The medicinal properties of narcissus plants are attributed to the presence of Amaryllidaceae alkaloids. Elicitors, such as nanoparticles, are employed to enhance the production of secondary metabolites through signaling and the generation of reactive oxygen species. We investigated the effects of Fe3O4 nanoparticles (Fe3O4 -NPs), trans-cinnamic acid (tCA), and L-phenylalanine (L-Phe) precursors on various physiological and biochemical parameters in Narcissus tazetta L., with a particular focus on alkaloid content. Fe3O4 - NPs treatment significantly increased photosynthetic pigments, secondary metabolites (including alkaloids, phenolic compounds), total soluble carbohydrates, and polysaccharides. The activities of antioxidant enzymes (SOD, CAT, POD), phenylalanine ammonia-lyase (PAL), and tyrosine ammonia-lyase (TAL) enhanced under Fe3O4-NPs treatment. While tCA treatment led to an increase in H-2 O-2 level, L-Phe treatment decreased it. Both elicitors influenced the plant's metabolism, promoting primary and secondary metabolites. The augmentation of photosynthetic pigment content and antioxidant enzyme activity induced by tCA and L-Phe treatments appeared to improve alkaloid production. Furthermore, in silico studies using Molegro Virtual Docker (MVD) software were conducted. The third structures of PAL and N4OMT enzymes were designed by Modeler software and the ligand structures were designed using ChemDraw (R) (Fe3O4) and Atomsk (precursors) software. Docking results revealed that the Fe3O4 -NPs and precursors interacted with the active sites of PAL and N4OMT, two enzymes involved in alkaloid biosynthesis in narcissus, exhibiting varying binding energies and impacting their activities. The Fe3O4 /N4OMT and L-phenylalanine/PAL complexes had higher free energies of binding. Our results indicated that Fe3O4-NPs and precursors significantly affected the gene expressions of PAL and N4OMT. The highest levels of PAL (after 96 h) and N4OMT (after 24 h) expressions were obtained in tCA + Fe3O4-NPs and L-Phe + Fe3O4-NPs treatments. In summary, the application of Fe3O4-NPs, tCA, and L-Phe demonstrated the potential to activate the production of secondary metabolites, including alkaloids, by modulating the plant's metabolic pathways.
