Phenotypic and metabolic responses to ozone of two Fraxinus excelsior provenances

Environmentally-induced phenotypic variations in plants are considered to be functional responses that maximize fitness in variable environments. Provenances of a species may have evolved mechanisms to cope with stress and to complete their life cycles under hostile conditions. In particular, the magnitude of metabolic alterations in response to stress correlates with sensitivity/tolerance. Many phenylpropanoid compounds play a crucial role in plant defense against several environmental stressors such as tropospheric ozone (O3). Leaf physiological and metabolic traits of two Italian provenances [Piedmont (P) and Tuscany (T)] of Fraxinus excelsior (two-year-old plants) exposed to O3 (150 ppb, 5 h d-1 , 35 consecutive days) were investigated to verify if the geographical gradient affects O3 sensitivity. Analysis of visible injuries, chlorophyll a fluorescence and phenylpropanoids were performed. In P plants exposed to O3, the onset of visible injuries [widespread chlorosis which developed in minute (1–2 mm Ø) roundish dark-reddish necrosis scattered among the leaf veins of the adaxial surface of completely expanded leaves] occurred after 12 days of treatment; in treated T plants it occurred after 21 days from the beginning of the exposure. Throughout the whole period of exposure, chlorophyll a fluorescence analysis has been applied with success in a high-throughput phenotyping system. Differential responses to the pollutant between provenances were observed in terms of physiological responses. Negative effects of O3 on PSII were evident in P plants [the variable and maximal fluorescence ratio and the maximum quantum yield of PSII (ΦPSII) decreased throughout the whole period]. By contrast in T, these effects were shown intermittently and plants recovered at the end of the treatment (ΦPSII remained unchanged). Differential responses to the pollutant between provenances were observed also in terms of metabolic responses. A non-targeted metabolomics approach was applied using HPLC, detecting 18 phenolic compounds in both provenances and other de novo peaks (potential phytoalexins) in O3-elicited plants. The application of this approach provides a wider perspective of metabolic responses to O3 and supports the discovery of stress tolerance-specific metabolic phenotypes. Specific metabolite indicators were identified. Among hydroxycoumarins (Fraxinus-specific compounds), only scopoletin showed differential responses, decreasing only in T plants (-53%). Still among species-specific compounds, verbascoside decreased in comparison to controls only in T plants (-70%); oleuropein increased up to more than 8-fold only in P. Also trans-chalcone (present in the middle of the flavonoids biosynthesis pathway) increased up to 4 times over the controls only in treated P plants. Flavonoids were affected by O3 only in T plants: rutin (a quercetin glycoside) decreased by 56%; isoquercetrin and quercetrin showed higher values than controls (+152% and +120%). Among the hydroxibenzoic acids, O3 exposure decreased the syringic only in P plants (-48%). Also the hydroxycinnamic acids, placed at the beginning of the coumarin biosynthetic pathways, showed differential responses to the pollutant: p-coumaric increased only in T plants (+126%). We can conclude that: (i) the phenotypic variability and the geographical gradient affect the O3 response mechanisms, (ii) P plants activated different mechanisms (in terms of visible injury and PSII functionality) in comparison to T, (iii) P treated plants showed the highest biosynthesis within some specific phenolic metabolites (i.e. oleuropein, transchalcone), and (iv) metabolomics is a diagnostic tool that provides a powerful means to gain a better understanding of structural and physiological responses to O3.