• Sage
• Ozone

Tropospheric ozone (O3) is a major air pollutant causing negative effects on health of living organisms, as well as the third most powerful anthropogenic greenhouse gas. Despite numerous legislatory attempts aimed to control emissions of its precursors, O3 is still among the major air pollutants worldwide, especially in areas with elevated temperatures such as the Mediterranean basin, where it frequently exceeds the World Health Organization (WHO) guideline average values (i.e., 8 hours a day at most above 100 μg m-3).
Four decades of research on O3-vegetation effects have shown that excessive uptake of this gas and the consequent accumulation of reactive oxygen species (ROS) induce detrimental effects such as reduction of photosynthesis and growth, partial stomatal closure, cell dehydration, excessive excitation energy, accelerated leaf senescence, and appearance of chlorotic/necrotic leaf injuries, that overall result in reduction of plant yield and productivity. However, plants possess a forceful and multifarious antioxidant system composed of enzymatic reactions (e.g., superoxide dismutase, catalase and ascorbate peroxidase) and non-enzymatic compounds (e.g., ascorbic acid and glutathione), which are involved in detoxification, removal and/or neutralization of ROS overproduction due to biotic and abiotic stresses.
During the last decades, medicinal and aromatic plants have been extensively studied and found to be excellent sources of bioactive and health-promoting molecules. Most of these compounds are also beneficial for the plant itself by their significant role in plant resistance. Salvia officinalis L. (sage) is one of the most well-known aromatic herbs. Native of southern Europe, it is largely cultivated in the Mediterranean countries due to its high ability to cope with environmental stressors. Since the impact of O3 on medicinal plants remains poorly understood, the overall scope of this research work was to investigate the responses of S. officinalis to a single pulse and a chronic exposure of O3.
Firstly, S. officinalis plants were investigated to assess the role of signalling molecules, phytohormones and transcription factors under a single pulse of O3 (200 ppb of O3 for 5 h). The O3 concentration adopted in this experiment induced only a transient oxidative burst. Furthermore, signalling molecules resulted as good mediators of cell survival by providing better antioxidant defences and regenerating active reduced forms, whereas phytohormones were not involved in the perception and transduction of O3 stress. WRKY trancription factors seemed to act as promoter elements of the apoplastic response to ROS generation, regulating oxidative protection and providing O3-stress tolerance. Overall, this first dataset suggested that sage evolved several biochemical mechanisms to cope with such adverse environmental conditions.
Following the results came out from this first experiment, the antioxidant mechanisms adopted by S. officinalis under the same single pulse O3 exposure (i.e., 200 ppb of O3 for 5 h) were also investigated. This study demonstrated that O3 induced some photosynthetic impairments in sage leaves during the exposure, but plants quickly recovered after the fumigation, confirming the tolerance of this species to the pollutant. Indeed, slightly higher levels of lipid peroxidation were only observed at the end of the exposure, and the antioxidant capacity was overall increased by the pollutant. The antioxidant response seemed to be finely regulated by the activation/suppression of specific antioxidants at the different times of analysis, both during and after the exposure. A key role in the antioxidant response seemed to be played by the Halliwell-Asada cycle. Actually, another major scope of the present study was to test the capability of full-range (350-2500 nm) reflectance spectroscopy to rapidly and non-destructively characterize responses of asymptomatic sage leaves to the single pulse of O3. 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 maintained under charcoal-filtered air (i.e., controls) from those exposed to O3.
The main objective of the third experiment was to give a thorough description of the effects of an O3 pulse at a lower concentration (120 ppb for 5 h) on the phenylpropanoid metabolism of S. officinalis, 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 of the phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and rosmarinic acid synthase (RAS) activities (i.e., key enzymes of phenolic biosynthetic pathways) 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 the exposure, whereas an enhanced RAS activity occurred at the end of the treatment and at the recovery time, suggesting that specific phenylpropanoid 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.
Finally, the S. officinalis-O3 interaction was investigated from a different angle than previous experiments. Eliciting of plants consists in the application of chemical, physical and biological factors inducing stressful conditions to trigger defence mechanisms and thus the production of various bioactive compounds and phytochemicals with beneficial health effects. Thus, a final study was performed to weekly assess the effects of a chronic O3 exposure (120 ppb of O3 for 36 consecutive days, 5 h day-1) on the phenolic and volatile organic compounds (VOC) profiles of sage leaves, also relating the compositions of these compounds with the changing antioxidant activity induced by the air pollutant. The effects of O3 exposure on the VOC composition, yield and antioxidant capacity of essential oils (EOs) obtained from sage leaves were also evaluated. We identified 17 phenolic compounds in S. officinalis leaves (phenolic acids and flavonoids), and 62 and 56 VOCs emitted by leaves and EOs, respectively (monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, two oxygenated sesquiterpenes and nine non-terpene derivatives). Ozone exposure resulted in an overall accumulation of phenolic compounds. In terms of VOCs, O3 mainly decreased the emission of monoterpenes, while increased the production of sesquiterpenes and non-terpene derivatives (from both leaves and EOs). These O3-induced accumulations were mainly triggered during the first weeks of exposure whereas they disappeared at the last time of analysis, suggesting that sage plants finally lost their ability or interest in investing in this response strategy, and indicating that reaching too high doses of O3 resulted in slow inhibition of secondary metabolites. The antioxidant capacity of all tested extracts resulted increased by O3 exposure. These outcomes support our speculation on the application of O3 for a limited period (i.e., a maximum of four weeks, at the investigated concentration) as a potential tool to improve the quality of sage leaf extracts.