Specialized metabolites constitute a major antioxidant system involved in plant defence against environmental constraints, such as tropospheric ozone (O₃). The objective of this experiment was to give a thorough description of the effects of an O₃ pulse (120 ppb, 5 h) on the phenylpropanoid metabolism of sage, at both biochemical and molecular levels. Variable O₃-induced changes were observed over time among the detected phenylpropanoid compounds (mostly identified as phenolic acids and flavonoids), likely because of their extraordinary functional diversity. Furthermore, decreases in the phenylalanine ammonia-lyase (PAL), phenol oxidase (PPO), and rosmarinic acid synthase (RAS) activities were reported during the first hours of treatment, probably due to an O₃-induced oxidative damage to proteins. Both PAL and PPO activities were also suppressed at 24 h from the beginning of exposure, whereas enhanced RAS activity occurred at the end of treatment and at the recovery time, suggesting that specific branches of the phenolic pathways were activated. The increased RAS activity was accompanied by the up-regulation of the transcript levels of genes like RAS, tyrosine aminotransferase, and cinnamic acid 4-hydroxylase. In conclusion, sage faced the O₃ pulse by regulating the activation of the phenolic biosynthetic route as an integrated defence mechanism.

Advancements in techniques to rapidly and non-destructively detect the impact of tropospheric ozone (O₃) on crops are required. This study demonstrates the capability of full-range (350–2500 nm) reflectance spectroscopy to characterize responses of asymptomatic sage leaves under an acute O₃ exposure (200 ppb for 5 h). Using partial least squares regression, spectral models were developed for the estimation of several traits related to photosynthesis, the oxidative pressure induced by O₃, and the antioxidant mechanisms adopted by plants to cope with the pollutant. Physiological traits were well predicted by spectroscopic models (average model goodness-of-fit for validation (R²): 0.65–0.90), whereas lower prediction performances were found for biochemical traits (R²: 0.42–0.71). Furthermore, even in the absence of visible symptoms, comparing the full-range spectral profiles, it was possible to distinguish with accuracy plants exposed to charcoal-filtered air from those exposed to O₃. An O₃ effect on sage spectra was detectable from 1 to 5 h from the beginning of the exposure, but ozonated plants quickly recovered after the fumigation. This O₃-tolerance was confirmed by trends of vegetation indices and leaf traits derived from spectra, further highlighting the capability of reflectance spectroscopy to early detect the responses of crops to O₃.

Antioxidants and phytohormones are hallmarks of abiotic stress responses in plants. Although it is known that they can offer cell protection or accelerate programmed cell death (PCD) depending on the level of stress, the involvement of these metabolites in stress acclimation is still not fully elucidated. Here, we showed the role of antioxidants and phytohormones in Salvia officinalis tolerance to long-term ozone (O₃) exposure (120 ppb for 36 days, 5 h day⁻¹). Salicylic acid (SA) content was increased under O₃ throughout the whole experiment (+150%, as average compared with control), being required to maintain the cellular redox state and potentiate defense responses. This accumulation was induced before the production of ethylene (ET), suggesting that ET was controlled by SA during O₃ exposure to modulate the magnitude of chlorosis formation and the cell redox balance (by regulating ascorbate and glutathione levels). The synthesis and/or regeneration of these antioxidants did not protect membranes from lipid peroxidation, as demonstrated by the accumulation of malondialdehyde (+23% as average). However, these processes of lipid oxidation did not include the synthesis of the membrane breakdown products, as confirmed by the unchanged values of jasmonic acid, thus indicating that this compound was not involved in the regulation of PCD strategies.

To investigate how plants cope with multi-stress conditions, we analyzed the biochemical and molecular changes of Vitis vinifera leaves subjected to single or sequential double stresses (infection by Botrytis cinerea (Bc) and ozone (O₃, 100 ppb for 3 h) treatment). In Bc⁺/O₃⁻ leaves, the hydrogen peroxide (H₂O₂) induction (observed at 12 and 24 h from the end of treatment (FET)) triggered a production of ethylene (Et; +35% compared with Bc⁻/O₃⁻ leaves), which was preceded by an increase of salicylic acid (SA; +45%). This result confirms a crosstalk between SA- and Et-related signaling pathways in lesion spread. The ozone induced an early synthesis of Et followed by jasmonic acid (JA) and SA production (about 2-fold higher), where Et and SA signaling triggered reactive oxygen species production by establishing a feedback loop, and JA attenuated this cycle by reducing Et biosynthesis. In Bc⁺ + O₃⁺ leaves, Et peaked at 6 and 12 h FET, before SA confirmed a crosstalk between Et- and SA-related signaling pathways in lesion propagation. In O₃⁺ + Bc⁺ leaves, the H₂O₂ induction triggered an accumulation of JA and Et, demonstrating a synergistic action in the regulation of defence reactions. The divergence in these profiles suggests a rather complex network of events in the transcriptional regulation of genes involved in the systemic acquired resistance.