate genes of PgWNK that could significantly respond to methyl jasmonate regulation and conducted an in-depth study of their expression patterns to explore the impact of these candidate genes on the variation in triterpenoid ginsenoside content. We identified 32 WNK genes in ginseng that were unevenly distributed across 11 of the 24 chromosomes in the ginseng genome. GO functional annotation and enrichment analysis of the PgWNK genes revealed their involvement in numerous biological processes, including ginseng growth, development, and biosynthesis of secondary metabolites. Through expression network analysis, we further understand that there are complex interactions among members of the PgWNK gene family. In addition, 17 non-biological stress response components related to hormone response, growth, and development were predicted in the promoter area of the PgWNK gene. At the same time, we studied the gene expression of PgWNK after MeJA treatment and found that the expression related to ginsenoside biosynthesis was significantly increased as the regulation time was prolonged. Following MeJA regulation, the content of five protopanaxatriol-type ginsenosides (Re, Rf, Rg1, Rg2, and Rh1) increased owing to the adjustment of PgWNK05 gene expression. Therefore, our results provide an important theoretical foundation for understanding the molecular characteristics, genetic evolution, genetic functions, expression patterns, and non-biological coercion of the WNK gene family in ginseng. The underlying research on the biological synthesis of ginsenosides provides a foundation and theoretical support.