The HD-Zip gene family plays a vital regulatory role in plant growth and development, but its function in plant secondary metabolism remains to be further elucidated. This study therefore investigated the involvement of the HD-Zip gene family in ginsenoside biosynthesis and identified the genes that were highly correlated. Through correlation analysis between gene expression and ginsenoside content, heatmap and co-expression network analyses, and association analysis between Single Nucleotide Polymorphism (SNP) variations and ginsenoside content, the PgHDZ01 gene was identified as the core candidate gene highly associated with ginsenoside biosynthesis. Subsequently, this gene was shown to significantly respond to Methyl Jasmonate (MeJA) treatment and correlated with changes in ginsenoside content. Further functional verification using Virus-Induced Gene Silencing (VIGS) in Panax ginseng (Ginseng) seedlings and gene overexpression in ginseng hairy roots confirmed the positive regulatory role of PgHDZ01 in ginsenoside biosynthesis. Moreover, its overexpression led to a significant increase in the content of ginsenoside Rb1, and its expression was positively correlated with the change in Rb1 content. The results of this study provide a reference for the comprehensive analysis of the functions of the HD-Zip gene family and the regulatory mechanisms of ginsenoside synthesis. The VIGS method used in this study also provides a new approach for the verification of functional genes in ginseng. The results demonstrate the critical role of PgHDZ01 in regulating ginsenoside biosynthesis, deepening our understanding of the underlying mechanisms of ginsenoside biosynthesis and offering a potential candidate gene for molecular breeding applications.

MADS-box genes are essential for plant development and secondary metabolism. The majority of genes within a genome exist in a gene family, each with specific functions. Ginseng is an herb used in medicine for its potential health benefits. The MADS-Box gene family in Jilin ginseng has not been studied. This study investigated the evolution and structural and functional diversification of the PgMADS gene family using bioinformatics and analyzed gene expression through methyl jasmonate (MeJA) regulation. The results revealed that the evolution of the PgMADS gene family is diverged into ten clusters of a constructed phylogenetic tree, of which the SOC1 cluster is the most prevalent with a higher number of PgMADS genes. Despite their distinct evolutionary clusters, a significant number of members contains common conserved motifs. The PgMADS gene family was functionally differentiated into three primary functional categories, biological process, molecular function, and cellular component. Their expression is variable within a tissue, at a developmental stage, and in cultivars. Regardless of the diversity of the functions of PgMADS genes and evolution, their expression correlated and formed a co-expression gene network. Weighted gene co-expression network analyses identified hub genes that could be regulating ginsenoside biosynthesis. Interestingly, the family also is involved in MeJA regulation. These findings provide a valuable reference for future investigations on PgMADS genes.

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.