Molecular and biochemical insights into tryptophan-induced indole alkaloid biosynthesis in Isatis tinctoria L. root cultures

Abstract This study was carried out to investigate the effects of tryptophan (Trp) application on indigotin (IND) and indirubin (INR) production, as well as on the expression levels of Isatis tinctoria Tryptophan Synthase Alpha (It-TSA) and Cytochrome P450 ( CYP79B2) genes involved in their biosynthesis, in Isatis tinctoria L. root cultures. Three-week-old root cultures were treated with Trp at concentrations of 100, 200, and 300 mg/L for 48 h, after which the IND and INR contents in the harvested roots were quantified. Concurrently, changes in the expression levels of It-TSA and CYP79B2 genes, which are responsible for indole alkaloid biosynthesis, were determined. The findings revealed that all analyzed parameters significantly varied depending on the Trp concentration. Notably, the 200 mg/L Trp treatment resulted in both the highest accumulation of IND and INR and the maximum expression levels of the target genes. A striking positive relationship was found between the increase in gene expression and metabolite accumulation. These results clearly demonstrated that Trp acts as an effective precursor for increasing secondary metabolite production in I. tinctoria root cultures. By providing crucial data for optimizing the production of plant-derived valuable metabolites through in vitro techniques, this study makes valuable contributions to the field of plant biotechnology. Graphical abstract