Enhancement of indigotin and indirubin production in root cultures of <i>Isatis</i> species by H₂O₂: biochemical and molecular responses

Isatis species are prominent in pharmaceutical and industrial sectors due to their rich indole alkaloid content. This study examined the effects of hydrogen peroxide (H2O2) treatments on the syntheses of indigotin (IND) and indirubin (INR), antioxidant enzyme activities, and the expression of TSA and CYP79B2 genes involved in the biosynthesis of IND and INR in root cultures of four Isatis species I. constricta, I. ermenekensis, I. floribunda, and I. tinctoria. Intracellular H2O2 contents were also assessed. For this aim, H2O2 was applied at concentrations of 100, 200, 300, and 400 mu M for 24 h to root cultures. The results indicated that when used appropriate concentrations, H2O2 applications increased IND and INR accumulation in roots and the concentrations that provide the highest metabolite production also vary according to the species. H2O2 elicitation significantly changed the expression of TSA and CYP79B2 genes and antioxidant enzyme activities depending on species and H2O2 concentration, reflecting dynamic oxidative stress responses. These findings revealed that H2O2 has significant potential to enhance alkaloid biosynthesis in Isatis root cultures through oxidative signaling pathways, providing a foundation for the sustainable in vitro production of pharmaceutically valuable compounds. This approach is important as it offers a scalable and environmentally friendly alternative for commercial applications by reducing dependence on natural populations.