Molecular and Biochemical Characterization of Coumarins and Inulin Biosynthetic Pathways in Chicory (Cichorium intybus) Callus Cultures under Nitric Oxide and Sucrose Treatments

This investigation delves into the modulation of coumarin and inulin biosynthesis in Cichorium intybus callus cultures by sodium nitroprusside (SNP), a nitric oxide donor, and sucrose. These bioactive compounds are acclaimed for their health-promoting properties, including antioxidant, anti-inflammatory, antimicrobial, anticancer, prebiotic, and immunomodulatory activities. Employing a molecular and biochemical methodology, the study examines the expression and enzymatic activity of phenylalanine ammonia-lyase (PAL), hydroxycinnamoyl-CoA transferase (HCT), sucrose : sucrose 1-fructosyltransferase (1-SST), and fructan : fructan 1-fructosyltransferase (1-FFT). Quantitative analysis revealed that a synergistic treatment of 30 µM SNP and 30 g/L sucrose yielded the highest concentrations of coumarins, specifically scopoletin and esculin, and inulin. This condition also correlated with the peak expression and activity of the aforementioned enzymes. Conversely, a treatment of 60 µM SNP and 0 g/L sucrose resulted in the lowest compound levels and enzymatic expression and activity. Additionally, SNP and sucrose treatments influenced the growth and morphological characteristics of chicory callus, inducing variations in color, shape, size, and texture. The agglomerative hierarchical clustering represented three different clusters for sucrose concentrations and three clusters for SNP levels so that SNP at 0 and 60 µM were placed in one cluster. Heat map analysis revealed that HCT1, PAL, esculin, and inulin possessed the maximum changes upon SNP. The study posits the potential application of SNP as an elicitor and sucrose as both an energy source and signaling molecule to enhance the production of these bioactive compounds in chicory, both in vivo and in vitro, for industrial and medicinal applications. Furthermore, this research contributes to the understanding of the molecular and biochemical mechanisms governing the biosynthesis of coumarins and inulin in chicory.