Q. robur is a broadleaf tree species widely distributed in Europe growing in mixed and deciduous forest ranging from sea level up to 1200 m a.s.l. (Ülker et al., 2018). According to Ellenberg (1988), the defensive actions of Q. robur against drought stress are caused by fast regulation of transpiration rates and stomatal conductance and a low susceptibility of water embolism in the xylem (Van Hees, 1997).

Fourteen 2-year-old Q. robur seedlings were planted in 7 L pots in March 2019. The substrate consisted of one-thirds of soil used by the city gardeners for city trees in Vienna and two-thirds of quartz sand to improve drainage. The plants were fertilized once after planting (universal fertilizer Novatec, Compo, Münster, Germany) and from thereon kept well-watered in a greenhouse at near-ambient light (80 % to 90 % of photosynthetically active radiation) (Lak et al., 2020). The trees were moved from a greenhouse in Tulln into another close-by greenhouse in Vienna 2 weeks prior to the experiments. Dust was removed from the leaves by showering the trees before starting the drought stress.

For the biochemical reference assays, 8 trees of the initial 14 were used: four well-watered plants (C) and four well-watered plants receiving 100 ppb ozone for 1 h one time (OS) inside the enclosures. The remainder (six plants) were used for BVOC emission measurements, CO2 and H2O gas exchange measurements, and biochemical assays. Hereby, we were left with three replicates under drought stress (DS) and three replicates exposed to drought stress and ozone (DS × OS). The drought stress was initiated for all six plants 10 d before the VOC measurements started and was maintained by keeping the soil water content at 4 vol %–5 vol % using a soil moisture probe (FieldScout TDR100, 20 cm probe depth, Spectrum 105 Technologies, UK), whereas 100 % field capacity was 13.4 vol %. With the start of VOC measurements, we stopped watering the previously drought-stressed trees to further increase drought stress.

The plants were moved from the greenhouse to an indoor climate chamber (Fitotron Weiss Gallenkamp, UK) 24 h before the experiment started. Thereafter trees were kept in the climate chamber for the remainder of the experiment and were only placed into the branch enclosures during the gas exchange measurements. The branch enclosures were situated next to the climate chamber in a climatized laboratory exhibiting the same environmental conditions as in the climate chamber. The climate conditions during the first day of experiment were kept at 25 ∘C, ∼ 60 % relative humidity (RH), and ∼ 1000 µmol m2 s-1 photosynthetically active radiation (PAR) at canopy top, to adapt to constant air temperature. To continuously increase the drought stress, the plants were not watered and the humidity in the climate chamber was decreased to 40 % RH and temperature was increased to 30 ∘C after the first day. The same temperature conditions were also present in the climatized laboratory, where the plants were placed in the enclosures at an RH of 32 % and 30 ∘C. Overall light conditions remained constant during the day, with lights off during the night.

To study the effect of ozone exposure of trees during increasing drought, the six trees mentioned above were separated into two groups: three trees were drought stressed and fumigated with 100 ppb O3 (DS × OS) inside the enclosure for 1 h each day after the daily measurement of BVOCs. The other three trees were drought stressed but not fumigated with ozone (DS).

At the end of the experiment leaves were harvested for leaf area and enzyme analysis. Values of the enzymatic activity of C and OS were compared to DS and DS × OS to investigate the effect of ozone fumigation.

Throughout the increasing drought stress, tree leaf gas exchange (CO2 and H2O) and BVOC emissions were measured for two sets, DS and DS × OS, over a 7 d period, one in the morning and one in the afternoon alternating daily. The plants were taken out of the climate chamber and kept inside the custom-made plant enclosures (Fig. 1; TC-400, Vienna Scientific Instruments GmbH, Alland, Austria) for 2–3 h each day in order to measure their CO2 and H2O exchange along with key physiological parameters (soil moisture and stem water potential). After the measurements inside the enclosures, the plants were moved back to the climate chamber until the next measurement session. The plant enclosures covered most of the plant material excluding a few leaves (about seven on each tree) to allow determination of stem water potential (SWP). Each day, one leaf was wrapped in aluminum foil and placed in a plastic bag for equilibrating to SWP (Williams and Araujo, 2002). After darkening for 30 min the leaf was cut off and SWP was measured by using a Scholander pressure bomb (Soilmoisture Equipment Corp., Goleta, CA, USA).

The four custom-made plant enclosures (12 L) were lined with PTFE and sealed on top with 55 × 60 cm PET bags. The plant enclosures were continuously flushed with 10 L min-1 of ambient outside air that was previously passed through a cold trap to remove water and an activated carbon filter (360 m3 h-1, Prima Klima Trading, Radnice, CZ) to remove VOCs and O3. This resulted in 32 % RH air and ∼ 370 ppm CO2 entering the enclosures (experimental conditions in Appendix A, Table A1). The flow rate of 10 L min-1, tested during the experiment set-up prior to the actual experiments, assured that no condensation of water occurred in the tubing and enclosures as well as resulted in a slight overpressure preventing the entry of room air into the enclosures. Three of the enclosures were used to measure the air–gas exchange of the plants and the fourth enclosure was kept empty as a reference to allow continuous monitoring of the air entering the enclosures. Trees inside the enclosure were LED-irradiated with a mean PAR value of 1374 µmol m-2 s-1 at canopy top (Eckel Electronics, Trofaiach, Austria) during daytime when the exchange measurements were performed. During night, trees were kept in the dark. Leaf temperature was monitored in each enclosure by placing a calibrated (±0.1 ∘C) thermocouple (type k, PTFE IEC wire; Labfacility Ltd, Bognor Regis, West Sussex, UK) on the abaxial side of a mature mid-canopy leaf.

An automated valve system allowed the consecutive analysis of air exiting each enclosure for 5 min each, leading to a 20 min cycle through the four enclosures. Before inserting the three trees into the enclosures, background measurements of the empty enclosures were carried out. After inserting each plant into one enclosure, the plant was allowed to acclimatize for approximately 2 h, and the following 40–60 min of data were analyzed to determine plant CO2 assimilation, transpiration, and BVOC emissions rates. After the measurements, the trees of DS × OS were fumigated for 1 h with 100 ppb of ozone each day.

CO2 and H2O mixing ratios in the air leaving the enclosures were measured using a CIRAS-3 SC PP system (Amesbury, MA, USA), which was factory calibrated 3 months before the measurement campaign. Ozone measurements before and after the enclosures were conducted continuously in all enclosures with an ozone monitor (six-channel ozone monitor BMT 932, BMT Messtechnik, Berlin, Germany). BVOC measurements were made using a proton-transfer-reaction time-of-flight mass spectrometer (PTR-Tof-MS, PTR-TOF 6000X2, IONICON Analytik GmbH, Innsbruck, Austria; Graus et al., 2010) operated at 350 V drift voltage, ion funnel settings of 1 MHz and 35 V amplitude as well as 35 VDC, and 2.5 mbar drift pressure. These settings are comparable to an E/N of 100 Td in a PTR-TOF 8000 with no ion funnel (Markus Müller, IONICON Analytic GmbH, personal communication 2019). The drift tube temperature was 100 ∘C. Full PTR-Tof-MS mass spectra were collected with a time resolution of 1 s and up to a mass-to-charge ratio m/z of 547 amu. The instrument background was characterized daily during calibrations and in the third empty enclosure that was flushed with background air. Backgrounds were measured every 20 min for 5 min. Humidity-dependent dynamic calibrations of VOCs using a standard gas mixture (Apel Riemer Environmental Inc., Broomfield, CO, USA), containing 15 compounds (Table A2) with different functionality distributed over a mass range of 33–137 amu, were performed daily. Daily measured sensitivities based on compounds in a calibration standard varied on the order of 8 %–20 % depending on the compound. This lies within the combined calibration uncertainties of the gas standard and dilution setup using two flow controllers. Whenever a compound was not contained in the calibration standard, we applied a compound-specific sensitivity using procedures described by Cappellin et al. (2012). The PTR-Tof-MS data were analyzed using the PTR-TOF Data Analyzer v4 software (Müller et al., 2013) and customized MATLAB scripts to obtain volume mixing ratios in the enclosures. The PTR-Tof-MS instrument has a high enough mass resolution to obtain isobaric formulas, minimizing potential interferences compared to quadrupole mass spectrometers. Strictly speaking measurements represented here are characterized by the isobaric formulas. The instrument was run in H3O+ mode, detecting isoprene (IS), at m/z 69.070 [(C5H8)H+], the sum of monoterpenes (MTs) at m/z 137.133 [(C10H16)H+], the major fragment at m/z 81.070 [(C6H8)H+], and the sum of sesquiterpenes (SQTs) at m/z 205.195 [(C15H24)H+] and m/z 149 [(C11H16)H+]. The identity of isoprene and monoterpenes was additionally confirmed by gas chromatography–mass spectrometry (GC–MS) measurements. The sum of GLVs presented in this study was monitored on m/z of 83.085, 85.101, 99.080, 101.096, and 143.107, representing 2-hexenal and 3-hexenal [(C6H10O)H+], 3-hexenol [(C6H12O)H+], 1-hexanol [(C6H14O)H+], 3-hexenol [(C6H12O)H+], and hexenyl acetate [(C8H14O2)H+], respectively (Beauchamp et al., 2005; Giacomuzzi et al., 2016; Portillo-Estrada et al., 2017). The correspondence of these ions to GLV has been demonstrated by previous studies (e.g., Fall et al., 1999; Karl et al., 2001, 2005). Shikimate BVOCs were tentatively assigned to benzene as m/z 79.054 [(C6H7)H+], phenol as m/z 95.050 [(C6H7O)H+], methyl salicylate (MeSa) as m/z 153.055 [(C8H9O3)H+], and eugenol as m/z 165.092 [(C10H13O2)H+] (Brilli et al., 2011, 2016; Tasin et al., 2012; Maja et al., 2014; Giacomuzzi et al., 2016; Portillo-Estrada et al., 2017; Yener et al., 2016; Misztal et al., 2015). Emissions of IS, MT, and SQT were standardized to 1000 µmol m2 s-1 PAR and 30 ∘C (ISS, MTS, SQTS) using the Guenther et al. (1993) algorithm for ISS and Geron et al. (1994) for MTS and SQTS.

1ISS=ISCL×CT2CL=αcL1L1+α2×L23CT=expCT1T-TsRTST1+expCT2T-TMRTST4MTS=MTexpβT-Ts5SQTS=SQTexpβT-Ts Here IS, MT, and SQT are emission rates normalized by leaf area at sampling temperature T (K) and sampling PAR flux L (µmol m-2 s-1) at half plant height; α =0.0027, cL1 =1.066, R =8.314 J K-1 µmol-1, cT1 =95,000 J µmol-1, cT2 =230000 J µmol-1, TM =314 K, β =0.1, and TS =303.15 K (Guenther et al., 1993; Geron et al., 1994).

Mass flow of air (W), transpiration rate (E), net photosynthesis (A), and stomatal conductance (gS) were calculated accordingly (CIRAS-3 Operation Manual V. 2-01, PP-Systems, 2018). 6W=V060×103×122.414×104amolm-2s-1 7E=W×eout-einP-eout[molm-2s-1] 8A=-Cout-Cin×W+Cout×Eµmolm-2s-1 9eleaf=6.1365×expTleaf×17.502Tleaf+240.9710rs=eleaf-eoutE×P-eleaf+eout/2-rbm2smol-111gS=1rs×103[mmolm-2s-1] Here V0 is the volume air flow, a is the leaf area, ein is the partial water vapor pressure of the air entering the enclosures, eout is the partial water vapor pressure inside the enclosure, (P) is the atmospheric pressure, Cin in the concentration of CO2 entering and Cout is the concentration exiting the enclosure, eleaf is the saturation vapor pressure at leaf temperature (Tleaf), rs is the stomatal resistance, and rb is the boundary layer resistance to water vapor transfer, which was assumed to be zero according to the recommendations of the manufacturer (CIRAS-3 Operation Manual V. 2-01, PP-Systems, 2018).

The ratio of the sum of carbon lost in the form of BVOC (CBVOCs) vs. the uptake of carbon from net photosynthesis (CA) was calculated according to Pegoraro et al. (2004), with the BVOCs used to calculate CBVOCs given in Table A3.

After 7 d, finishing the emission measurements, all leaves were harvested immediately, imaged with a flatbed scanner (Epson Expression 10 000XL, Epson, Japan), and analyzed with the PC program WinFOLIA 2013 Pro (Regent Instruments Inc., Qúebec, Canada) to determine the leave surface area. About 80 % of the leaves' fresh mass was shock-frozen and crushed in liquid nitrogen for biochemical assays (Sect. 2.3). About 20 % of the leaves per plant were dried for 3 d in a drying room at 40 ∘C to determine dry weight to an accuracy of ±0.001 g for the calculation of enzyme activity and specific leaf area (SLA) (Table A4).

For the interpretation of the emissions of GLVs and shikimate volatiles, enzymatic activities were analyzed additionally to better understand the effect of ozone fumigation during a situation of severe drought. Using foliar materials collected after the 7 d period of emission measurements (Sect. 2.2) and stored at -80 ∘C until analysis, peroxidase and antioxidant capacity and phenol content (TPhe) were measured. These properties provide additional insights into the response of GLV and shikimate emissions as products of the metabolic process of the enzymatic activity (Betz et al., 2009).

Values from plants after 7 d of increasing drought (DS × OS, DS) were compared to well-watered control plants (C) and a well-watered set of plants that received ozone fumigation once (OS).

For measurements of peroxidase activities, 0.5 g plant material, 0.25 g Polyclar AT (Serva Electrophoresis, Heidelberg, Germany), and 0.25 g quartz sand (Sigma-Aldrich, Steinheim, Germany) were homogenized in a mortar with 3 mL 0.1 M potassium phosphate buffer (pH 6.0). After removal of solid compounds by centrifugation at 4 ∘C and 10 000 ×g for 10 min, 400 µL of the supernatant was subjected to gel chromatography with Sephadex G-25 medium (GE Healthcare, Chicago, IL, USA) to remove low-molecular-weight compounds. Peroxidase activity was determined according to the Worthington Manual (1972). Briefly, the enzyme assay contained in a final volume of 1110, 1095 µL buffer 0.1 M potassium phosphate buffer +0.003 % (v/v) H2O2 (pH 6.0), 5 µL enzyme preparation, and 10 µL 1 % (w/v) o-dianisidin (Sigma-Aldrich-Aldrich, Vienna Austria) in MeOH.

The activity was determined by measuring the extinction at 460 nm on a DU-65 spectrophotometer (Beckman Instruments, Brea, CA, USA) in intervals of 30 s for a period of 6 min. The activity was calculated from the slope in the initial linear portion of the reaction progress curved using an extinction coefficient of 1.13 ×104 M-1 cm-1 for oxidized o-dianisidine (Worthington manual, 1972). The protein content was determined by a modified Lowry procedure (Sandermann and Strominger, 1972) using bovine serum albumin as a standard. All measurements were performed in two technical replicates.

For the determination of the antioxidant capacity and the TPhen, the material was lyophilized and homogenized by grinding to a fine powder in a mortar. A total of 0.25 g of the lyophilized powder was extracted with 3 mL distilled water for 1 h in a cooled water bath during sonication. After centrifugation for 5 min at 4 ∘C and 10 000 ×g, the supernatant was filtered through a Chromafil AO-20/25 polyamide filter (Roth, Karlsruhe, Germany).

The TPhen was determined as described (Wootton-Beard et al., 2011) with some modifications. Briefly, 100 µL of the aqueous solution was mixed with 6 mL distilled water and 500 µL Folin–Ciocâlteu reagent (Sigma-Aldrich, Vienna, Austria) (1:1 v/v with distilled water). After equilibration for 8 min, 1.5 mL 20 % Na2CO3 (w/v) and 1.9 mL distilled water were added, and the mixture was incubated at 40 ∘C for 30 min. The TPhen was obtained by measuring the absorbance of the mixture at 765 nm using a freshly prepared standard curve obtained with gallic acid. The results were expressed as microgram gallic acid equivalents per gram of sample. All measurements were performed in technical triplicates.

The in vivo antioxidant activity was determined with Saccharomyces cerevisiae ZIM 2155 as model system following the procedures described in Slatnar et al. (2012), which estimates intracellular oxidation by fluorometric measurements using the ROS-sensitive dye 2′,7′-dichlorofluorescin (H2DCF). A total of 100 µL of the aqueous samples was incubated with 10 mL yeast suspension at their stationary phase in phosphate-buffered saline (PBS, Merck KGaA, Darmstadt, Germany) at a density of 108 cells per suspension at 28 ∘C and 220 rpm for 2 h. After a centrifugation step at room temperature for 5 min at 14 000 × g, the pellet was washed three times with 50 mM potassium phosphate buffer (pH 7.8) and was finally resuspended in nine volumes of 500 µL 50 mM potassium phosphate buffer (pH 7.8) and incubated for 10 min at 28 ∘C and 220 rpm in the dark. After addition of 10 µL H2DCF (1 mM stock solution in 96 % ethanol), the mixture was incubated for further 30 min at 28 ∘C and 220 rpm. The fluorescence of the yeast cell suspensions was measured at a GloMax^®Multi Microplate Reader (Promega, Walldorf, Germany) using excitation and emission wavelengths of 490 and 520 nm, respectively. Values of fluorescence intensity were measured against a blank, in which the sample was replaced with water. Data are expressed as relative fluorescence intensity, where the values obtained with the blank are defined as 1. Values lower than 1 indicate a higher antioxidant activity than the blank (Slatnar et al., 2012). All measurements were performed in two technical replicates.

Emission rates, physiological parameters, means, and standard deviation were calculated with MATLAB (MATLAB and Statistics Toolbox Release 2017a; The MathWorks, Inc., Natick, MA, United States). All leaf gas exchange (CO2 and H2O) and BVOC flux measurements collected over the 7 d period for the set DS and DS × OS were aggregated into four ranges of SWP (R1: 0.00 to -1.40 MPa; R2: -1.45 to -2.85 MPa; R3: -2.90 to -4.30 MPa; R4: -4.35 to -6.00 MPa) to perform statistical analysis using the Wilcoxon rank sum test. To test for significant differences in the biochemical markers, a one-way ANOVA test was used. For both tests p values below 0.05 were considered significant.

SWP was measured daily and used as a drought stress indicator to study the evolution of Q. robur under continuously increasing drought condition. All six trees began the experiment with a high to moderate mean SWP of -0.9 MPa (Brüggemann and Schnitzler, 2002) and reached low values on the order of -5.5 MPa after 7 d of continuously increasing drought stress. Mean and standard deviation of stomatal conductance, net photosynthesis, leaf temperature, and SWP as well as notes for statistically significant differences are summarized in Table 1 for the four drought stress ranges defined in Sect. 2.4. The mean stomatal conductance (gS) of DS × OS was 20.2 mmol m-2 s-1 in R1 and decreased to 6.8 mmol m-2 s-1 in R2 (Table 1). For DS it was 42.4 mmol m-2 s-1 in R1 and decreased to 6.6 mmol m-2 s-1 in R2. For both sets the reduction of gS and SWP between R1 and R4 was significant (p value 0.02 and 0.05 for DS and DS × OS, respectively). R1, shown in Fig. 2a, includes values of trees fumigated with ozone (DS × OS) from the first and the second days of analysis, because, for this set, SWP had not changed much during these 2 d. Differently, for DS, R1 includes only measurements of the first day. The values collected during the second day of analysis, for the set DS, is assigned to R2, because we observed a decrease in SWP between the first and second days of measurement. This shows that trees of DS × OS closed their stoma quickly at higher stem water potential after the first ozone fumigation session and confirms what was reported in other studies that moderate ozone concentrations can induce partially closed stomata (Khatamian et al., 1973; Farage et al., 1991; Wittig et al., 2007). A partial stomatal closure prevented excessive water loss through stomatal openings (Pinheiro and Chaves, 2011; McDowell et al., 2008; Allen et al., 2010) during drought stress and enhanced the closure with ozone, allowing DS × OS plants to better survive the increased drought. Kobayashi et al. (1993) consider the interactive effects of O3 and drought stress using a growth model of soybeans, finding that ozone fumigation reduces or postpones drought stress, similar to the findings of this experiment.

Figure 2b shows a decrease in net photosynthesis (A) with the increase in the stress for both sets, especially between R1 and R2, whereas the values in R3 and R4 are close to zero. In R1, A presented the same differences exposed for gS between the sets. Our results are different from the findings of Tjoelker et al. (1995) and Paoletti (2005), where stomatal conductance and photosynthesis are shown to decouple at moderate ozone exposure due to direct damage to biochemical carboxylation, caused by chronic ozone exposure.

The ratio of CBVOCs and CA is shown in Fig. 3. IS, the dominant BVOC (averagely 96 % of the total emissions), and mean standardized IS emissions of DS × OS-treated plants were consistently higher in all SWP ranges compared to DS alone (Fig. 4), thus showing the difference between DS and DS × OS in CBVOCs/CA in the highest SWP ratio range. Initially, at low drought stress (R1), 3 %–7 % of the assimilated carbon was lost as emitted BVOC, which matches findings in other studies (Sharkey et al., 1991; Baldocchi et al., 1995; Monson and Fall, 1989; Fang et al., 1996), showing that ∼ 2 % of carbon assimilated is lost as IS (CIS/CA) under unstressed conditions and at 30 ∘C. As CO2 assimilation rate decreased quickly and BVOC emission (especially isoprene emission) stayed elevated, the ratio of lost vs. fixed carbon increased to 20 % for DS and 16 % for DS × OS in R2. Pegoraro et al. (2004) reported a carbon loss on the order of 50 % for SWP of -2 MPa, in a drought experiment with Quercus virginiana. In R3, the increasing stress corresponded to ratios of 0.7 and 1.03 for DS and DS × OS, respectively. Alternative carbon sources for isoprene biosynthesis under drought stress are thus proposed for DS × OS. For example, extra-chloroplastic origin or chloroplastic starch (Karl et al., 2002; Kreuzwieser et al., 2002; Funk et al., 2004; Affek and Yakir, 2003; Schnitzler et al., 2004; Rosenstiel et al., 2003) can sustain carbon sources for isoprene production. At very high drought stress (R4) this ratio decreased again to 0.4 in DS and 0.8 in DS × OS.

To give a general overview on BVOC emissions for both sets, Fig. 5a and b show the total mass spectra ranging from 40–220 amu for the first and last days of measurement for DS and DS × OS, respectively. Figure 5c shows relative changes of the mass spectra between the first and last days of measurements. The mass range 80–110 amu, hosting many mass-to-charge ratios associated with GLVs, showed the strongest difference between the two sets. Plants exposed to ozone and drought stress (DS × OS) exhibited smaller increases in this mass range compared to drought-stressed (DS) plants. Changes in emissions or lack thereof for IS, MT, SQT, and stress-related BVOCs are investigated in further detail below and are summarized in Table 2.