*4.3. SNP and Nonenzymatic Antioxidants during Salt Stress in C. erythreae*

The accumulation of endogenous proline content under salinity conditions can be considered as a marker of plant stress tolerance [14]. Increased proline content during exposure to NaCl-induced stress has been documented in numerous plant species including centaury [38]. The results obtained during this investigation showed that the application of all SNP pretreatments increased the proline content in centaury shoots grown on NaCl, similar to those grown on <sup>1</sup> <sup>2</sup>MS medium, in comparison to both control groups (Figure 5). In addition, it was noted that all SNP concentrations in treatments were positively correlated with increased proline content. It is obvious that SNP alone, as a potential stressogenic factor, further induced proline accumulation in centaury shoots, likely with enhanced activity of proline-synthesizing enzymes, together with a reduction in proline catabolism

under stress conditions [68]. On the contrary, pretreatments including combinations of SNP and NaCl together, reduced free proline content in centaury shoots. Many studies indicated that NO is involved in proline metabolism during stress conditions but the detected effects were different. Some reports revealed increased proline content in SNPtreated *Lactuca sativa* [66], *Pisum sativum* [69] and *Brassica chinensis* [70] under saline stress. Conversely, reduced proline content, as a consequence of SNP pretreatment, was detected in cucumber [23] and *Brassica rapa* [71] under salt stress. All of these results imply that enhanced proline content is not always essential for plant stress tolerance response because the accumulation of this osmolyte does not always correlate with better plant responses, as in case of NaCl-treated centaury shoots. In addition, considering that synthesis of different osmolytes is an "energetically expensive" process, it is possible that centaury activates other mechanisms with lower energy demands, for example, efficient ions compartmentalization to achieve salinity tolerance [72].

Phenolic compounds belong to the group of secondary metabolites that participate in numerous physiological processes in plants; one of those roles is ROS scavenging under various environmental stresses [73]. Although in most plant species total phenolic content increased under high salinity, there are reports describing decreased phenol content in *Phaseolus vulgaris* and *Schizonepeta tenuifolia* grown under salt stress conditions [74,75]. The same result was observed in NaCl-treated centaury shoots (Figure 6a). The effect of SNP on the total phenolic content increase under NaCl stress conditions was previously documented in mangrove species *Aegiceras corniculatum*, wheat, sunflower, and apple [27,56,76,77]. A similar result was detected in centaury shoots pretreated with SNP and then grown on <sup>1</sup> <sup>2</sup>MS medium or medium supplemented with NaCl. Treatments with all SNP concentrations also increased total phenolic content, while the highest increment among all the treatments was recorded in centaury shoots after combination treatments with all SNP concentrations and NaCl together. During abiotic stress, NO can increase the activity of phenylalanine ammonia-lyase (PAL) and consequently enhance phenolic compounds biosynthesis [17]. The increased activity of the PAL enzyme could be the reason for the increased total phenolic content in centaury shoots after exposure to SNP.

Due to its ability to react with antioxidants, the DPPH radical is a good indicator of the antioxidant capacity of plants [78]. The results obtained in this work showed that, in control conditions, centaury shoots grown on a medium supplemented with NaCl had decreased antioxidant capacity in comparison to shoots grown on a NaCl-free medium (Figure 6b). This result is in accordance with the previous reports where decreased DPPH concentration under NaCl-induced stress in cucumber, sage, spinach, henbane and flax was described [23,79–82]. In order to investigate the changes in centaury antioxidant capacity, the influence of SNP pretreatments on DPPH concentration was tested. The results showed that, in general, all SNP pretreatments increased DPPH concentration in centaury shoots. The largest DPPH concentration was detected in shoots grown on a combination medium supplemented with SNP and NaCl together. These changes in DPPH concentrations, based on their free radical scavenging capacities, positively correlated with total phenolic content in centaury shoots. Furthermore, in several medicinal herbs and selected species of wild vegetables, total phenolic amounts were also significantly correlated with antioxidant capacity [83,84].

#### *4.4. SNP and Enzymatic Antioxidants during Salt Stress in C. erythreae*

Various stress conditions can induce ROS production, which leads to a change in enzyme activity in order to maintain homeostasis in plant cells. Antioxidant enzymes that play a significant role in removing ROS forms and protecting plant cell structures from oxidative stress, include SOD, CAT and POX [16]. Increased SOD, CAT and POX activities under NaCl stress have been documented in many species including sunflower and oilseed rape [56,64]. In this work, decreased activities of SOD, CAT and POX were observed in centaury shoots grown under stress conditions caused by NaCl. Although unexpected, the same results were also reported in halophytic species *Salvadora persica*, date palm and the oil-seed crop *Brassica juncea* [60,61,67]. The positive effect of SNP on the activity of SOD, CAT and POX was previously confirmed in citrus seedlings, wheat and lentil under salinity stress [28,29,85]. The application of SNP increased SOD activity in centaury shoots grown under NaCl, as well as in shoots grown on NaCl-free medium in comparison to the corresponding control groups (Figure 7a). The highest SOD activity was recorded after 50 μM SNP pretreatment while increased SNP concentration decreased SOD activity in centaury shoots. The same trend was also observed in cotton seedlings [25]. The application of SNP pretreatments also increased CAT activity in centaury shoots grown under NaCl-induced stress conditions as well as in shoots grown on nutrient media without NaCl (Figure 7b). The highest CAT activity was determined after the application of 250 μM SNP pretreatment. It can be concluded that SNP stimulated CAT activity in centaury shoots, which has also been observed in tomato and sunflower [22,56]. As in the case of CAT, the same pattern in POX activity was observed. SNP pretreatments increased POX activity, with the highest activity recorded after 250 μM SNP pretreatment (Figure 7c). Similar results were recorded in cotton and sunflower plants grown under salinity stress conditions [25,56]. It is known that the addition of signaling molecules such as NO and hydrogen sulfide (H2S), stimulates the activity of antioxidant enzymes [86]. The role of NO in salt tolerance has been studied in numerous plant species, and there is evidence that the application of NO donors protects plants from salt stress by increasing antioxidant enzyme activity [21]. All the results suggest that NO mitigates the salt-induced oxidative stress by enhancing the activity of enzymatic antioxidants, thus improving centaury's tolerance to salt stress caused by NaCl.
