Melatonin Influences Terpenoid Indole Alkaloids Biosynthesis and 5′ Upstream-Mediated Regulation of Cell wall Invertase in Catharanthus roseus

Melatonin is a well-known hormone, found ubiquitously in plants and has been known to impart several beneficial traits. Although its role in influencing therapeutically significant specialized metabolites has been studied, not much is understood in medicinal plants, including Catharanthus roseus. C. roseus is a valuable plant source of anti-neoplastic terpenoid indole alkaloids (TIAs) vincristine and vinblastine, and most studies have focused on the TIAs. However, understanding the sucrose metabolism, which provides the carbon backbone for the biosynthesis of specialized metabolites would provide a holistic understanding of the metabolic and regulatory interconnections between both processes. This report provides the first evidence on the role of melatonin as an elicitor of TIAs and CWIN (Cell wall invertase; a key sucrose metabolizing enzyme with pleiotropic functions) in C. roseus. Besides, a potential relationship among melatonin, CWIN, and TIA biosynthesis was observed via CrSNAT gene silencing (a key rate-limiting gene in melatonin biosynthesis) and post-translational CWIN repression approaches. Further, the cis-based regulation studies using transgenic C. roseus calli and Nicotiana benthamiana involving the 5′ upstream regions of CrCWIN2 identified potential melatonin-responsive regions that showed binding by the CrERF transcription factors (TFs: CrERF1b and CrERF5). Silencing the CrERFs showed reduced accumulation of the key TIAs along with their biosynthetic genes and CWIN (transcript and activity). Based on our findings, it could be inferred that melatonin acts as an elicitor of CWIN and TIA biosynthesis and influences the 5′ upstream-mediated regulation of CrCWIN2 possibly via ERFs: CrERF1b and CrERF5. Taken together, our study provides scope for future elicitation-based medicinal crop-improvement programs to achieve enhanced therapeutic productivity.