De novo transcriptomic analysis reveals salicylic acid-induced biosynthesis of vinblastine and vincristine in undifferentiated Catharanthus roseus callus

Catharanthus roseus is a pharmaceutically important medicinal herb known for being the sole natural source of the anticancer dimeric alkaloids vinblastine and vincristine, synthesized via the complex monoterpenoid indole alkaloid (MIA) biosynthetic pathway. The in-planta accumulation of these alkaloids is exceedingly low, primarily due to their intrinsic cytotoxicity and the strict spatiotemporal regulation of MIA biosynthesis, which spans at least four distinct cell types (epidermis, internal phloem associated parenchyma, laticifers, and idioblasts) and five intracellular compartments (chloroplast, vacuole, nucleus, endoplasmic reticulum, and cytosol). Previous studies demonstrated that only differentiated multiple shoot cultures of C. roseus can biosynthesize vinblastine and vincristine, whereas undifferentiated systems such as cell suspensions, callus, and hairy roots lack the necessary cellular compartmentalization required for dimeric MIA synthesis. However, limited evidence suggests that abiotic elicitors may activate the latent biosynthetic machinery in callus tissues. In the present study Salicylic acid (SA) elicitation significantly enhanced MIA biosynthesis in C. roseus callus cultures. RNA-seq analysis of callus cultures treated with potassium chloride (3 g/l, 5 g/l), Salicylic Acid (5 mM, 15 mM), and succinic acid (5 mM, 15 mM) treatments identified 12,555 DEGs under SA 5 mM, with strongest activation of MIA pathway genes including TDC, SLS, STR, LAMT, G10H, CrHL2, and late-stage vindoline enzymes. qRT-PCR confirmed 3–8-fold induction of key genes mirroring RNA-seq trends. SA treatments uniquely activated PRX1 peroxidase isoforms essential for vindoline-catharanthine coupling to vinblastine/vincristine. These findings demonstrate SA's superior efficacy in enhancing C. roseus MIA biosynthesis compared to salt or metabolic stress, enabling the biosynthesis of vinblastine and vincristine in an undifferentiated system previously thought to lack this capacity. These findings establish a framework for elicitor-based enhancement of anticancer alkaloid production.