Effects of exogenous NO on antioxidant system of Taxus plants under simulated acid rain stress

Taxus is a famous medicinal and landscape tree species. The aim of this study was to explore the effect of exogenous nitric oxide (NO) on the resistance of Taxus plants to acid rain stress and to identify Taxus species with strong acid rain resistance by principal component analysis and comprehensive evaluation. In this study, sodium nitroprusside (SNP) was used as the exogenous NO donor. The effects of different SNP solution concentrations on the antioxidant systems of three Taxus species subjected to simulated acid rain stress (pH = 3.0) were compared. In order to achieve this goal, we determined the rate of O production, the ASA and GSH contents in leaves of three Taxus plants (Taxus mairei, Taxus chinensis, and Taxus yunnanensis). At the same time, the active leaves of some antioxidant enzymes (SOD, POD, CAT, APX and GR) were determined. For Taxus chinensis plants subjected to acid rain stress, treatment with an SNP concentration of 0.25 mmol·L led to the most significant improvements in the antioxidant system. For Taxus mairei and Taxus yunnanensis, the treatment with the SNP concentration of 0.50 mmol·L was best for improving their antioxidant systems under stress. Meanwhile, Taxus chinensis had the strongest resistance to simulated acid rain, followed by Taxus mairei and Taxus yunnanensis.


Introduction
Taxus is a genus in the Taxaceae family, which mostly consists of evergreen shrub species. Most Taxus species have a beautiful tree shape and strong germination ability. Taxus plants is well known for its high value in the treatment of various cancers (Fei et al., 2019). They grow quickly, are well adapted to their environment and are easy to trim and shape (Rathore et al., 2019). Taxus is an economically important and an endangered gymnosperm genus and Taxus is an important ornamental plant for horticulture and gardening (Fei and Tang, 2018). Therefore, Taxus species have been increasingly used in urban landscaping. Rainwater acidification is considered to be one of the most serious environmental problems affecting transboundary natural environments. Due to the interaction of these acids with other components in the atmosphere, protons are released. This proton release increases the acidity of the soil and the utilization of toxic heavy metals, reduces 2 soil fertility, and ultimately negatively affects the growth and productivity of gardens and crops (Singh and Agrawal, 2008). Nitric oxide (NO), as a signaling molecule, usually interacts with plant hormones and other endogenous molecules during early plant growth and development (Freschi and Luciano, 2013). Many reports have indicated that the application of exogenous NO donors confers tolerance to various abiotic stresses (Mirza et al., 2010;Hasanuzzaman et al., 2011;Hasanuzzaman et al., 2013). Sodium nitroprusside (SNP) is an exogenous NO donor. In this study, the effects of SNP on the antioxidant substance content and antioxidant enzyme activity of Taxus mairei, Taxus chinensis and Taxus yunnanensis were analyzed under acid rain stress (pH = 3.0). Choosing Taxus species with strong acid rain resistance is of great significance for the future promotion and application of Taxus in landscape beautification. It is also of practical significance to explore the effect of exogenous NO on mitigating the effects of acid rain stress on Taxus plants.

Experimental setup and treatment conditions
The experiment was conducted in the greenhouse of the Botanical Garden of the College of Horticulture and Gardening, Yangtze University, China. In November 2016, 200 2a seedlings of each of three species, Taxus mairei, Taxus chinensis, and Taxus yunnanensis, were introduced from outside of the university.
A mixed matrix, with a loam-to-fine sand-to-wood chips ratio of 3:1:1, was used to plant the seedlings in. The seedlings were planted in a flower pot with an inner diameter of 10 cm and placed in a greenhouse with normal water and fertilizer management. On August 9, 2018, approximately 180 robust Taxus mairei, Taxus chinensis and Taxus yunnanensis seedlings were selected. The seedlings were randomly divided into six groups, as shown in Table 1. Four times (on August 9, 11, 13, and 15), in the evening, each group of seedlings was sprayed with SNP concentrations of 0 mmol·L -1 (CK1), 0 mmol·L -1 (CK2), 0.10 mmol·L -1 (N1), 0.25mmol·L -1 (N2), 0.50mmol·L -1 (N3) and 0.75mmol·L -1 (N4). The control groups (CK1 and CK2) were sprayed with clear water. A repeat was set for every 10 plants, each treatment was repeated three times, and the leaves were evenly sprayed. The purpose of CK1 was to provide a control for CK2, to show to what extent the simulated acid rain solution with a pH of 3.0 could damage the Taxus plants.
Acid rain stress treatment began on August 20, and 500mL of simulated acid rain solution (Ju et al., 2017) (pH = 3.0) was sprayed evenly on the seedling leaves of each stress group (CK2, N1, N2, N3 and N4). An equal volume of simulated acid rain with a pH of 5.6 was used as a control (CK1). The leaves were sprayed once a week, and the SNP solutions of various concentrations were used to prevent decomposition and deterioration. Normal water and fertilizer management was maintained throughout the test period. All the treatments ended on September 10, 2018, and samples were taken after 10 days of growth. An appropriate amount of functional leaves was collected and stored at -80 °C in an ultra-low temperature refrigerator.

Antioxidant system index measurements
The superoxide dismutase (SOD) activity was determined through the aorta-anisidine method (Durak et al., 1993). The activity of peroxidase (POD) was determined via the guaiacol method (Rufino et al., 2008).The formation rate of superoxide radical (O2 -) was measured using the hydroxylamine oxidation method (Karpets and Kolupaev, 2018). The procedure given by Huang et al. (2005) was used for the determination of 3 ascorbic acid (ASA) contents. The guaiacol method was used to measure the catalase (CAT) activity (Durak et al., 1999). Ascorbate peroxidase (APX) activity was measured using a 2 mL system measuring instrument (Gadelha et al., 2017). The reduced glutathione (GSH) contents were determined using spectrophotometry (Cao et al., 2018). The glutathione reductase (GR) activity was calculated by measuring the decrease in absorbance at 340 nm caused by the dehydrogenation of NADPH (Gadelha et al., 2017).

Data analysis
The SAS 10.0 statistical software was used to process data for the analysis of variance (ANOVA) and Duncan multiple comparison tests. The Origin 2020 software was used for plotting, and the principal component analysis (PCA) was used to comprehensively evaluate the three Taxus plants.

Results
Effect of SNP on the rate of O 2production in leaves of Taxus spp. under acid rain stress As shown in Figure 1A, after simulated acid rain treatment, the O 2generation rate in the leaves of Taxus mairei, Taxus chinensis and Taxus yunnanensis was significantly higher than in the CK1 group (p < 0.05). The O 2generation rate in Taxus mairei and Taxus chinensis leaves first decreased and then increased as the SNP concentration increased. The O 2generation rate in Taxus yunnanensis leaves decreased with increasing SNP solution concentration. Compared with CK2, the production rate of O 2in the leaves of all three species was significantly lower in the groups sprayed with SNP solution (p < 0.05). The Taxus mairei plants in the N3 group displayed the most significant reduction (p < 0.05) in O 2generation compared to those in the CK2 group, with a 36.85% reduction. The Taxus chinensis plants in the N2 group displayed the most significant reduction (p < 0.05) in O 2generation compared to those in the CK2 group, with a reduction of 26.00%. For Taxus yunnanensis, the N4 treatment (except N1) led to the most significant decrease in O2generation at 25.48% (p < 0.05). The results show that the application of simulated acid rain with a pH of 3.0 can significantly (p < 0.05) increase the rate of O 2production in the leaves of the three Taxus species. Spraying with SNP concentrations of 0.50 mmol·L -1 , 0.25 mmol·L -1 and 0.75 mmol·L -1 can significantly (p < 0.05) reduce the O 2production rate in the leaves of Taxus mairei, Taxus chinensis and Taxus yunnanensis plants subjected to acid rain stress, respectively.
Effects of SNP on SOD, POD and CAT activities in leaves of Taxus spp. under acid rain stress After simulated acid rain treatment (pH = 3.0), the SOD ( Figure 1B), POD ( Figure 1C) and CAT ( Figure 1D) activity in the leaves of all three Taxus species were significantly lower than after CK1 treatment (p < 0.05). Upon the addition of SNP, the SOD activity of Taxus mairei generally increased with increasing SNP solution concentration, while the POD and CAT activity first increased and then decreased. The activities of SOD and CAT in the leaves of Taxus chinensis increased first and then decreased as the concentration of SNP increased. While the activity of POD showed a downward trend. The activity of SOD in Taxus yunnanensis leaves decreased with increasing SNP concentration, the POD activity first increased and then decreased. While the CAT activity showed a downward trend. For the Taxus mairei plants, the activities of SOD, POD and CAT were significantly higher (p < 0.05) than CK2. The N3 treatment had the most significant effect on the activities of SOD, POD and CAT in Taxus mairei (p < 0.05), with increases of 63.59%, 126.30% and 41.65 %, respectively. The SOD and POD activities (N1, N2 and N3) in Taxus chinensis leaves have been significantly increased (p < 0.05), N4 has been significantly decreased (p < 0.05). The SOD activity of Taxus chinensis leaves increased significantly (p < 0.05) after N3 treatment, with an increase of 37.69% compared to after CK2. The POD activity of Taxus chinensis leaves increased significantly (p < 0.05) after N2 treatment, with an increase of 34.50% compared to after CK2. The leaves of Taxus yunnanensis plants subjected to N1 treatment displayed the highest SOD activity, subjected to N3 treatment displayed the highest POD activity and those subjected to N4 treatment displayed the highest CAT activity (p < 0.05), with increases of 37.69%, 123.95% and 29.19%, respectively. The results showed that the application of simulated acid rain with a pH of 3.0 can significantly (p < 0.05) reduce the activities of SOD, POD and CAT in the leaves of the three Taxus species. After the SNP solutions were sprayed at concentrations of 0.50 mmol·L -1 , 0.50 mmol·L -1 and 0.10 mmol·L -1 , the leaf SOD activity in stressed Taxus mairei, Taxus chinensis and Taxus yunnanensis plants, respectively, increased significantly (p < 0.05). After the SNP solutions were sprayed at concentrations of 0.50 mmol·L -1 , 0.10 mmol·L -1 and 0.50 mmol·L -1 , the leaf POD activity in stressed Taxus mairei, Taxus chinensis and Taxus yunnanensis plants, respectively, increased significantly (p < 0.05). When SNP solutions were sprayed at 0.50 mmol·L -1 , 0.25 mmol·L -1 and 0.75 mmol·L -1 , the leaf CAT activity instressed Taxus mairei, Taxus chinensis and Taxus yunnanensis plants, respectively, could be significantly increased (p < 0.05). Different lowercase letters indicate a significant difference (p<0.05), same the below. CK1 represents a control group (simulated acid rain at a pH of 5.6), as does CK2 (simulated acid rain at a pH of 3.0). The other treatments involved simulated acid rain (pH = 3.0) with 0.10 mmol·L -1 (N1), 0.25 mmol·L -1 (N2), 0.50 mmol·L -1 (N3) and 0.75 mmol·L -1 (N4) SNP solution concentrations.

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Effects of SNP on the ASA and GSH contents in leaves of Taxus spp. under acid rain stress After simulated acid rain treatment (pH = 3.0), the contents of ASA ( Figure 2A) and GSH ( Figure 2B) in leaves of the three species were significantly lower than after CK1 treatment (p < 0.05). The ASA content of Taxus mairei leaves showed an upward trend with increasing SNP concentration, the GSH content increased at first and then decreased. As the SNP concentration increased, the contents of ASA and GSH in the leaves of Taxus chinensis increased first and then decreased. The content of ASA in the leaves of Taxus yunnanensis generally increased first and then decreased, and the content of GSH showed an upward trend. The ASA and GSH contents in the leaves of the three Taxus species were significantly higher (p < 0.05) after SNP treatment than after the CK2 treatment. For Taxus mairei, the ASA and GSH contents were most significantly increased in the N3 treatment (p < 0.05), at 27.52% and 108.77% higher than in the CK2 group, respectively. The contents of ASA (N1, N2 and N3) and GSH in the leaves of Taxus chinensis were the most significantly increased after N2 treatment (p < 0.05), with increases of 80.94% and 91.45%, respectively. For Taxus yunnanensis, the most significant increase in ASA content was observed in the N3 group (p < 0.05), with an increase of 37.88%. Meanwhile, the Taxus yunnanensis leaf GSH content was most significantly increased by the N4 treatment (p < 0.05), with an increase of 62.85%. The results show that the application of simulated acid rain with a pH of 3.0 can significantly (p < 0.05) reduce the leaf ASA and GSH contents in the three Taxus species. After spraying the stressed Taxus mairei, Taxus chinensis and Taxus yunnanensis plants with SNP concentrations of 0.50 mmol·L -1 , 0.25 mmol·L -1 and 0.50 mmol·L -1 , respectively, the leaf ASA content increased significantly (p < 0.05). When SNP concentrations of 0.50mmol·L -1 , 0.25mmol·L -1 and 0.75mmol·L -1 were applied to stressed Taxus mairei, Taxus chinensis and Taxus yunnanensis plants, respectively, the leaf GSH content could also be increased significantly (p < 0.05).
Effects of SNP on APX and GR activities in leaves of Taxus spp. under acid rain stress As shown in Figure 2C and 2D, after simulated acid rain treatment (pH = 3.0), the leaf APX and GR activities of the three Taxus species were significantly lower than after CK1 treatment (p < 0.05). Upon application of SNP, the leaf APX activity in the three Taxus species first increased and then decreased with increasing SNP concentration. For Taxus mairei, the leaf APX activity was significantly higher in the N1, N2 and N3 groups than in CK2 (p < 0.05), the most significant increase (42.94%) was observed after the N3 treatment (p < 0.05). The leaf APX activity of Taxus chinensis was significantly higher (26.13%) in the N3 group than in the CK2 group (p < 0.05). The APX activity in the leaves of Taxus yunnanensis was significantly higher in the N3 and N4 groups than in CK2 (p < 0.05), the N4 treatment had the most significant effect, leading to an increase in APX activity of 53.45%. With increasing SNP solution concentration, the GR activity of Taxus mairei and Taxus yunnanensis increased, while that of Taxus chinensis showed a downward trend. The leaf GR activity of Taxus mairei was significantly higher in the N1, N2 and N3 groups than in CK2 (p < 0.05), the N3 treatment had the most significant effect, leading to an increase in GR activity of 43.56%. The GR activity of the leaves of Taxus chinensis was significantly higher in the N1, N2, N3 and N4 groups than in CK2 (p < 0.05). The N1 treatment had the most significant effect, leading to an increase in GR activity of 56.45%. For Taxus yunnanensis, the leaf GR activity was significantly higher in the N2, N3 and N4 groups than in CK2 (p < 0.05), the N4 treatment had the most significant effect, leading to an increase in GR activity of 129.15%. The results show that the application of simulated acid rain with a pH of 3.0 can significantly (p < 0.05) reduce the APX and GR activities of the leaves in all three Taxus species. When SNP solutions were sprayed on stressed Taxus mairei, Taxus chinensis and Taxus yunnanensis plants at concentrations of 0.50 mmol·L -1 , 0.50 mmol L -1 and 0.75 mmol·L -1 , respectively, the leaf APX activity of the significantly increased (p < 0.05). When SNP solutions were sprayed on stressed Taxus mairei, Taxus chinensis and Taxus yunnanensis plants at concentrations of 0.50 mmol L -1 , 0.10 mmol L -1 and 0.75 mmol·L -1 , respectively, the leaf GR activity significantly increased (p < 0.05). Comprehensive evaluation of plant resistance to acid rain stress in different treatment groups As shown in Table 2, the resistance coefficient (α) differed for the different indicators of the same treatment and for the same indicators of different treatments. Therefore, it is difficult to reasonably compare the resistance to acid rain stress under different treatments and to draw a conclusion with high reliability. The results of evaluating acid rain resistance using a single index are different, which shows that the acid rain resistance of the plants is not determined by a character index. It is thus one-sided to evaluate the acid rain resistance using a certain index, so principal component analysis (PCA) was used to evaluate the acid rain resistance. The PCA was carried out for the resistance coefficient (α) of different treatments (Figure 3). Then, the comprehensive evaluation (D) of the acid rain resistance of each treatment was calculated using PC1 and PC2. The D value was then used to rank the acid rain resistance of the different treatments (Table 3). The results showed that the best SNP solution concentrations for improving the stress resistance of Taxus mairei, Taxus chinensis and Taxus yunnanensis, were 0.50 mmol·L -1 , 0.25 mmol·L -1 and 0.75 mmol·L -1 , respectively. As shown in Figure 3D, the results of PCA analysis on the resistance coefficients of different Taxus plants under CK2 treatment were carried out. As above, we can also get the ranking of the resistance of different Taxus plants to simulated acid rain ( Table 4). The comprehensive evaluation results showed that Taxus mairei and Taxus yunnanensis had the strongest resistance to simulated acid rain under N3 treatment, and Taxus chinensis had the strongest resistance to simulated acid rain under N2 treatment. Meanwhile, Taxus chinensis had the strongest resistance to simulated acid rain, followed by Taxus mairei and Taxus yunnanensis.

Discussion
When plants are subjected to external stress, many reactive oxygen species (ROS) are produced. These ROS accumulate in plant cells as the stress continues or increases, which causes damage to the cells (Wen et al., 2020;Zhang et al., 2020). The stress stimuli caused by unfavorable environmental factors can activate the antioxidant defense system within plant cells. The activation of the antioxidant defense system helps the plant to survive the unfavorable environment and alleviates the damage suffered under stress due to ROS (Anjaneyulu and Chopra, 2004). In this experiment, after the simulated acid rain stress (pH = 3.0) was applied to the leaves of the Taxus species, the O 2production rate in the leaf cells of all three Taxus species increased. This result is consistent with the results of Kamrun et al. (2016), who investigated the salt tolerance of mung bean seedlings. Furthermore, the simulated acid rain stress inhibited the metabolism of the protective antioxidant enzyme system of the leaves of three Taxus species. The activities of SOD, CAT, APX, GR and other protective enzymes were significantly reduced by the stress (p < 0.05). These results are consistent with those of the study by Seckin et al. (2009), in which wheat root antioxidant enzyme activity was investigated under stress. The reduced activities of CAT, APX and GR were significantly negatively correlated with the increase in O 2production rate (p < 0.05). This result was similar to the salt stress tolerance of maize (Kholova et al., 2010). In the present study, it was also found that the contents of ASA and GSH in the cells of plants subjected to acid rain stress were significantly lower (p < 0.05) than in the CK1 treatment. This may have been due to the production rate of antioxidant substances in the plant being far less than the rate of oxidation.
After spraying different concentrations of SNP solution on the stressed plants, the activities of SOD, CAT, APX and GR in the Taxus plants significantly increased (P < 0.05). This demonstrated that exogenous 9 NO application can reactivate the activity of antioxidant enzymes in plant leaves. This result is in accordance with the research results of Egbichi et al. (2014), who investigated the effect of exogenous NO on the antioxidant capacity of soybeans treated with cadmium. In addition, NO can also be directly combined with O2to produce ONOO -, which is less toxic to plant cells than O 2-, thereby reducing the damage caused to plant cells by oxidative stress (Lehotai et al., 2011;Long et al., 2020;Piacentini et al., 2020). In this study, it was found that treatment with specific concentrations of SNP solution significantly increased the antioxidant enzyme activity of the Taxus species (p < 0.05). This may have been related to the direct participation of NO in partially quenching O 2-, which would eliminate the inhibition of antioxidant enzyme activity caused by high O2concentrations. NO also has a certain regulatory effect on the synthesis of some substances in plant leaves. For example, NO can promote the synthesis of GSH in plant leaves. As an antioxidant, GSH can reduce the amount of ROS within plant cells (Lin et al., 2020;Sun et al., 2015). In addition, NO can play a role in activating APX activity, which is essential for removing H2O2 (Egbichi et al., 2014;Yun et al., 2020). In the present study, after treatment with different concentrations of SNP solution, the content of GSH and ASA in the leaves of the Taxus plants subjected to acid rain stress increased significantly (p < 0.05). These results are consistent with those of the study by Gadelha CG et al. (2017), which focused on the salt tolerance of tree seedlings. Through further research, it was found that after spraying specific concentrations of SNP solution, the O 2production rate in the leaf cells of the Taxus species decreased to varying degrees, this decrease was significant at specific SNP concentrations (p <0.05). Therefore, as the O 2production rate in the cells decreased significantly (p < 0.05), the content of ROS in the cells also decreased significantly, and the peroxidation of the cell membrane system was alleviated (Chen et al., 2015).
A comprehensive analysis of the effect of different SNP concentrations on the antioxidant system indexes of Taxus species subjected to simulated acid rain was performed. The O 2generation rate in the leaf cells of Taxus mairei and Taxus yunnanensis was found to be significantly associated (negative correlation) with CAT activity, GR activity and GSH content (p < 0.05). This shows that antioxidant enzymes play a more important role in the antioxidant defense system of Taxus mairei and Taxus yunnanensis than antioxidant substances. It may be that these enzymes removed a large amount of H2O2 in the leaves, and accelerated the conversion rate of O 2to H2O2, so that a large amount of O 2was converted into H2O2 and removed in the form of H2O2. However, the rate of O 2production in the Taxus chinensis leaf cells was significantly negatively correlated with GR activity, and GSH and ASA content (p < 0.05). This indicated that compared with the antioxidant enzymes, the antioxidant substances played a more important role in Taxus chinensis. In the leaves of the Taxus species, the removal of antioxidants is the main reason. One possible mechanism underlying this effect is that O 2is converted into H2O2 under the action of antioxidants, and then under the combined action of SOD, GR, ASA and GSH, H2O2 is eradicated from the plant cells.

Conclusions
In this study, the effect of exogenous NO on the antioxidant system of Taxus seedling leaves under simulated acid rain stress was investigated. We explored the ability of exogenous NO to improve the tolerance of Taxus plants subjected to acid rain stress and identified Taxus species with strong acid rain tolerance. For Taxus chinensis, treatment with an SNP solution concentration of 0.25 mmol·L -1 led to optimal improvement of the antioxidant system. For Taxus mairei and Taxus yunnanensis, the treatment with 0.50 mmol·L -1 SNP was best for improving the antioxidant systems of the plants. Meanwhile, Taxus chinensis had the strongest resistance to simulated acid rain, followed by Taxus mairei and Taxus yunnanensis.