Comparative transcriptomic analysis and genome-wide identification provide insights into the potential role of fungal-responsive MAPK cascade genes in tanshinones accumulation in Salvia miltirrohiza

Ann Abozeid^1*Xinru Du¹Lan Zhang¹Furui Yang¹Jianxiong Wu²Lin Zhang¹Qi Cui¹Zongqi Yang^1*Dongfeng Yang^1*

• ¹Zhejiang Sci-Tech University, Hangzhou, Zhejiang, China
• ²China Agricultural University, Beijing, Beijing Municipality, China

Salvia miltiorrhiza is a well-known Traditional Chinese Medicine (TCM) for its bioactive tanshinones that are used to treat various diseases and have high antimicrobial properties. Previous studies have shown that tanshinones accumulation in S. miltiorrhiza was shown to be significantly induced by fungal elicitors. Mitogen-activated protein kinase (MAPK) s, which play critical roles in plant-microbe interactions and cellular processes, are known to regulate the accumulation of antimicrobial metabolites. In this study, we aimed to identify MAPK families in S. miltiorrhiza and screen SmMAPKs for candidates involved in fungal elicitor-mediated tanshinones accumulation. Through genome-wide analysis, we identified 17 MAPKs, 7 MAPKKs, and 22 MAPKKKs genes in S. miltiorrhiza, which were distributed across 9 chromosomes. Phylogenetic analysis classified SmMAPKs into two subgroups, TEY and TDY, similar to Arabidopsis MAPKs, while all SmMAPKKKs clustered under the MEKK subfamily. Cis-acting elements analysis revealed that most SmMAPK genes are associated with stress and phytohormone responses, suggesting their involvement in defense mechanisms. To investigate the role of MAPK s in tanshinones accumulation, hairy roots of S. miltiorrhiza were treated with two fungal elicitors, yeast extract and Aspergillus niger, for 1 and 4 days. HPLC analysis demonstrated that both elicitors significantly promoted the accumulation of tanshinones, particularly cryptotanshinone and dihydrotanshinone. Comprehensive transcriptomic analysis, followed by Pearson correlation coefficient analysis, revealed a strong positive correlation between tanshinones content and SmMPK4 and SmMPKK5, while negative correlations were observed with SmMPKKK6, SmMPKKK11, and SmMPKKK20. The presence of defense-related cis-acting elements in the promoter regions of SmMPK4, SmMPKK5, SmMPKKK6, SmMPKKK11, and SmMPKKK20 further supports their involvement in fungal elicitor-mediated tanshinones accumulation. This study provides critical insights into the regulatory roles of SmMAPK genes in tanshinones accumulation in S. miltiorrhiza in response to fungal elicitors. These findings have potential applications in enhancing tanshinones production for medicinal purposes, offering a foundation for further research into the molecular mechanisms underlying tanshinones biosynthesis.