Despite the increasing identification of long non-coding RNAs (lncRNAs) in plants, their roles in specialized metabolism remain largely unexplored, particularly in medicinal and aromatic plants. In this study, we investigated the regulatory roles of lncRNAs in the monoterpene indole alkaloid (MIA) biosynthesis pathway of Catharanthus roseus, a plant renowned for producing the anti-cancer compounds, vinblastine and vincristine. Candidate lncRNAs associated with MIA accumulation were identified using a systems biology approach and subsequently validated through transient overexpression. Among the candidates, Crlnc440 was negatively associated with MIA biosynthesis. Its transient overexpression in C. roseus leaves led to significant reductions in vindoline (23%), vinblastine (33%), anhydrovinblastine (27.7%), and serpentine (18%), accompanied by the downregulation of key MIA biosynthetic genes (D4H, DAT, PRX1, and SS) and the master regulator MPK6. Conversely, Crlnc343 and Crlnc1096 were positively associated whit MIA biosynthesis. Overexpression of Crlnc343 resulted in significant increases in vinblastine (142%), anhydrovinblastine (175%), and catharanthine (64.5%). Similarly, Crlnc1096 overexpression elevated vinblastine (151%), anhydrovinblastine (35%), and catharanthine (30%). All three lncRNAs exhibited organ-specific expression patterns, and their differential expression under methyl jasmonate conditions was identified through transcriptomic data analysis. This study suggests the diverse roles of lncRNAs in regulating specialized metabolism and highlights their potential as promising targets for metabolic engineering to enhance the production of pharmacologically valuable alkaloids.

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