Ozone (O3) has become one of the major pollutants in near-surface air (Paoletti and Cudlin 2012). With the acceleration of industrialization and urbanization, tropospheric O3 has risen about 0.5%–2% annually in the twenty-first century (Feng et al. 2015). In most areas of China, the average O3 concentration is more than 50–60 ppb per day for 8 h (from 9:00 to 17:00) from late spring to autumn harvest period (Wang and Mauzerall 2004). It has been well reported that elevated O3 causes serious harm to human, animal and plant species (Feng et al. 2014; Izuta 2017).

As a gaseous strong oxidizer, O3 enters the leaf tissue through stomata and dissolves into the water solutions in the apoplast (Esposito et al. 2009; Li et al. 2017), and then is transformed into reactive oxygen species (ROS) (Dizengremel et al. 2009; Frei et al. 2010; Cotrozzi et al. 2016). The ROS attacks the cell membrane, induces membrane lipid peroxidation (Li et al. 2017), increases the levels of malondialdehyde, decreases cell membrane stability and ultimately leads to programmed cell death (Zhang et al. 2017b; Dumont et al. 2017). Furthermore, it can inhibit plant net assimilation, change assimilates distribution, slow down plant growth, and reduce crop yield (Fiscus et al. 2005; Singh and Agrawal 2017).

In order to alleviate O3-caused damage on sensitive plants, a range of protectants have been evaluated, including antioxidants (Zhang et al. 2017a), anti-senescence agents (Navakoudis et al. 2003), growth regulators (Paoletti et al. 2011), flavonoids and polyamines (Didyk and Blum 2011), fungicides (Hassan et al. 2007). Many studies have found that the application of ethylene diurea (EDU) is highly effective on decreasing O3-caused damage (Manning et al. 2011; Agathokleous 2017; Tiwari 2017). However, synthetic protectants may contaminate environment and agricultural products, and may also cause dose-dependent toxicity of natural vegetation (Didyk and Blum 2011). Moreover, some of these substances are expensive because they are hard to manufacture. It has been reported that leaf water extract from aromatic plants such as French marigold (Tagetes patula) and basil (Ocimum basilicum) can alleviate O3 damage to some extent (Blum and Didyk 2006, 2007). We postulated that other aromatic plant species, whose leaf water extract usually contain flavonoids, could also mitigate O3 damage. So far, the information about this area is still very limited.

The objectives of this study therefore were: (1) to compare mitigating effects of leaf water extract from three aromatic plant species on O3-caused injury, (2) to reveal physiological mechanisms related to the different mitigating effect. This study can provide the basis for screening environmental friendly and low-cost reagent resistance to O3 pollution.

Materials and Methods

Seeds of snap beans (Phaseolus vulgaris L. ‘Jiangjunyoudou’) were bought from Harbin Academy of Agricultural Science, Harbin, CN. On 10 July 2016, the seeds were sown in plastic pots (10 cm in diameter) filled with a mixture of vermiculite and peat (1:2, v:v). Seedlings were cultivated in a greenhouse at the Horticultural Experiment Station (HES) of Northeast Agricultural University (NEAU, 45°74′15″N, 126°73′14″E), Harbin, CN. To avoid water deficiency, the seedlings were irrigated every day to field capacity throughout the experiment.

Leaf water extract of Tagetes patula L. (TP), Pelargonium hortorum Bailey (PHB), and Plectranthus hadiensis var. tomentosus (PHT) were prepared according to Huang et al. (2015). Fresh fully expanded and healthy leaves of plants in each species were sampled (25 g). The samples were cut into small pieces, then put into flask filled with 125 mL of distilled water (CK) for 48 h in a dark room. After filtration, a mother liquid (200 g L−1) was obtained and stored in a refrigerator at 4°C.

Six open-top chambers (OTCs, 1 m in diameter and 1 m in height) were constructed with transparent polymethyl methacrylate plates for treatments. On 23 July 2016, 20 potted seedlings were placed in each OTC for 5 days’ adaptation [the air was charcoal-filtered (CF) and O3 concentration was < 5 ppb] at the HES of NEAU. During the experiment, the daily average max/min relative humidity and temperature in the OTCs were 63/27% and 29/18°C, respectively. The daytime average photosynthetic photon flux density (PPFD) inside the OTCs was about 850 µmol (photon) m−2 s−1. After adaptation every five plants in each OTC were sprayed with diluted leaf water extract (20 g L−1, 10 mL for each plant) of TP, PHB, PHT, or CK (10 mL for each plant), respectively. After 12 h three OTCs were ventilated with O3, where concentration was maintained at 150 ± 20 ppb for 3 days (from 8:30 to 16:30, 8 h day−1), and the other three chambers were treated with CF air. Every day, when the fumigation was finished, the plants were sprayed again until the end of the experiment (16:30, on 31 July 2016). Ozone was produced by an O3 generator (CF-KG1, Beijing Sumsun EP Hi-Tech, Beijing, CN). In order to maintain the target value, O3 concentrations at the plant height were monitored using an O3 analyzer (Model 202, 2B Technologies Inc., Boulder, Colorado, USA).

After 3 days of exposure, two plants of each spraying treatment from each of six OTCs (O3 and CF air, n = 3) were randomly selected for observation. Visible injuries on the first pair of fully expanded leaves in each plant were observed and recorded by three surveyors independently using a 5% and 1% step scale when visible injury was above and below 5%, respectively (Paoletti et al. 2009). Then the values were averaged as visible injury of the plant.

Instantaneous gas exchange was measured on the first pair of fully expanded leaves from 8:00 to 11:30 on the first day after O3 exposure using a portable photosynthesis system (Li-6400, Li-Cor, Lincoln, NE, USA). Two plants of each spraying treatment from each of six OTCs were randomly selected. The light intensity and the air temperature inside the leaf chamber were set to 1200 µmol (photon) m−2 s−1 and 35°C, respectively. Ambient CO2 concentration (410 ± 5 µmol mol−1) was used as reference. Net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E), and the ratio of intercellular/ambient CO2 concentration (Ci/Ca) were calculated.

The first pair of fully expanded leaves of snap bean were sampled and cut into tiny pieces, then mixed for the following measurements.

Fresh leaf sample (0.2 g) was put into test tubes containing 20 mL of CK at 25°C. After 24 h, electrolyte leakage (EL) was measured using a conductivity meter (Delta 326, Mettler-Toledo, Switzerland) as EL1. Then tubes were maintained in boiling water bath (100°C) for 20 min. After cooling to 25°C, EL was measured again as EL2. The relative conductivity was calculated according to Liu et al. (2016) as: \(E{L_1}/E{L_2} \times 100\% .\)

Fresh leaf sample (0.2 g) was ground and homogenized with trichloroacetic acid (10%). The homogenates were centrifuged at 4000×g for 10 min. The supernatants (1 mL) were mixed with 5 mL of thiobarbituric acid (0.6%) and then maintained in boiling water bath for 15 min. After cooling, the mixture was centrifuged at 4000×g for 10 min. The absorbance of supernatants was measured using a UV–Visible spectrophotometer (T6 New Century, CN) at 450, 532, and 600 nm. The malondialdehyde (MDA) content was calculated according to Hao et al. (2004) as: \(\left[ {6.45 \times \left( {O{D_{532}} - O{D_{600}}} \right) - 0.56 \times O{D_{450}}} \right]/0.2.\)

Leaf pigment content was measured according to Sui et al. (2017). Fresh leaf sample (0.3 g) was ground with quartz sand, calcium carbonate powder, and ethanol (95%, v/v) in a dark room, then was filtered to 25 mL brown volumetric flask. The final constant volume was kept at 25 mL by ethanol. Absorbance was measured using a UV–Visible spectrophotometer (T6 New Century, CN) at 649 and 665 nm. Contents of chlorophyll (Chl) a, Chl b, and total Chl were calculated.

Fresh leaf sample (0.5 g) was homogenized in ice-cold mortar using 2 mL of 50 mmol L−1 phosphate extraction buffer (PBS) (pH 7.8). The residue was rinsed three times with 1 mL of PBS. Then the homogenate was centrifuged at 12,000×g for 15 min at 4°C. After that the supernatant was used to determine enzyme activities of superoxide dismutase (SOD), peroxidases (POD), catalase (CAT) and the content of superoxide anion (O2•−). Activity of SOD was based on the methods of Syeed et al. (2011). One unit of SOD activity (U) was defined as the amount of enzyme that inhibited 50% of nitro blue tetrazolium (NBT) photoreduction and the absorbance was read at 560 nm. SOD activity was expressed as U g−1 fresh weight (FW). POD activity was determined according to the method described by Wang et al. (2014) with minor modifications. The enzyme extract (0.1 mL) was mixed with 100 mL PBS (50 mmol L−1, pH 6.0) containing 56 µL guaiacol and 38 µL 30% H2O2, and the increase in absorbance at 470 nm was monitored for 4 min. Unit of POD activity expressed the amount of enzyme oxidizing 1 µmol (guaiacol) g−1 min−1 FW. CAT activity was estimated based on the method reported by Diaz-Vivancos et al. (2008), which measures the decline of H2O2 at the maximum absorption at 240 nm. One unit was defined as the amount of enzyme catalyzing the decomposition of 1 µmol (H2O2) g−1 min−1 FW. The content of O2•− was determined according to the method described by Tian et al. (2003). The extract (1 mL) was mixed with 1 mL hydroxylamine hydrochloride (10 mmol L−1), then water bathed (25°C) for 20 min. Absorbance was determined at 530 nm.

The experimental design had three blocks, each block contained one CF air and one O3 chamber, each chamber had four kinds of spraying treatment, and each treatment contained five replicate plants. All data were analyzed using SPSS (v.12, SPSS, Chicago, IL, USA). Normal distribution was checked by Kolmogorov–Smirnov test. Two-way analysis of variance (ANOVA) was used to identify the effect of O3 treatment (Treatment), leaf water extract (Extract), and their interactions (ns not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Post-hoc Duncan’s test was used to compare means of each parameter among different treatments. Relative change of each parameter was expressed as percentage difference between O3-exposed (O3) plants and CF air plants, \(\left( {{O_3} - CF} \right)/CF \times 100\% .\)

Results and Discussion

Acute O3 fumigation induced interveinal chlorotic and necrotic lesions on the adaxial surface of snap bean leaves (Fig. 1). This symptom is consistent with other studies on snap bean, although the cultivars for test are different (Guidi et al. 2009; Paoletti et al. 2014). This result means that ‘Jiangjunyoudou’ is also a sensitive cultivar. The average percentage of visible injured surface over the first pair of fully expanded leaves was significantly lower after spraying leaf extract compared with CK (Table 1). The most effective extract was that of PHT (− 93.09% of injury relative to CK), followed by PHB (− 68.68%) and TP (− 31.53%).

Fig. 1
figure 1

Leaf visible symptoms of P. vulgaris L. ‘Jiangjunyoudou’ under charcoal-filtered air (CF, 0 ppb) or elevated ozone (O3, 150 ppb). Plants sprayed with CK (a), leaf extract of TP (b), of PHB (c), or of PHT (d) in CF air; plants sprayed with CK (e), leaf extract of TP (f), of PHB (g), or of PHT (h) under O3

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Table 1 Visible injury on the first fully expanded leaves in plants of P. vulgaris L. ‘Jiangjunyoudou’ sprayed with CK, leaves extract of TP, of PHB, of PHT under elevated ozone (O3, 150 ppb) and charcoal-filtered air (CF, 0 ppb)
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Significant interactions between O3 treatment and extract spraying were found in the Pn, gs, E, and Ci/Ca of snap bean (Fig. 2). Compared with plants in CF air Pn, gs and E were significantly decreased, while Ci/Ca increased under O3 fumigation. The significant inhibition of Pn and the slight increase of Ci/Ca induced by O3 fumigation demonstrated that the decline of Pn was mainly caused by non-stomatal factors, probably biochemical processes. These results are in accordance with previous researches (Zhang et al. 2010; Sui et al. 2017). Leaf extract spraying reduced the O3-caused loss of Pn. The relative loss of Pn was the lowest in PHT (− 27.20%), followed by PHB (− 45.91%), TP (− 69.11%), and CK (− 76.10%). The trend of Pn under O3 fumigation was in accord with that of visible injury.

Fig. 2
figure 2

Net photosynthesis (Pn, a), stomatal conductance (gs, b), the ratio of intercellular/ambient CO2 concentration (Ci/Ca, c), and transpiration rate (E, d) of P. vulgaris L. ‘Jiangjunyoudou’ sprayed with CK, leaf extract of TP, of PHB, or of PHT exposed to charcoal-filtered air (CF, 0 ppb) or elevated ozone (O3, 150 ppb) for 3 days (8 h per day). Each value is mean of six replicates, error bars represent + SE. Different letters show significant differences among bars (Duncan’s test, p ≤ 0.05). Results of two-way ANOVA are shown in the inset

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Ozone fumigation significantly decreased the contents of Chl a, Chl b and total Chl (Fig. 3).This result is consistent with other studies (Bagard et al. 2015; Zhang et al. 2017b). The decrease of Chl probably partially caused the reduction of Pn. Leaf extract spraying had no significant effect on the O3-caused decrease of Chl. These results indicated that the mitigating effect of leaf extract on the loss of Pn was not due to the change of Chl.

Fig. 3
figure 3

Contents of chlorophyll (Chl) a (a), Chl b (b), total Chl (c) of P. vulgaris L. ‘Jiangjunyoudou’ sprayed with CK, leaf extract of TP, of PHB, or of PHT exposed to charcoal-filtered air (CF, 0 ppb) or elevated ozone (O3, 150 ppb) for 3 days (8 h per day). FW was the FW of the sample. Each value is mean of six replicates, error bars represent + SE. Different letters show significant differences among bars (Duncan’s test, p ≤ 0.05). Results of two-way ANOVA are shown in the inset

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Significant interaction between O3 treatment and extract spraying was observed in EL and MDA (Fig. 4). Ozone significantly increased EL and MDA content. The significant increases of EL and MDA content were indicative of plasma membrane damage caused by O3 fumigation (Liu et al. 2015). The relative increase of EL and MDA was the lowest in PHT (0.07% and 4.03%, respectively), followed by PHB (3.31% and 13.03%, respectively), TP (5.72% and 21.46%, respectively) and CK (10.23% and 21.09%, respectively). This result indicated that extract of PHT could significantly protect the plasma membrane from the ROS attack. Responses of EL and MDA to O3 had a similar pattern to that of visible injury. These results confirmed that the observed visible injuries were due to, at least in part, cell membrane damage.

Fig. 4
figure 4

Electrolyte leakage (EL, a) and malondialdehyde contents (MDA, b) of P. vulgaris L. ‘Jiangjunyoudou’ sprayed with CK, leaf extract of TP, of PHB, or of PHT under charcoal-filtered air (CF, 0 ppb) and elevated ozone (O3, 150 ppb) for 3 days (8 h per day). FW is the fresh weight of the sample. Each value is mean of six replicates, error bars represent + SE. Different letters show significant differences among bars (Duncan’s test, p ≤ 0.05). Results of two-way ANOVA are shown in the inset

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The interactions between O3 treatment and extract spraying on O2•−, SOD, POD and CAT were significant (Fig. 5). Ozone fumigation significantly increased O2•− contents relative to plants in CF. Leaf extract spraying inhibited the increases of O2•− contents. The relative increase of O2•− in the PHT-sprayed plants was the lowest (4.03%), followed by PHB-sprayed plants (15.61%), TP-sprayed plants (20.71%), and CK (33.63%). SOD, POD and CAT can cooperate with each other to remove ROS such as O2•− produced by plant under stress (Pan et al. 2006). As the first line of defense, SOD is the key to remove O2•− in cells and convert O2•− into H2O2, which can be scavenged by POD and CAT (Liu et al. 2015; Xu et al. 2015). It has been observed that O3 fumigation increased the SOD activity (Zhang et al. 2010), while in the current study the activity of SOD was decreased by elevated O3. This result might be due to increased concentrations of other antioxidants, which had higher ability to scavenge superoxide radicals (Daripa et al. 2016). It has been reported that the leaves of PHT and TP contain lots of flavonoids and phenolic compounds (Blum and Didyk 2006, 2007; Mothana et al. 2010; Li et al. 2014; Rijo et al. 2014; Daripa et al. 2016), which probably played key roles in scavenging the O2•−. Leaf water extract of TP has been proved to alleviate O3 damage (Blum and Didyk 2006, 2007; Daripa et al. 2016), which is in accordance with our results. PHT and PHB extracts had higher ability to scavenge the O2•− than TP suggesting their higher contents or activities of antioxidants. Furthermore, in plants treated with PHT extract the relative decrease of SOD activity was lower than that in the other extract treatments after O3 fumigation. This result suggested that the PHT extract could maintain the SOD activity to a certain degree. This may be another reason why the plant sprayed with PHT extract had lower content of O2•− than other extract treatments. Under O3 fumigation, POD activity significantly increased in PHT-sprayed and PHB-sprayed plants, but did not change in TP-sprayed plants and CK. Compared with plants in CF air, the activity of CAT was significantly increased by O3 fumigation. The relative increases of CAT activity in PHT-sprayed and PHB-sprayed plants were lower than those in TP-sprayed plants and CK. These results showed that POD played more important roles in scavenging ROS in PHT-sprayed and PHB-sprayed plants after O2•− was converted to H2O2. In TP-sprayed or CK plants, H2O2 was scavenged depending on the increase of CAT.

Fig. 5
figure 5

Superoxide anion content (O2•−, a) and SOD (b), POD (c), CAT (d) activities of P. vulgaris L. ‘Jiangjunyoudou’ sprayed with CK, leaf extract of TP, of PHB, or of PHT under charcoal-filtered air (CF, 0 ppb) and elevated ozone (O3, 150 ppb) for 3 days (8 h per day). FW is the fresh weight of the sample. Each value is mean of six replicates, error bars represent + SE. Different letters show significant differences among bars (Duncan’s test, p ≤ 0.05). Results of two-way ANOVA are shown in the inset

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In summary, an acute O3 fumigation caused significant visible foliar injury, impaired the plasma membrane, reduced the content of Chl, increased the content of MDA and O2•−, thus decreased photosynthesis. Spraying with extract of PHT, PHB or TP ameliorated the negative effects of O3. PHT proved the best ability to mitigate the damages caused by O3, followed by PHB and TP.