Ecophysiological and phytochemical response to ozone of wine grape cultivars of Vitis vinifera L.

Abstract Vitis vinifera sensitivity to tropospheric ozone (O3) has been evidenced in several studies. In this work, physiological and metabolic effects of O3 on two wine cultivars of V. vinifera (i.e. Maturano and San Giuseppe) have been studied. Moreover, chlorogenic acid (CGA) production, in consideration of its importance in the biosynthetic pathway of polyphenols and as antioxidant, has been investigated. Maturano cultivar resulted more sensitive to O3, as evidenced by the gas exchange reduction at the early stage of treatment, and by the increase in Ci/Ca and the decoupling of net photosynthesis and the stomatal conductance at the end of the treatment. Unexpectedly, O3 did not activate stilbene production. Ozone induced an early CGA decrease, significantly more consistent in cv. Maturano, and an increase after 8 days, more consistent in cv. S. Giuseppe. These results suggest that CGA could be considered a biochemical marker of O3-induced stress in V. vinifera.


Introduction
human health greatly depends by the nutritional environment, which nowadays means the quality of food, directly or indirectly derived from plants. Several factors can influence the quality of cultivated plants both at the pre-and post-harvest stages. Effects of human activity on environment, such as atmospheric pollution, play a central role causing damages in food productivity and quality, which results in decrease in market value, profit, occupation and quality of life. Among pollution agents, increasing attention concerns tropospheric ozone (O 3 ). O 3 in high percentages can increase cell oxygen reactivity destabilising the delicate molecular equilibrium, evident in subsequent damage of several structures. Climate in Southern Europe is especially favourable to the formation of O 3 with visible damage on several plants, including fruit trees and other crops (Manes et al. 2003). In the case of O 3 pollution, plant reaction to oxidative stress appears focused on increasing production of detoxificant agents, such as enzymes (e.g. superoxide dismutase and peroxidase) and small molecules (e.g. ascorbic acid, glutathione and polyphenols) (diao et al. 2014). The effects of the O 3 oxidation stress can be observed at macroscopic, microscopic and biochemical levels. In particular, Vitis vinifera sensitivity to O 3 stress has been evidenced in several studies (Fumagalli et al. 2001;Saitanis 2003) with enhanced senescence of the leaves, reduction in net assimilation and severe reduction in carbohydrate import. The present study was performed on two Italian cultivars of V. vinifera, i.e. San Giuseppe (red grape) and Maturano (white grape), autochthonous cultivars of the latium region. The aim of this work was to study the effects of O 3 stress in ozone vinifera l., trying to explain the mechanism of action at the cultivar level. The effects of oxidative stress of O 3 fumigation have been investigated at different levels, including gas exchanges, stomatal conductance and sub-stomatal CO 2 concentration, photosynthesis performance, tissue damages and metabolic responses especially concerning the phenolic content.

Ecophysiological measurements and O 3 injury
In Figure 1, the trend of gas exchanges measured in Maturano and San Giuseppe before fumigation (day 0) and during the fumigation period (days 1-10) has been shown. A decreasing trend of stomatal conductance (gs) and net assimilation (Pn) is evident in the fumigated plants with respect to the control plants of both cultivars (Figure 1(a)-(d)); however, the white grape cultivar Maturano shows a significant gas exchange reduction already after the first day of fumigation (AOT40 = 845 ppb h), while in the red grape cultivar San Giuseppe, significant O 3 effects on gs and Pn appeared only after the third day of fumigation (AOT40 = 2554 ppb h). The Ci/Ca ratio also increased significantly (p < 0.001) in Maturano plants compared to the control, although the mean values were never higher than 0.80 during the whole experiment ( Figure 1(e)). In San Giuseppe, instead, Ci/Ca values were slightly, but significantly, lower in fumigated than in control plants, suggesting that mesophyll photosynthetic capacity was not altered by O 3 (Figure 1(f )). As previously reported for other species (Salvatori et al. 2013), this indicates that the gas exchange reduction observed in this cultivar can be ascribed to an O 3 -induced stomatal limitation, that is considered as an ozone avoidance mechanism, since it decreases the stomatal O 3 flux into the leaves (Manes et al. 2007;Salvatori et al. 2013). Moreover, while in San Giuseppe, stomatal conductance and photosynthesis were strictly coupled during the whole experiment, fumigated Maturano plants showed a marked decoupling of gs and Pn at the end of the treatment (after 10 dOF, AOT40 = 8586 ppb h), indicating that the mechanism of stomatal regulation was impaired by O 3 (löw et al. 2007). The analysis of chlorophyll fluorescence confirms this finding. In fact, while no significant O 3 effect on the JIP test parameters was detected in both cultivars after 1 day of fumigation (Figure 2(a) and (b)), from dOF3, a different response was evident between the two cultivars (Figure 2(c) and (d)). Maturano plants showed a significant reduction in the quantum yield of electron transport between PS II and PS I (φ E0 = ET 0 /ABS) and a slight, non-significant reduction in the photosynthetic performance index on absorption basis PI ABS ( Figure  2(c)). San Giuseppe plants showed instead an opposed behaviour (significant decrease in φ E0 and increase in PI ABS, slight non-significant increase in PItot) (Figure 2(d)). In particular, the upregulation of PI ABS and PItot is known as an early response to oxidative stresses, and indicates an increased efficiency of the reduction in end acceptors downstream of PSI (namely ferredoxine and NAdP+) (Bussotti et al. 2011). This increased PSI activity suggests the involvement of alternative electron transport pathways, i.e. cyclic and/or pseudocyclic (Asada 1999). These cycles generate a large proton gradient across the thylakoid membrane,  thus enhancing the xanthophyll-cycle-controlled downregulation of PS II; moreover, they detoxify photochemically produced ROS in chloroplasts, and supply additional ATP. The latter is particularly required during plant stress response, and can be also used for detoxification and repair processes of O 3 injury (Mereu et al. 2011).
At the end of treatment (dOF 10), both cultivars showed a significant reduction in the maximum quantum yield of primary photochemistry φ p0 , in φ E0 and in PI ABS ; as a typical O 3 response, this was followed by the increase in the amount of energy dissipation per active RC (dI 0 /RC), and in the energy absorption and trapping per reaction centre (ABS/RC and TRo/RC, respectively, Figure 2(e) and (f)) (Salvatori et al. 2013). Noticeably, a difference between the two cultivars was again evident in this date, with Maturano showing a marked increase in F 0 and a significant decrease in total Performance Index (PItot), suggesting the occurrence of a structural damage to PS II reaction centres (Bussotti et al. 2011), while in San Giuseppe, these two parameters showed no variation, thus confirming the highest resistance of the latter cultivar to O 3 stress. Finally, the analysis of visible foliar symptoms showed, in both cultivars, the appearance of foliar macroscopic alterations attributable to the action of O 3 after 6 days of fumigation, in the form of dots on the upper side of the leaf (stippling), indicating the areas of cell death in the palisade parenchyma (data not shown). The percentage of injured leaves per plant, after 6 days of fumigation, was 9.52 and 21.6% in Maturano and S. Giuseppe plants, respectively (data not shown). At the end of the fumigation, however, no significant difference between the cultivars was evident in the extent of injury (30 and 35% of injured leaves per plant in Maturano and S. Giuseppe, respectively), thus confirming that visible foliar O 3 symptoms cannot be considered a reliable indicator for biomass and productivity losses (Salvatori et al. 2013).

Chemical study
Phenolic content of the grapes, including stilbenes, is crucial not only for plant biology but also in humans for many biological activities of these compounds (Giovannelli et al. 2014).
The hPlC and hPTlC analyses did not show a detectable production of stilbenes as resveratrol and viniferins in both the V. vinifera cultivars San Giuseppe and Maturano in response to ozone treatment (data not shown). This is an unexpected result, as it is well known that stilbenes are usually overproduced in response to numerous types of biotic and abiotic stress such as fungal infections (Botrytis cinerea, Plasmopara viticola, etc.) (Gindro et al. 2012), UV radiations (Fritzemeier & Kindl 1981) or heavy metal contamination (Cai et al. 2013). however, in accordance with our results, mild ozone treatments, as those using ozonated water, which are effective in the control of microbial growth, did not induce stilbenoid accumulation in grapes (González-Barrio et al. 2006). hPTlC analysis (Figure 3(a)) revealed that chlorogenic acid (CGA) is constitutively accumulated in the two cultivars at similar concentrations (1.6-4.7 mg/g dW in San Giuseppe and 1.5-3.7 mg/g dW in Maturano). In the first 192-h post-treatment, a significant decrease in the content of CGA was observed. This decrease was much more evident in Maturano in comparison with San Giuseppe (Figure 3(b) and (c)). In Maturano, the greatest difference in CGA content between control and treated plants was observed 24 h after the beginning of the O 3 treatment [difference in concentration (dC) 1.4 mg/g dW] ( Figure  3(B)). In San Giuseppe, the decrease in CGA concentration was less pronounced and the maximum difference between treated and control plants was observed 192 h (8 days) after the treatment (dC 0.6 mg/g dW) (Figure 3(c)). In both cultivars, at long experimental . asterisks indicate statistically significant differences between control and treated plants (p ≤ 0.05). each data represent the mean of three independent determinations ± sd. times, it was observed a reversal of the trend, i.e. the concentration of CGA increased up to exceed the concentration present in the controls. The main difference between controls and treated plants was observed in the cultivar San Giuseppe 216 h (9 days) post-treatment (dC 0.8 mg/g dW). CGA has antioxidative properties and it is involved in the response to abiotic stresses such as high light (Izquierdo et al. 2011), heavy metals (Kováčik et al. 2008), UV-B (Kondo & Kawashima 2000). Recently, it was also demonstrated that CGA concentration approximately doubled in response to O 3 stress (Oksanen et al. 2007). In our results, CGA under ozone treatment decreased in both cultivars in the first 8 days and then increased in the next days. Such decrease in CGA not necessarily indicates a decrease in its biosynthesis because the content of any metabolite results from the equilibrium between its production and its consumption. Then, it is highly probable that CGA biosynthesis increases in response to O 3 stress, already in the first days of fumigation, but its direct consumption, as antioxidant for scavenging ROS or precursor to other phenolic compounds, exceeds its biosynthesis (Pasqualini et al. 2003, Correia andJordão 2015). In support of our hypothesis, it has been recently highlighted a novel antioxidant function for the CGA and it has been assessed its possible role in abiotic stress tolerance (Grace & logan 2000). Moreover, in Acer saccharum leaves, the concentration of three flavonoids, i.e. avicularin, isoquercitrin and quercitrin, was significantly increased by ozone exposure suggesting that change in phenolics in leaves might be considered as biochemical indicator of tropospheric O 3 exposure (Sager et al. 2005).

Conclusion
Multiple effects of the ozone treatment in two V. vinifera cultivars have been evaluated, and physiological and chemical response to ozone exposure was assessed. From the ecophysiological point of view, the white grape cultivar Maturano resulted more sensitive to O 3 , as highlighted by the early stomatal closure and downregulation of the photochemical process, as well as by the decoupling of stomatal conductance and photosynthesis and the occurrence of a structural damage to PS II reaction centres at the end of the experiment. differently from what expected, ozone treatment did not activate stilbene production, but influenced the CGA content in both cultivars. Compared with the controls, a decrease in CGA in the treated samples was detected in both cultivars, and a recovery was observed after 8 days. The decrease in CGA not necessarily indicates a decrease in its biosynthesis. It is highly probable that CGA is consumed or as antioxidant or as precursor to other phenolic compounds. The results suggest that CGA leaf content could be considered a biochemical marker of O 3 -induced stress in V. vinifera.

Disclosure statement
The authors declare that they have no conflict of interest.

Funding
This research was supported by grant from the Sapienza University of Rome (Italy) [grant number C26A10R2CK].