**3. Results**

### *3.1. The Effect of SNP on Photosynthetic Pigments Content during Salt Stress in C. erythreae Shoots*

The centaury shoots successfully survived four weeks on <sup>1</sup> <sup>2</sup>MS media supplemented with different combinations of SNP (0, 50, 100 or 250 μM) and/or NaCl (150 mM). Control centaury shoots grown on NaCl-free medium developed the usual rosette morphology and dark green oval leaves (Figure 2). Pretreatments with 50 and 100 μM SNP altered the color of the leaves to light green. Pretreatment with 250 μM SNP caused leaf tip curling and desiccation as well as yellowing of the leaves and chlorosis of the entire shoot. After this pretreatment, and especially in combination with SNP, and NaCl, the highest number of yellow leaves was observed. Unlike other pretreatments/treatments, only after the pretreatment with 250 μM SNP, most centaury shoots did not spontaneously develop roots.

Leaf chlorosis is one of the most common symptoms of stress caused by NaCl due to decreased photosynthetic pigments and is also an important indicator of the physiological state of the plants. Therefore, the content of photosynthetic pigments was determined in two control groups of shoots that were grown on <sup>1</sup> <sup>2</sup>MS NaCl-free medium throughout the whole experimental period, then in medium supplemented with 150 mM NaCl, as well as in shoots grown on different SNP pretreatments and NaCl and/or SNP treatments. In the second control group of centaury shoots not exposed to SNP during the pretreatment, NaCl decreased total *Chl* content ~21% in comparison to the first control group grown on <sup>1</sup> <sup>2</sup>MS NaCl-free medium (Figure 3a). In addition, pretreatment with 50 μM SNP significantly decreased the total *Chl* in shoots grown on NaCl-free medium in comparison to the control group of shoots grown on the same medium. Conversely, the combination of 50 μM SNP pretreatment and then treatments with NaCl and 50 μM SNP, increased total *Chl* content ~20% in comparison to the control group of shoots grown on NaCl-supplemented medium, as well as in comparison to the control shoots from the appropriate treatment. Pretreatment with 50 μM SNP in combination with treatment including NaCl and 50 μM SNP together, also reduced total *Chl* content to the lowest level in this experimental group. The application of 100 μM SNP in the pretreatment, did not lead to significant changes in total *Chl* content in comparison to the control group centaury shoots grown on medium with NaCl. The 250 μM SNP pretreatment did not show any positive effects, and decreased total *Chl* content in comparison to the control group of shoots grown on medium with NaCl. It was interesting to note that the lowest total *Chl* content was detected in centaury shoots exposed to treatments including both NaCl and SNP after the appropriate SNP

pretreatments. It was also found that increased SNP concentrations in the pretreatment were negatively correlated with decreased total *Chl* content after the corresponding SNP and NaCl treatments.

The effect of the different SNP pretreatments and NaCl and/or SNP treatments on total carotenoid content is shown in Figure 3b. In the control groups of century shoots, NaCl decreased the total carotenoid content 27% in comparison to the control group of shoots grown on NaCl-free medium. Pretreatment with 50 μM SNP halved the total carotenoid content in shoots grown on NaCl-free medium in comparison to the control group of shoots grown on the same medium. Conversely, pretreatments with 150 and 250 μM SNP did not significantly change the total carotenoid content in comparison to the control group of shoots grown on NaCl-free medium. In shoots grown on medium supplemented with NaCl, pretreatments with 100 and 250 μM SNP increased the total carotenoid content 31 and 52%, respectively, in comparison to control group of shoots grown on the same medium. In addition, in comparison to the control group, the application of 100 and 250 μM SNP as pretreatments in combination with the same SNP concentrations in the treatments, influenced a significant increase in total carotenoid content. Furthermore, pretreatments with 100 and 250 μM SNP, followed by treatments with the same SNP concentrations and NaCl together, resulted in a significant increase in total carotenoid content in comparison to the control group of shoots grown on medium supplemented with NaCl.

**Figure 2.** *Centaurium erythraea* shoots after four weeks of cultivation on different SNP pretreatments, NaCl and/or SNP treatments.

#### *3.2. The Effect of SNP on Oxidative Stress Biomarkers during Salt Stress in C. erythreae Shoots*

The effect of different SNP pretreatments and NaCl and/or SNP treatments on level of lipid peroxidation in centaury shoots was determined by monitoring the MDA concentration (Figure 4a). In the control group cultured on medium supplemented with NaCl, a decrease in MDA concentration (15%) was observed in comparison to the control group grown on NaCl-free medium. All SNP pretreatments significantly reduced MDA concentrations in the centaury shoots grown on NaCl-supplemented medium, especially the 50 and 250 μM SNP pretreatments, where the MDA concentrations were reduced to 56 and 52%, respectively, in comparison to the control group grown on NaCl. Treatments with 50 and 250 μM SNP decreased MDA concentration, while 100 μM SNP did not significantly change the MDA concentration in comparison to both control groups. A significant increase in lipid peroxidation was observed after treatments with a combination of 50 or 100 μM SNP with 150 mM NaCl. In addition, the highest degree of lipid peroxidation, compared to all treatments tested, was detected after the treatment using 250 μM SNP and 150 mM NaCl.

**Figure 4.** The effect of different SNP pretreatments and NaCl and/or SNP treatments on MDA (**a**) and H2O2 (**b**) concentrations in *C. erythraea* shoots. Data represent mean ± standard error. Bars marked with a different letter are significantly different from the control according to the LSD test (*p* ≤ 0.05).

Since lipid peroxidation is one of the consequences of oxidative stress, H2O2 concentration was also determined as a marker of the degree of plant cell oxidative damage (Figure 4b). The two control groups had approximately the same H2O2 concentrations. Pretreatments with 50, 100 or 250 SNP concentrations increased H2O2 in shoots grown on NaCl-free medium by about 233, 75 and 71%, respectively, in comparison to the control centaury shoots grown on NaCl-free medium and by about 173, 131 and 177%, respectively, in comparison to control shoots grown on NaCl medium. Treatment with 50 μM SNP did not significantly change the H2O2 concentration in comparison to both control groups. Conversely, treatments with 100 and 250 μM SNP significantly increased H2O2 concentration in comparison to both control groups. The same pattern was also observed in all SNP treatments in combination with NaCl.

#### *3.3. The Effect of SNP on Nonenzymatic Antioxidants during Salt Stress in C. erythreae Shoots*

The centaury control shoots grown under unstressed and NaCl-stressed conditions in vitro has similar free proline contents (Figure 5). After pretreatment with 50, 100 or 250 μM SNP, increased proline content (38, 50 and 52%, respectively) was observed in shoots grown on <sup>1</sup> <sup>2</sup>MS nutrient medium in comparison to the control group of shoots grown on the same medium. Only pretreatment with 50 μM SNP resulted in a significant increase in proline content (32%) after NaCl treatment in comparison to the control group of centaury shoots grown on medium supplemented with NaCl. Increased SNP concen-

trations, using the same concentration in pretreatments and in following treatments, was positively correlated with increased proline content in comparison to both control groups. However, treatments with all SNP concentrations showed lower levels of proline content in comparison to the corresponding treatments control. On the other hand, pretreatments with 50 and 100 μM SNP followed by treatments with the same SNP concentrations and NaCl together, decreased proline content to the control values of stressed shoots, while the lowest proline content, lower than in both control groups, was detected in centaury shoots grown on treatment with 250 μM SNP and NaCl together.

**Figure 5.** The effect of different SNP pretreatments and NaCl and/or SNP treatments on proline content in *C. erythraea* shoots. Data represent mean ± standard error. Bars marked with a different letter are significantly different from the control according to the LSD test (*p* ≤ 0.05).

The amount of total phenolic compounds in centaury shoots exposed to different SNP pretreatments and/or treatments was determined (Figure 6a). In the control centaury shoots grown on NaCl-free medium, similar total phenolic content was detected, in comparison to shoots grown on NaCl-supplemented medium. Pretreatments with 50 and 100 μM SNP in NaCl-free medium did not significantly change the amount of total phenolic content in comparison to the corresponding control group, while pretreatment with 250 μM SNP increased the amount of total polyphenols by about 23%. Conversely, all applied SNP pretreatments (50, 100 and 250 μM) caused significant increase in the total phenolic content (29, 69 and 82%, respectively) in shoots grown on medium supplemented with NaCl in comparison to control shoots grown on the same medium. In addition, the application of all SNP concentrations in the pretreatments and treatments, increased the total phenol content in comparison to control shoots grown on NaCl, but these levels still did not exceed the values recorded in control shoots grown on NaCl-free medium. The same pattern was observed after all treatments that included the combinations of 50 or 100 μM SNP and NaCl. The only exception was the combination of 250 μM SNP and NaCl, where an increase of about 26% was observed in comparison to control shoots grown on <sup>1</sup> <sup>2</sup>MS medium.

The influence of the different SNP pretreatments and NaCl and/or SNP treatments on the antioxidant capacity of centaury shoots is presented on Figure 6b. In control conditions, the addition of NaCl decreased the DPPH concentration by 28% in comparison to shoots grown on <sup>1</sup> <sup>2</sup>MS medium. In comparison to control shoots grown on NaCl-free medium, pretreatments with 50 and 100 μM SNP did not significantly change DPPH concentrations while pretreatment with 250 μM SNP significantly increased DPPH in shoots grown on the same medium. Under the conditions of salt stress caused by NaCl, pretreatments with all SNP concentrations (50, 100 and 250 μM) shown an increase in the degree of DPPH reduction by 11, 17 and 31%, respectively, in comparison to the corresponding control group. Treatments with 50 and 100 μM SNP did not significantly alter DPPH concentrations and both values were similar to control shoots grown on <sup>1</sup> <sup>2</sup>MS and NaCl-free

medium, respectively. Only treatment with 250 μM SNP, significantly increased DPPH concentration in comparison to both control groups, but still at the level of control shoots within the same treatment. Using the combination treatments containing NaCl and 50 or 100 μM SNP, an increased DPPH was detected in comparison to control shoots grown on NaCl, but DPPH concentration was not changed in comparison to the second group of shoots grown on <sup>1</sup> <sup>2</sup>MS medium. Among all treatments tested, the most significant degree of DPPH reduction, in comparison to both control groups, was recorded in shoots grown on media supplemented with NaCl and 250 μM SNP.

**Figure 6.** The effect of different SNP pretreatments and NaCl and/or SNP treatments on total phenolic content (**a**) and DPPH concentration (**b**) in *C. erythraea* shoots. Data represent mean ± standard error. Bars marked with a different letter are significantly different from the control according to the LSD test (*p* ≤ 0.05).

#### *3.4. The Effect of SNP on Enzymatic Antioxidants during Salt Stress in C. erythreae Shoots*

In the control groups of shoots grown in the presence of NaCl, SOD activity was decreased by about 18% in comparison to control shoots grown on NaCl-free medium (Figure 7a). In shoots grown on <sup>1</sup> <sup>2</sup>MS medium, the 50 and 100 μM SNP pretreatment increased SOD activity by about 34 and 24%, respectively, while pretreatment with 250 μM SNP did not significantly changed SOD activity in comparison to control shoots grown on the same medium. In shoots grown on medium supplemented with NaCl and previously pretreated with 50, 100 and 250 μM SNP, the same pattern was observed. SOD activity was increased by 88 and 71% after the application of 50 and 100 μM SNP, respectively, while after 250 μM SNP treatment, SOD activity was similar to control shoots. The application of the SNP treatments caused an increase in SOD activity in comparison to both control groups. However, it was interesting to note that the increasing SNP concentrations were inversely correlated with increasing SOD activity. The highest SOD activity among all the treatment combinations was recorded in shoots grown on NaCl and 100 μM SNP. By increasing the SNP concentration to 250 μM along with the NaCl treatment, the SOD activity decreased to the control level of the corresponding treatment and the control shoots grown on <sup>1</sup> <sup>2</sup>MS or medium supplemented with NaCl.

**Figure 7.** The effect of different SNP pretreatments and NaCl and/or SNP treatments on SOD (**a**), CAT (**b**) and POX (**c**) activities in *C. erythraea* shoots. Data represent mean ± standard error. Bars marked with a different letter are significantly different from the control according to the LSD test (*p* ≤ 0.05).

Similar to the SOD activity, in control conditions, CAT activity was also decreased (by about 55%) in shoots grown on NaCl medium (Figure 7b). Pretreatment with 50 μM SNP did not significantly change CAT activity in comparison to both control groups. Treatment with 100 μM SNP, individually or together with NaCl, significantly increased CAT activity. In the shoots grown on NaCl-free medium, a significant increase in CAT activity was recorded only after 250 μM SNP pretreatment. At the same time, this is the highest recorded CAT activity in the centaury shoots after all applied treatments, and represents an increase of 143% in comparison to control shoots grown on <sup>1</sup> <sup>2</sup>MS medium.

Similar to SOD and CAT activities in the control groups, POX activity was also decreased (by approximately 17%) in shoots grown on NaCl medium (Figure 7c). No significant changes in POX activity were observed in shoots pretreated with 50 or 100 μM SNP and grown on both MS and NaCl-free media. Pretreatments with 50 or 100 μM SNP and furtherr culture on media with the same SNP concentrations and NaCl together, increased POX activity). A significant increase in POX activity was observed after pretreatment with 250 μM SNP and all further treatments. Thus, POX activity was tripled in centaury shoots pretreated with 250 μM SNP and further grown on NaCl-free medium, in comparison to control shoots grown on the same medium. The same pattern was also observed in shoots grown on NaCl and control shoots grown on the same medium but not treated with SNP. Similar POX activity changes were detected in shoots treated only with 250 μM SNP. In comparison to all applied treatments, the highest POX activity was recorded in centaury shoots grown on medium supplemented with 250 μM SNP and NaCl together.

#### **4. Discussion**

Although in nature, centaury inhabits mountain slopes, dry grasslands, scrublands and saline soils, investigations of centaury's response to stressful conditions in vitro are still at the beginning stages. The role of the widely used NO donor, SNP, on plant tolerance to salt stress conditions is usually demonstrated after foliar treatment or using nanoparticles [53]. In this work, the effect of exogenously applied SNP, alone or in combination with NaCl, on several biochemical parameters of centaury shoots grown in vitro was investigated.

#### *4.1. SNP and Photosynthetic Pigments during Salt Stress in C. erythreae*

Due the importance of photosynthesis, as a key physiological process in plants, the effect of different SNP pretreatments and NaCl and/or SNP treatments on the concentration of photosynthetic pigments of centaury was determined. This work demonstrated that total *Chl* content was significantly decreased in control shoots grown on NaCl in comparison to the other control group of shoots grown on <sup>1</sup> <sup>2</sup>MS medium (Figure 3a). These results are in accordance with the results previously obtained in centaury shoots grown during NaClcaused salt stress in vitro [37,39]. The lowest SNP concentration applied at pretreatment (50 μM) shown a positive effect on total *Chl* content in centaury leaves during salt stress. Conversely, the highest SNP concentration (250 μM) decreased total *Chl* content to levels lower than the control group of shoots grown on NaCl. These results could be expected because in addition to oxidative stress, centaury shoots were also exposed to higher intensity of nitrosative stress. The positive effect of SNP on total *Chl* content under stress conditions caused by NaCl was also confirmed in cotton, red raspberry, barley, sunflower and wheat [25,28,54–56]. It was interesting to note that SNP pretreatments did not increase total *Chl* content in comparison to the control group of shoots grown on NaCl-free medium. However, some reports showed that SNP treatment increased total *Chl* content in cotton and raspberry plants grown under salt stress in comparison to control conditions [25,54]. In summary, it can be assumed that a lower SNP concentration had a positive effect on *Chl* preservation by promoting the synthesis, regeneration and/or inhibiting its degradation but also promoting the mechanisms that remove ROS, and the ability of SNP to improve the K+/Na+ ratio [25,27].

The results presented in this work showed that NaCl had negative effect on total carotenoid content in centaury shoots grown in control conditions (Figure 3b). Decreased carotenoid content was also recently reported in centaury shoots under salt stress in vitro [39]. In centaury shoots treated with NaCl, the application of SNP pretreatments resulted in increased total carotenoid content in comparison to control group of shoots grown on medium also supplemented with NaCl. The highest total carotenoid content was observed after treatment with 250 μM SNP and NaCl together. These findings, describing the positive effect of SNP on carotenoid content, correspond with published results from cotton, red raspberry and sunflower plants [25,54,56]. It is quite possible that carotenoids, as non-enzymatic antioxidants, prevent or minimize the oxidative damage induced by NaCl. The latest research proposed increased carotenoid content as a marker of salt tolerance [57]. Accordingly, it can be concluded that SNP increased centaury's tolerance to salt stress.

#### *4.2. SNP and Oxidative Stress Biomarkers during Salt Stress in C. erythreae*

In control conditions, a decrease in MDA content was observed in centaury shoots during salt stress in comparison to shoots grown on NaCl-free medium (Figure 4a). This result is unexpected because, theoretically, exposure to salt stress should increase the degree of lipid peroxidation. It is possible that the duration and/or level of stress intensity were not sufficient. However, similar results were also recorded in the halophyte species *Prosopis strombulifera* and *Salvadora persica*, as well as in soybean and a salt-tolerant cultivar of date palm, where no significant changes in MDA content under NaCl-induced stress was detected [58–61]. Pretreatments with 50 and 250 μM SNP decreased MDA content in NaCl-treated centaury shoots in comparison to both control groups. The effect of SNP on the reduction of MDA content was also shown in other plant species such as cotton, wheat, apple and lentil [25,27–29]. The most interesting results, in terms of lipid peroxidation, were obtained in centaury shoots treated with SNP and NaCl together. The highest MDA content was obtained with the application of 50 μM SNP and NaCl whereas the lowest recorded rate of lipid peroxidation was obtained with the application of 250 μM SNP and NaCl. According to certain studies, SNP application can reduce the activity of lipoxygenases and thereby reduce the degree of lipid peroxidation. In addition, NO has the ability to remove peroxyl radical and prevent further oxidative damage [62,63]. However, at low concentrations, NO, together with O2 •−, forms peroxynitrite, which has the ability to initiate lipid peroxidation [17,53].

In control conditions, salt stress caused a slight increase in H2O2 content in comparison to shoots grown on NaCl-free medium (Figure 4b). On the same media, pretreatments with all SNP concentrations induced significant H2O2 production in centaury shoots, with higher H2O2 content after salt stress. This result can be explained by considering H2O2 not only as oxidative stress marker, but also as a signaling molecule that is important for the establishment of salinity tolerance [64,65]. The application of all SNP concentrations, alone or in combination with NaCl, reduced H2O2 content in centaury shoots after all tested treatments. This reduction may be responsible for the induction of antioxidant defense system to scavenge H2O2. These results are in accordance with SNP application reducing the H2O2 content in cucumber, lettuce, wheat, brown mustard and lentil [23,28,29,66,67].
