Abstract
The responses of Hypericum perforatum root cultures to chitosan elicitation had been investigated through 1H-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.
Highlights
The development of alternative methods to whole plant cultivation for the production of pharmaceutically valuable compounds of commercial interest is an issue of considerable socio-economic importance
Root cultures characterized by a high growth rate and active secondary metabolism provides an efficient system to produce great amounts of secondary metabolites highly valued in pharmaceutical industry (Sivakumar, 2006)
To increase the time period of chitosan treatment, maintaining the root culture in the exponential growth phase, a necessary condition to assume the metabolic steady-state, the culture medium was renewed at day 4 and chitosan elicitation was performed at day 8 of the growth curve
Summary
The development of alternative methods to whole plant cultivation for the production of pharmaceutically valuable compounds of commercial interest is an issue of considerable socio-economic importance. One of the major obstacles to the use of organ cultures for the pharmaceutical industry is the low yield of the metabolites of interest. For this reason, several strategies have been adopted to improve the production of plant-derived secondary metabolites such as two-phase culture system, genetic transformation, metabolic and bioreactor engineering (Georgiev et al, 2012; Tocci et al, 2012; Wilson et al, 2014; Simonetti et al, 2015). Chitosan treatment has been shown to increase the production of xanthones in H. perforatum root cultures (Tocci et al, 2011, 2012, 2013a)
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