Expression analysis of Cell wall invertase under abiotic stress conditions influencing specialized metabolism in Catharanthus roseus

Catharanthus roseus is a commercial source for anti-cancer terpenoid indole alkaloids (TIAs: vincristine and vinblastine). Inherent levels of these TIAs are very low, hence research studies need to focus on enhancing their levels in planta. Since primary metabolism provides precursors for specialized-metabolism, elevating the former can achieve higher amounts of the latter. Cell Wall Invertase (CWIN), a key enzyme in sucrose-metabolism catalyses the breakdown of sucrose into glucose and fructose, which serve as carbon-skeleton for specialized-metabolites. Understanding CWIN regulation could unravel metabolic-engineering approaches towards enhancing the levels of TIAs in planta. Our study is the first to characterize CWIN at gene-expression level in the medicinal plant, C. roseus. The CWINs and their inter-relationship with sucrose and TIA metabolism was studied at gene and metabolite levels. It was found that sucrose-supplementation to C. roseus leaves significantly elevated the monomeric TIAs (vindoline, catharanthine) and their corresponding genes. This was further confirmed in cross-species, wherein Nicotiana benthamiana leaves transiently-overexpressing CrCWIN2 showed significant upregulation of specialized-metabolism genes: NbPAL2, Nb4CL, NbCHS, NbF3H, NbANS, NbHCT and NbG10H. The specialized metabolites- cinnamic acid, coumarin, and fisetin were significantly upregulated. Thus, the present study provides a valuable insight into metabolic-engineering approaches towards augmenting the levels of therapeutic TIAs.