Licochalcone A (LCA) is a characteristic compound in licorice Glycyrrhiza inflata and is widely utilized in pharmaceutical and cosmetic industries. However, the biosynthetic pathway and regulatory mechanisms of LCA remain poorly understood. In this study, we first found the accumulation of LCA is induced by methyl jasmonate (MeJA). Given that MYB transcriptional factors are well-documented as key regulators of flavonoid biosynthesis, we identified a total of 147 GiR2R3-MYB genes in G. inflata, which were classified into 28 subgroups. The chromosome distributions, sequence characteristics, gene structures, duplication events and cis-acting elements were also investigated. Through integrated analysis of GiR2R3-MYBs expression patterns across different tissues and under MeJA treatment, along with phylogenetic relationship, we identified GiMYB76—a MeJA-inducible MYB transcription factor—as a potential regulator of LCA accumulation. Functional validation showed that transgenic hairy roots overexpressing GiMYB76 exhibited a significant increase in LCA content. DAP-seq analysis of GiMYB76 revealed potential target genes involved in flavonoid biosynthesis regulation. Subsequent promoter activity assay verified that GiMYB76 can bind to the promoter and activate the expression of GiCHS4. Consistently, overexpression of GiCHS4 in G. inflata hairy roots also significantly enhanced LCA production. This study not only clarifies that GiMYB76 transcriptionally activated GiCHS4 to promote LCA biosynthesis but also provides valuable insights for basic research on licorice and the development of related industries.

The evolutionarily conserved mitogen-activated protein kinase (MAPK) cascades relay extracellular signals into cells, triggering a variety of cellular responses. We previously revealed NtMPK4 as a positive regulator of nicotine biosynthesis; however, its upstream regulation remains unclear. Here, we characterized a MAPK cascade, comprising NtMEKK1b, NtMPKK2a, and NtMPK4, that promotes nicotine biosynthesis. This signaling module transduces external cues, including jasmonate and pathogen elicitors such as flg22, into post-translational modifications that enhance transcriptional activity and pathway gene expression. NtMPKK2a physically interacts with and phosphorylates NtMPK4 in vivo, confirming its role as an upstream kinase. RNAi-mediated silencing of NtMPKK2a significantly reduced the expression of nicotine pathway genes and decreased nicotine accumulation, whereas induced-overexpression of NtMPKK2a upregulated nicotine pathway genes and increased nicotine contents in tobacco hairy roots. Overexpression of NtMPKK2a in tobacco cells enhanced the transactivation activity of a NIC2-locus Ethylene Response Factor NtERF221 on Putrescine N-methyltransferase (NtPMT) promotor, further supporting its role in promoting nicotine biosynthesis. Furthermore, we identified NtMEKK1b, a tobacco MEKK that interacts with NtMAPKK2a in yeast cells. Knock-down of NtMEKK1b in transgenic tobacco plants attenuated the expression of nicotine pathway genes and reduced nicotine contents, whereas induced-overexpression of NtMEKK1b upregulated gene expression and nicotine accumulation. Our findings uncover a previously uncharacterized MAPK cascade module, NtMEKK1b-NtMPKK2a-NtMPK4, that regulates nicotine biosynthesis, highlighting the importance of posttranslational regulation in nicotine biosynthesis.