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Hypericum perforatum L. is a medicinal plant commonly used worldwide for the treatment of mild and moderate depression due to its wide range of bioactive compounds. H. perforatum regenerated roots have been proposed as an efficacious in vitro system to biosynthesize pharmaceutically useful secondary metabolites. In the present study, a metabolomic platform, which integrates an nuclear magnetic resonance (NMR)-based metabolic profiling and analysis of variance-simultaneous component analysis (ASCA), has been applied in order to characterize the changes of the primary and secondary metabolism of H. perforatum regenerated roots induced by an achieved high biomass density in a confined growth environment or in response to chitosan treatment.The ASCA modelling applied to NMR-based metabolic profiling allowed to recognize the effects due to biomass growth rate changes and chitosan treatment. With an high biomass density, associated to a decelerating biomass growth rate, the levels of tryptophan, fructose, shikimic acid, and epicatechin increased, whereas γ-aminobutyric acid and histidine decreased. In response to chitosan elicitation, the biomass growth was arrested and valine, isoleucine, glutamine, γ-aminobutyric acid, fructose, sucrose, polyunsaturated fatty acids, epicatechin, xanthones, dimethylallyl-pyrophosphate, and stigmasterol levels increased, while histidine levels decreased. The metabolic profiling of regenerated roots shows how the cultures respond to different stress conditions: production of epicatechin in response to high biomass density and production of epicatechin, xanthones and isoprenoids in response to chitosan-treatment. This approach can be applied to define suitable protocols to produce the desired secondary metabolites with different bioactivities.

The responses of Hypericum perforatum root cultures to chitosan elicitation had been investigated through (1)H-NMR-based metabolomics associated with morpho-anatomical analyses. The root metabolome was influenced by two factors, i.e., time of culture (associated with biomass growth and related "overcrowding stress") and chitosan elicitation. ANOVA simultaneous component analysis (ASCA) modeling showed that these factors act independently. In response to the increase of biomass density over time, a decrease in the synthesis of isoleucine, valine, pyruvate, methylamine, etanolamine, trigonelline, glutamine and fatty acids, and an increase in the synthesis of phenolic compounds, such as xanthones, epicatechin, gallic, and shikimic acid were observed. Among the xanthones, brasilixanthone B has been identified for the first time in chitosan-elicited root cultures of H. perforatum. Chitosan treatment associated to a slowdown of root biomass growth caused an increase in DMAPP and a decrease in stigmasterol, shikimic acid, and tryptophan levels. The histological analysis of chitosan-treated roots revealed a marked swelling of the root apex, mainly due to the hypertrophy of the first two sub-epidermal cell layers. In addition, periclinal divisions in hypertrophic cortical cells, resulting in an increase of cortical layers, were frequently observed. Most of the metabolic variations as well as the morpho-anatomical alterations occurred within 72 h from the elicitation, suggesting an early response of H. perforatum roots to chitosan elicitation. The obtained results improve the knowledge of the root responses to biotic stress and provide useful information to optimize the biotechnological production of plant compounds of industrial interest.