Results
Determination of extreme halophyte characteristic of S. hieraciifolia
Morphological observations
It was observed that S. hieraciifolia grown on the MS media could survive under the excess salt stress condition (600 mM NaCl) though chlorosis. Conversely, T. parvula could withstand the 450 mM NaCl treatment but not the 600 mM NaCl treatment. The 300 mM NaCl caused shoot succulence and the shoot succulence increased in accordance with the enhanced NaCl concentration in S. hieraciifolia. Additionally, the shoot length was down while shoot number increased in the 300 mM NaCl treatment in S. hieraciifolia in comparison with the 0 mM NaCl treatment. S. hieraciifolia and T. parvula displayed optimal growth at 300 mM NaCl (Fig. 1 A-D).
Effects of salinity on relative water content
The relative water content (%) of S. hieraciifolia shoots significantly enhanced at all levels of salinity in comparison with the 0 mM NaCl treatment. High increases in RWC were determined in the 450 and 600 mM NaCl treatments. Conversely, in T. parvula, the RWC value decreased while salt concentration was increasing. The highest decrease was determined the shoots treated with 450 mM NaCl in T. parvula (Table 1).
 
Effects of salinity on lipid peroxidation
Lipid peroxidation enhanced in the shoots treated with 300, 450 and 600 mM NaCl, while 150 mM NaCl treatment had no significantly effect in comparison with 0 mM NaCl in S. hieraciifolia. However, in T. parvula, TBARS content was increased in all salt treatments compared to the 0 mM NaCl treatment and the highest TBARS content was observed in the 450 mM salt treatment. As mentioned above, the shoots of T. parvula lost their vitality in the 600 mM NaCl treatment. The rates of increase of TBARS content in both plants were same (1.1-fold) in the 300 mM NaCl treatments where observed the optimum growth. However, there were 2.9-fold and 3.8-fold increases in S. hieraciifolia and T. parvula in the 450 mM NaCl treatments, respectively (Table 1).
 
Effects of salinity on H2O2 content
H2O2 contents gradually increased in S. hieraciifolia and T. parvula while salt concentration was increasing. H2O2 contents and the rates of the increases observed in T. parvula were higher than those of S. hieraciifolia. For example, there were 2.4-fold increase in T. parvula and 1.8-fold in S. hieraciifolia in the 300 mM NaCl treated shoots as compared with non-saline conditions. Likewise, there were 8.3-fold and 4.4-fold enhancements in H2O2 contents of the shoots treated with 450 mM NaCl in T. parvula and S. hieraciifolia, respectively (Table 1).
Determination of shoot succulence in S. hieraciifolia
Fleshy shoots were observed at 300 mM and higher salt concentrations in Scorzonera hieraciifolia. Accordingly, the shoot succulence degree increased in the 300 mM NaCl treatment in comparison with the non-stressed shoots. The succulence degrees were high in the 450 and 600 mM NaCl treatments (Table 2). Likewise, the shoot thickness increased depending on salt concentration. The highest increase in the shoot thickness was observed in the 600 mM NaCl treated shoots. As compared with the 0 mM NaCl treatment, there were 2.4-fold and 9-fold enhancements in the thicknesses of shoots treated with 300 mM and 600 mM NaCl, respectively (Table 2).
The salt treatments also increased the plant area. The highest plant area was detected in plants treated with 300 mM NaCl (Table 2). There was 5.4-fold and 2.7-fold enhancements in the plant areas treated with 300 mM and 600 mM NaCl, respectively (Table 2).
Effects of salinity on osmoprotectants
Soluble sugar and proline contents
Both total soluble sugar and proline contents increased by the all salt treatments compared to the non-stressed shoots in Scorzonera hieraciifolia. Moreover, the highest increases in the contents of proline (2.6-fold) and total soluble sugar (1.4-fold) were observed in the 600 mM salt treated seedlings in comparison with the 0 mM NaCl treatment. There were 1.2-fold and 1.3-fold increases in proline and total soluble sugar contents of the shoots treated with 300 mM NaCl, respectively (Fig. 2A, B).
Ion contents
K+ ion concentration increased at all NaCl concentrations in comparison with the non-stressed shoots. K+ content was high in the 150 and 300 mM NaCl treated shoots. Na+ and Cl- contents increased gradually with the enhanced NaCl concentrations as compared to 0 mM NaCl treatment. The highest Na+ and Cl- contents were detected in the shoots exposed to 600 mM NaCl. Ca2+ and Mg2+ ion contents significantly increased in all salt treatments as compared with non-saline condition. The highest increases in Ca2+ and Mg2+ contents were determined in the 600 mM NaCl treated shoots and the rate of increase of both Ca2+ and Mg2+ was recorded by 3.3-fold (Table 3).
Effects of salinity on antioxidant system-related signal compounds
ROS levels
Superoxide (O2-) level in the shoot of S. hieraciifolia increased in parallel with the increasing salt level. The highest superoxide level was detected in the shoots treated with 600 mM NaCl (Fig. 3).
The changes in H2O2 level in the shoot tissues of S. hieraciifolia was also determined with the observation of light brown by DAB staining. We observed that all salt treatments increased H2O2 level in comparison with the non-stressed shoots (Fig. 4).
Ascorbate and glutathione contents
The total ascorbate concentration no significantly changed in the shoots treated with 150 mM NaCl. However, the 300 mM NaCl and further salt concentrations (450 and 600 mM) significantly increased the total ascorbate content in comparison with 0 mM NaCl treatment. As compared to the 0 mM NaCl treatment, there were 2.2-fold and 8-fold increases in ASC content of the shoots treated with 300 mM and 600 mM NaCl, respectively (Fig. 5A).
Total glutathione content gradually enhanced while salt concentration was increasing in S. hieraciifolia. There were 3.4-fold and 10-fold increases in GSH content of the shoots treated with 300 mM and 600 mM NaCl in comparison with the 0 mM NaCl treatment respectively (Fig. 5B).
Effects of salinity on antioxidant enzyme activities
The activities SOD and APX significantly enhanced while NaCl concentrations were increasing. The highest increases in the activities of SOD and APX were recorded in the 600 mM NaCl treatment (Fig. 6 A). The catalase activity increased in the 150 and 300 mM NaCl treatments but it decreased in the 450 and 600 mM NaCl treatments (Figure 6 B). There was no significant change in GPOD activity in the shoots treated with 150 mM NaCl, while the activity increased in 300 mM, (1.7-fold), 450 mM (3.4-fold) and 600 mM (4-fold) NaCl treatments as compared to the 0 mM NaCl treatment (Figure 6 B). As for glutathione reductase, the activity increased under the all salt stress conditions. The rate of the increase was high (2.5-fold) in the high (600 mM) salt treatment (Fig. 6 B).
 
Effect of salinity on antioxidant enzyme contents
The increases in the SOD activity were consistent with increases in Fe-SOD contents in S. hieraciifolia. Fe-SOD content increased in the all salt treatments compared to 0 mM NaCl treatment and, Fe-SOD content was high in the shoots treated with 450 and 600 mM NaCl (Figure 7). The protein content of catalase distinctively enhanced in the shoots treated with 300 mM NaCl. Similar to the CAT activity, there was a decrease trend in the shoots exposed to 450 mM NaCl. Also, CAT content no significantly changed in the 150 and 600 mM salt treatments (Figure 6). Glutathione peroxidase content increased in all salt treatments in comparison with the non-stressed shoots. GPX content was especially high in the 300 mM NaCl and upper salt treatments (Fig. 7). GR content increased in the shoots treated with 150, 300 and 450 mM NaCl compared to the non-stressed shoots. There was a significant increase in the GR protein content in the 300 mM treatment in S. hieraciifolia (Fig. 7).
Discussion
We detected that S. hieraciifolia displayed optimal growth at the 300 mM NaCl concentration, as similar to Salvadora persica, stem-succulent extreme-halophyte, reported by Aghaleh et al. (2009). S. hieraciifolia was able to survive, even at the 600 mM NaCl concentration in spite of chlorosis. Likewise, Aghaleh et al. (2009) recorded that succulent halophyte, Salvadora persica was able to survive at 600 mM salt, which is a dose higher than the concentration of salt in seawater. T. parvula also displayed optimal growth at the 300 mM NaCl but it was unable to tolerate the 600 mM NaCl concentration in MS media. Accordingly, Uzilday et al. (2015) showed that T. parvula grown in a media including peat moss, vermiculite and soil mix could withstand 300 mM NaCl treatment. These results indicated that the maximum salt concentration that could be tolerated by halophytes may depend on the plant species, the presence of succulent structures, severity of the stress treatments and media exposed to the stress.
The relative water content increased in accordance with increased salt concentrations in S. hieraciifolia with fleshy shoots. Conversely, RWC value decreased with increasing salt concentration in T. parvula. Our data in T. parvula is consistent with previous reports that RWC and osmotic potential decreased in extreme halophyte, Salsola crassa (Yildiztugay et al. 2014). Increase in RWC indicated that succulent extreme-halophytes could dilute excess salt in their succulent structures and they could promote a mechanism of salt tolerance.
Amount of thiobarbituric acid reactive substances that is the product of lipid peroxidation, and ROS levels in plant tissues are important parameters indicating the stress damage of plants (Gupta and Huang 2014). Uzilday et al. (2015) reported no significant change in membrane damage in shoots of T. parvula grown in saline soil (300 mM NaCl). However, our findings obtained in MS media showed that TBARS content and H2O2 level increased by enhanced salt concentrations in T. parvula, and the rates of the increases in T. parvula were higher than those of S. hieraciifolia. Likewise, Uzilday et al. (2015) showed that H2O2 content significantly increased by the 300 mM NaCl treatment but no changed by low salt (50 and 200 mM NaCl) treatments in T. parvula. In the light of all this information on morphological observations, RWC, TBARS and H2O2 content we can say that S. hieraciifolia can be capable of tolerating high salt conditions and thus it can have characteristic properties of extreme-halophytes like T. parvula.
We also determined shoot succulence in S. hieraciifolia by measuring changes in the shoot succulence degree, shoot thickness and plant area under increased salinity conditions. We observed the increases in these parameters. Likewise, Parida et al. (2016) reported that succulence degree increased when salinity was increasing in extreme-halophyte, Salvadora persica. Increased shoot thickness and plant area due to increased salt concentration supported the idea that S. hieraciifolia displayed a succulent shoot structure under salt stress.
            Plants supply osmotic adjustment via two processes under high saline environment: ion accumulation in the vacuole and synthesis of compatible solutes in cytosol (Zhou and Yu 2009; Chen and Jiang 2010; Shabala and Mackay 2011). Our results showed that proline content and total soluble sugars significantly increased in S. hieraciifolia shoots at all levels of salinity. Increased level of organic osmolytes in response to salt stress has been reported in many succulent halophytes. For instance, proline content increased in succulent extreme-halophyte, Salvadora persica under NaCl stress conditions (Parida et al. 2016). Likewise, Liang et al. (2018) suggested that the soluble sugar content of Arabidopsis overexpressing salt-related wheat TaSST gene was significantly higher than that of wild-type and the transgenic plants resisted external salt stress by accumulating soluble sugars. The regulated accumulation of total soluble sugar and proline with signaling functions may contribute to the osmotic adjustment to continue water uptake in S. hieraciifolia under excess salinity.
            Our findings showed that K+ content increased in all salt treatments and the increase was high in the 150 and 300 mM NaCl treated shoots. Levels of Na+ and Cl- ions also increased parallel with the increase in NaCl concentrations in the shoots. Moreover, we observed 3.3-fold increases in both Ca2+ and Mg2+ contents of the 600 mM NaCl treated shoots (Table 3). The same increase rate pointed out that Mg2+ may have a secondary messenger function similar to Ca2+ and affect uptake of other inorganic ions under salt stress in plants. Magnesium also led to the increase in the osmotic potential in plant tissues to maintain water uptake from medium to the tissues (Ahmad et al. 2013). Therefore, in the present study, the possible signaling role of Mg2+ reported earlier in animals has been expressed for the first time in plants. Additionally, based on the increases in Na+, K+, Ca2+, Mg2+ and Cl- contents, we can say that S. hieraciifolia can provide osmotic adjustment by using inorganic ions and induce water uptake under excess salinity.
            Our results showed that increased salinity enhanced the amounts of superoxide and the H2O2 as a sign that the shoots were subjected to oxidative stress. The highest superoxide and H2O2 levels were detected in the 600 mM NaCl treated shoots. Uzilday et al. (2015) showed that H2O2 concentration of T. parvula treated with 300 mM NaCl was significantly promoted. It was also reported that ROS level increased in extreme halophyte Salsola crassa by Yildiztugay et al. (2014). However, moderated ROS levels in the plant tissues can act signaling role for the induction of antioxidant system under excess salinity.
Cellular antioxidant system through the activation of non-enzymatic or enzymatic antioxidants is crucial for the maintenance of cellular redox homeostasis required for salt adaptation or tolerance response in different plants (Gupta and Huang 2014). There were 2.2-fold and 8-fold increases in ASC content of the shoots treated with 300 mM and 600 mM NaCl, respectively. ASC contributes to ROS-scavenging and salt tolerance (Gupta and Huang 2014). We observed that like ASC, GSH content increased by salt stress and there were 3.4-fold and 10-fold increases in GSH content in the 300 mM and 600 mM NaCl treatments, respectively. GSH has been reported to play a protective role in salt stress tolerance by maintaining redox status and plants adapted to stress conditions had higher GSH levels (Gupta and Huang 2014). Coherently with our results, it was reported that total ASC and total GSH contents significantly increased in extreme halophyte, Salsola crassa, treated with 1000-1500 mM NaCl (Yildiztugay et al. 2014). Therefore, we can say that the antioxidant signal compounds, ASC and GSH could activate the signaling network to stimulate the antioxidant system responses to provide salt tolerance to S. hieraciifolia.
Antioxidant enzymes such as SOD activity was increased under salt stress conditions in S. hieraciifolia. There were 1.6-fold and 2.4-fold enhancements in SOD activity of the shoots treated with 300 mM and 600 mM NaCl, respectively. Increase in SOD activity was recorded in T. parvula during salt stress (Uzilday et al. 2015). CAT activity enhanced in the shoots treated with 150 mM and 300 mM NaCl although the activity declined under high salt stress conditions. Similar to our results, Parida and Jha (2010) reported that CAT activity decreased under high salinity (400 mM and 600 mM NaCl) in succulent extreme-halophyte Salicornia brachiate. Increased CAT activity under salt stress indicated that CAT can be effective in scavenging of H2O2 in S. hieraciifolia. Moreover, GPOD activity increased by 1.7-fold at 300 mM NaCl and 4-fold at 600 mM NaCl in Scorzonera hieraciifolia. The activity of GR playing an important role in controlling endogenous H2O2 content through a redox cycle containing glutathione and ascorbate (Gupta and Huang 2014), increased under salt stress in S. hieraciifolia. There were 1.3-fold and 2.5-fold enhancements in the GR activities in the 300 mM and 600 mM NaCl treatments, respectively. Also, APX activity showed significant stimulation in all treatments of salinity and, the activity increased by 1.3-fold and 1.5-fold at 300 mM and 600 mM NaCl, respectively. In the light of these findings, we can say that succulent extreme-halophytes can improve their salt tolerance capacity through the powerful antioxidant enzyme system.
We also detected here the changes in the contents of some antioxidant enzymes. As known, in higher plants, SOD is divided into three groups: manganese SOD (Mn-SOD), iron SOD (Fe-SOD), and copper/zinc SOD (Cu/Zn-SOD), according to the different metal ions, bound by its auxiliary sites (Wang et al. 2016). Fe-SOD and GPX protein contents increased in the 300 mM NaCl treatments in comparison with the un-stressed treatment and the highest increases were recorded in the 600 mM NaCl treatment. CAT content was also high in the 300 mM NaCl treatment. The results point out the fact that the antioxidant enzyme system act as a defense arsenal for building an adaptive mechanism under high salinity in succulent extreme-halophytes. Also, GR protein content enhanced in all salt treatments except 600 mM NaCl treatment. Previous studies have reported that GR has a protective role against salinity-induced oxidative damage in different plant species (Zeng et al. 2018; Parida and Jha 2010). High antioxidant enzyme activities and the protein contents in S. hieraciifolia exposed to excess salinity showed that the plant can effectively induce the antioxidant system to survive excess salt. Moreover, there could be essential protective roles of antioxidant enzyme activities in conjunction with their content in the scavenging processes in S. hieraciifolia.
Conclusion
Our results showed that succulent Scorzonera hieraciifolia with antioxidant and anti-inflammatory activity may be extremely salt tolerant that could survive at excess salinity (600 mM NaCl) conditions. S. hieraciifolia displays optimal growth at 300 mM NaCl in the MS media. S. hieraciifolia, can display shoot succulence by up-taking water effectively. The combined accumulations of signal compounds involved in osmoregulation such as proline, total soluble sugar, calcium and magnesium can provide the exceptional salt tolerance to S. hieraciifolia. Also, ASC can contribute activation of ROS and Ca2+ signaling under salt stress in succulent extreme-halophyte Scorzonera hieraciifolia. The modified accumulation of ASC, GSH and other signaling compounds may trigger the propagation of salt tolerance mechanism. Therefore, the exceptional salt tolerance of succulent extreme-halophyte S. hieraciifolia is achieved by regulated accumulation of combined signal compounds, involved in osmoregulation and induction of antioxidant system. Concomitant increases in antioxidant enzyme activities, antioxidant enzyme contents and the shoot succulence in the salinized plants showed that S. hieraciifolia may use the comprehensive strategy when coping with salinity. 
 
Acknowledgments
This work was supported by the Research Unit of Karadeniz Technical University (Project No: FDK-2018-7712). We also thank to Prof. Dr. İsmail TURKAN for providing us with the seeds of Thellungiella parvula.
Author contributions
CA performed the all experiments and wrote the manuscript; RT analyzed all data, read and edited the manuscript.
Conflicts of interest
The authors declare that they have no conflict of interest.
ORCID
Cansu Altuntaş 0000-0001-94994269
Rabiye TERZİ 0000-0002-9328-166X