Montbretia flowers as a source of bioactive crocins: Biotechnology tools and delivery systems

Crocins are potent antioxidants with significant therapeutic potential, exhibiting anti-inflammatory, anticancer, and antidepressant properties. The ornamental plant <i>Crocos-mia x crocosmiiflora</i> is widely cultivated for its aromatic and vibrant flowers. In this study, we identified unique crocins as the primary pigments responsible for the flower's coloration. These metabolites predominantly consist of molecules with eight glucose units, followed by crocins containing six and seven glucose units. To elucidate the mo-lecular mechanisms underlying crocin biosynthesis in <i>C. x crocosmiiflora</i>, tran-scriptomic analysis was performed to identify key carotenoid cleavage dioxygenase (CCD) genes. Using <i>Crocus sativus</i> CsCCD2L gene as a bait, we identified a CCD transcript from the transcriptome data. Phylogenetic analysis revealed that the identified CCD belongs to the CCD2 subfamily, and it was designated as CroCCD2. Functional characterization of CroCCD2 was carried out using bacterial expression systems and <i>Nicotiana benthamiana</i> plants with a virus-mediated expression system. These experiments demonstrated that CroCCD2 efficiently converts the precursor zeaxanthin into crocetin, a key intermediate in crocin biosynthesis. Furthermore, we investigated the bioactivity of crocins and discovered that their anti-inflammatory effects depend on their vehiculation within exosomes or liposomes. This suggests that the transport mechanism is critical for the biological activity of crocins. Our findings highlight the specialization of CCD subfamilies in monocots and dicots for crocin biosynthesis and provide evidence of the anti-inflammatory activity of exosome-transported crocins. This study establishes a foundation for further research into the metabolic network of crocins in <i>C. x crocosmiiflora</i> and suggests that the CroCCD2 gene could be introduced into other crop plants to produce these bioactive apocarotenoids.