Oxidative burst and metabolome modifications inducted by elicitation with yeast extract in suspension cultures of Passiflora garckei

The aim of this thesis was to study the oxidative burst and metabolome modifications induced by elicitation with yeast extract in suspension cultures of Passiflora garckei. In order to observe early events induced by elicitation we first measured pH medium alkalinization and ROS and NO production. Secondly, we looked for a possible correlation between these events and metabolome changes, by administrating different combinations of ROS and NO donors. To perform such a complex study in a non model species, lacking genetic tools, we used a metabolomic approach, which can integrate different techniques, from NMR to HPLC-DA, LC-MS and FTIR microspectroscopy to obtain a qualitative and quantitative analysis on as many metabolites as possible. The treatment of P. garckei suspension cells with yeast extract caused an accumulation of certain secondary metabolites that starts early and reaches its maximum 24 hours after the treatment. Preliminary NMR analysis combined to SPEC18 preparation method allowed us to separate sugars from aromatic compounds thus revealing that controls differ from treated samples mainly in the phenolics region. Starting from NMR data, we applied the HPLC-DA and then the more sensitive HPLCMS in order to analyse the methanol extractable sub-metabolome of P. garckei cells. PCA analysis showed that treated samples and controls were still well separated into two groups and they differed because of a lower amount of free sugars and a higher amount of hydroxycinnamic acids, flavonoids and cyanogenic glucosides in treated samples. Most of metabolic changes induced in P. garckei elicitated cells are related to direct defence strategies e.g. flavonoids and cyanogenic glycosides and to passive defence, such as structural reinforcement of the cell wall with an increase in hydroxycynnamic acids. Besides HPLC-MS and NMR analysis of methanolic extracts, also in situ FTIR microspectroscopy analysis of dehydrated cells showed differences between control and treated samples. FTIR microspectroscopy allowed underlining cellulose changes after elicitation, not detectable with metabolomic approach on methanolic extracts. FTIR data were in line with the increase in Calcofluor fluorescence. The increase in cellulose reinforces “passive defence”, being cell wall the first barrier to environmental attacks. ROS and NO production resulted to be a direct consequence of yeast extract elicitation; moreover, ROS production influences NO accumulation and vice-versa, and the kinetics of ROS and NO production is directly related to different metabolic profiles. In detail, after YE administration, NO and ROS production followed a biphasic kinetics of accumulation, with a first maximum after 30 minutes and a second after 8 hours. This has already been reported for other cell lines. The novelty in the present work was to study whether combination of NO and ROS accumulation were involved in yeast-extract secondary metabolite accumulation. ROS and NO were then measured in absence of elicitation, after NO and ROS donors administration. The results showed that different combinations of donors cause both different kinetics of NO and ROS accumulation and different metabolic profiles. In particular, some of the combinations induced an effect very similar to that induced by yeast extract treatment, confirming that ROS and NO productions are interrelated, but also suggesting that they are necessary and sufficient to induce some defence responses, as metabolite accumulation Future perspectives on this topic could be the analysis of other fractions of P. garckei extracts, the identification of still “unidentified” metabolites and investigation of other events induced by elicitation, such as ion fluxes.