*2.7. Statistical Analysis*

One-way ANOVA with Tukey's post hoc comparison was applied to estimate the significance of the differences in (1) germination and growth parameters, (2) the activity of antioxidant enzymes and concentrations of proline in the roots, stem, and leaves, and (3) the salinity stress duration. A principal component analysis (PCA) was used to determine the correlation pattern between traits, and then Pearson's correlation coefficients were used for the correlation assessment. The statistical significance between treatments was agreed at the *p* < 0.05 level. For calculations, Statistica version 8.0 [37] and Canoco 5.0 [38] packages were used.

#### **3. Results**

### *3.1. Effect of Salinity on Two Growth Stages*

Salinity affected similarly on the germination and late-growth parameters of *T. pannonicum*. The differences between treatments were significant in all measured parameters except FMR (Tables 1 and S1). Salinity significantly reduced GP, GI, and GE and increased MGT. Germination was highest at 0 mM NaCl, and lowest at 800 mM NaCl (Table 1). The MGT was highest at 800 mM NaCl, but in the control, 200 mM, and 400 mM NaCl, we observed no statistically significant differences. Salinity also significantly reduced the parameters of late growth: SL, RL, FMT, and FMS. The stem was longest in the control (31.3 cm), and the roots were longest in the control and 200 mM NaCl (ca 8.3 cm). FMT was highest in the control and 200 mM NaCl (ca 24 g) and lowest at 400 and 800 mM NaCl (ca 20 g). We observed no significant difference in FMS and No.LR between the control and 200 mM and between 400 mM and 800 mM NaCl (Table 1). FMS was lowest at 400 and 800 mM NaCl (18 g and 16.7 g, respectively). FMR was not significantly affected by salinity.


**Table 1.** Effect of NaCl on the germination and growth parameters of *T. pannonicum*.

Average values (*n* = 3) with standard error (SE) are given. GP = germination percentage; GI = germination index; MGT = mean germination time; GE = germination energy; SL = shoot length; RL = root length; FMT = total fresh mass; FMS = shoot fresh mass; FMR = root fresh mass; No.LR = number of leaves in a rosette. Differences between groups based on Tukey's range test are marked by different letters and are significantly different at *p* ≤ 0.05.

#### *3.2. Effect of Salinity on Organ Stress Responses*

Salinity significantly affected the activity of antioxidant enzymes (APX and POD) and concentrations of salinity stress indicators (hydrogen peroxide and proline) in the roots, stems, and leaves of *T. pannonicum* (Table S2). The main effects of salinity on the organs, and the interaction effects between them, were significant for all measured parameters (Table 2). In all organs, salinity increased APX and POD activity. The activity of APX and POD was highest at 800 mM NaCl in all analyzed plant organs (Figure 1). In addition, the zymogram analysis indicated that CAT enzyme activity was also highest at 800 mM NaCl (Figure 2).

**Table 2.** Analysis of variance (mean squares) for the activity of antioxidant enzymes and salinity stress indicators in two organs (O) of *T. pannonicum* and four salt concentrations (S).


Average values (*n* = 3) with errors within group variance are given. APX = ascorbate peroxidase activity; POD = peroxidase activity; H2O2 = H2O2 concentration; P = proline concentration; df = degrees of freedom; \*\* = *p* ≤ 0.01.

**Figure 1.** Effect of NaCl on the activity of antioxidant enzymes—(**a**) APX and (**b**) POD, and salinity stress indicators—(**c**) H2O2 and (**d**) proline in the roots, stems, and leaves of *T. pannonicum*. Differences between groups based on Tukey's range test are marked by different letters and are significantly different at *p* ≤ 0.05. One unit of the enzyme activity was defined as the amount of enzyme causing a 0.001 change in absorbance per minute. Average values with standard error are given *n* = 3.

**Figure 2.** Zymogram of CAT enzyme activity in the roots (R), stem (S), and leaves (L) after 0 (control), 200, 400, and 800 mM NaCl treatment. Bands of catalase activity were marked as clear bands and their intensity corresponded with the activity of CAT (shown by the arrow) between organs.

Of all organs, the activity of APX and POD was lowest for the stem in the control (Figure 1a,b). The lowest activity of APX in the root and leaves was observed at 200 mM NaCl. The activity of POD for all organs was lowest in the control. We observed no significant difference in POD activity in the leaves in 200 and 400 mM NaCl (Figure 1b). The highest intensity of the bands in the CAT zymogram was observed for the roots after the 800 mM NaCl treatment. In addition, we detected one isoform of catalase in all NaCl treatments (Figure 2). The highest concentrations of H2O2 and proline were observed at 800 mM NaCl in all analyzed organs (Figure 1c,d). The lowest concentration of H2O2 between all organs was noticed for the stem in the control (18.2 μM). The lowest H2O2 concentration in the root and leaves was observed in 200 mM NaCl (Figure 1c). The

concentration of H2O2 in the root seems to be independent of the increasing salinity because no significant difference was observed between NaCl treatments. The amount of proline for all organs was lowest in the control (11.4 μg/mL for the root, 21.8 μg/mL for the stem, and 9.49 μg/mL for the leaves). For all organs, proline concentration increased with the growing salinity and was greatest in the leaves (Figure 1d).

#### *3.3. Effect of the Duration of Salinity on the T. pannonicum Stress Responses*

Based on the above result of our experiment, we investigated the effect of long- and short-term salt exposure on the enzyme activity, and the concentration of hydrogen peroxide and proline in the leaves as the main organ affected by salinity and the organ responsible for salt extrusion by the shedding of rosette old leaves saturated by salt [39]. Because the most significant effect of the salinity was obtained for 800 mM NaCl, we selected this concentration of salt for further analysis.

The duration of salinity exposure significantly affected the activity of antioxidant enzymes and salinity stress indicator concentrations (Table S3). We observed that salinity acts on analyzed parameters over different time scales (Figure 3). The activities of both APX and POD enzymes were similar for each analyzed timeframe, however, significant differences in the APX activity, between 5 h and 48 h, were observed (Figure 3a). The highest activity of APX and POD was observed 1 h after NaCl application (short-term salt stress) (26.2 U·mg−<sup>1</sup> for APX and 26.7 U·mg−<sup>1</sup> for POD) and 48 h (long-term salt stress) (32.3 U·mg−<sup>1</sup> for APX and 27.6 U·mg−<sup>1</sup> for POD). The highest concentrations of H2O2 and proline were at 48 h and in 5 days after NaCl application, respectively (Figure 3b,c). There were no significant differences in the salinity stress indicator concentrations in short-term salt stress (1 h, 3 h, and 5 h), nor in long-term salt stress (5 days and 7 days).

**Figure 3.** Effect of the duration of salinity exposure on the activity of antioxidant enzymes—(**a**) APX and POD, and salinity stress indicators—(**b**) H2O2, and (**c**) proline, in the leaves of *T. pannonicum*. Differences between groups based on Tukey's range test are marked by different letters and are significantly different at *p* ≤ 0.05. One unit of the enzyme activity was defined as the amount of enzyme causing a 0.001 change in absorbance per minute. Average values with standard error are given *n* = 3.

#### *3.4. Patterns of Correlation between Growth Stage, Organs, and Duration of Salinity Exposure*

All of the variables (germination and growth parameters, antioxidant enzymes, and salinity stress indicators in organs) were evaluated in the NaCl concentration, while biochemical parameters were also evaluated in the time scale using PCA. The response to salinity was dependent on the growth stage, organs, and duration of salinity stress, where the first ordination axis represents the salinity gradient (Figure 4a–c).

**Figure 4.** Result of the PCA analysis between: (**a**) salinity treatments and germination and growth parameters; (**b**) salinity treatments and stress responses in different organs; (**c**) duration of salinity exposure and stress responses in the leaves. Abbreviations as in Tables 1 and S2. The concentrations of NaCl (**a**,**b**) and duration of NaCl stress (**c**) are marked by red points.

We noticed a strong and statistically significant positive correlation between POD in the roots and the concentration of H2O2 in the stem (Pearson's r = 0.864) and a very strong positive correlation between APX in the roots and the concentration of H2O2 in the leaves (Pearson's r = 0.961). The concentration of proline was also correlated significantly with the activity of antioxidant enzymes, especially with APX activity. A very strong positive correlation was observed for the proline concentration in the stems and APX activity in the roots (Pearson's r = 0.990) and for the proline concentration in the leaves and APX activity

in the roots (Pearson's r = 0.985). The concentration of salinity stress indicators (H2O2 and proline) was positively correlated between organs. The strongest significant correlation was found for the concentration of proline in the stem and H2O2 in the leaves (Pearson's r = 0.981) (Figure 4b, Table S5). The activity of antioxidant enzymes and concentration of salinity stress indicators were strongly correlated with the type of salinity response early up to 5 h and late from 24 h, represented by the first PCA axis explaining ca 81% of the traits' variance (Figure 4c). The differentiation in the late response, represented by the second PCA axis, explained ca 18%. All analyzed parameters were positively correlated with the duration of salinity stress (Figure 4c, Table S6). The activities of APX and POD were significantly and very strongly correlated with each other (Pearson's r = 0.984). The positive correlation was high between APX and H2O2 (Pearson's r = 0.666), and moderate between POD and H2O2 (r = 0.532). The correlation between two salinity stress indicators (proline and H2O2) was also significant and moderate (r = 0.385; Table S6).

#### **4. Discussion**

#### *4.1. Salinity Affects Germination and Late Growth of T. pannonicum*

Germination is a crucial part of the plants' growth, but more so for halophytes, and salt tolerance usually varied between halophytes [40]. To survive in a saline habitat, halophytes required successful seed germination [41]. Most parameters of germination and growth were affected by salinity Tables 1 and S2). These results are in line with our previous studies on glycophytic species, e.g., fodder beet [42], sorghum [26], maize, millet, and oat [2]. In our study, the effective *T. pannonicum* seed germination was achieved in the control (0 mM NaCl) although the model plant is an obligatory halophyte. The negative effect of salinity was also visible in the reduction of the germination energy (Table 1), because reducing the osmotic potential of the solution inhibits the imbibition of water by the seeds. MGT parameter (determining the time for the seed to germinate) was almost the same in the control, 200 mM and 400 mM NaCl (Table 1) which can be an example of a *T. pannonicum*'s seed strategy for efficient germination even under high soil salinity. Salinity had a significant effect on germination time (Table 1), as in a study of *P. sativum* and *L. sativus* [14]. The higher NaCl concentrations lengthen the germination time until the seeds develop a tolerance and start to germinate. Faster and early germination under lower salinity confers an ecological advantage upon halophyte seedlings [40].

Our study has shown that salinity strongly reduces the growth parameters of threemonth-old plants, and maximum growth is obtained under non-saline and low-saline conditions (Table 1) as in a study by Geissler et al., 2009 [43], not all plant growth parameters were affected by salinity at the same level. The best growth parameters were observed in the control and 200 mM NaCl, which indicates the optimal concentration of NaCl for growth success. In addition, the total biomass of *T. pannonicum* was similar in 400 mM and 800 mM NaCl, however lower than in the control and 200 mM NaCl, indicating effective adaptation to higher levels of salinity. High variability of salinity in the *T. pannonicum* wet habitats promotes a broad spectrum of NaCl tolerance which was observed also by Ievinsh et al. [7]. They found *T. pannonicum* also in habitats with low salinity [7]. In addition, Karlsons et al. 2008 [44] demonstrated a higher decrease in the roots and leaves biomass of this species at 400 mM NaCl, compared with our studies. However, the adaptations to environmental stress can evolve within populations of the same species [45] and can be genetically established within a population as the result of local adaptation.

#### *4.2. Response to Salinity Depends on the Organs of T. pannonicum*

The result of our study demonstrated that the halophyte's organs do not simply tolerate high-salt conditions (Tables 2 and S2). All biochemical parameters (activity of APX and POD, concentration of H2O2 and proline) in the analyzed organs were affected by salinity. The higher salt tolerance of numerous halophytes is related to proper ROS homeostasis by the activation of their antioxidant systems under salt stress [22]. The highest activities of APX, POD, and CAT were observed at 800 mM NaCl in all analyzed plant organs and were greatest for the roots (Figures 1a,b and 2). The activities of APX and POD observed at 400 mM and 800 mM NaCl were varied between organs and revealed the following activity pattern: root > leaves > stem (Figure 1). Increased antioxidant activity has been observed in several salt-tolerant plants, indicating that antioxidants are an important factor in the salt stress response [46]. Enzymatic oxidative stress defenses to high salt concentration are more generally in the roots (such as CAT or APX activation), while root-specific antioxidant enzymes (i.e., SOD, and MDHAR) have also been found [47].

As shown in our study, the highest concentration of salinity stress indicators (H2O2 and proline) was observed at 800 mM NaCl in all analyzed organs (Figure 1c,d). Salt stress also generated oxidative stress in halophytes of the genus *Juncus* [48]. The highest efficiency of the antioxidant system in the root was the reduced H2O2 concentration in this organ which indicated that ROS scavenging mechanism is most effective in the roots, compared to the stems and leaves. An interesting observation is that proline was preferentially accumulated in the leaves (Figure 1d), however, in increasing NaCl concentrations, we noticed proline accumulation in all organs. Our results indicated that, in the context of proline accumulation, leaves are more salt-tolerant than other organs. Organ-specific accumulation of proline in the leaves of lupine and halophyte *K. virginica* was also observed by Rady et al., 2016 [18] and Wang et al., 2015 [49] and H2O2 in the leaves of rice [50].

#### *4.3. Response to Salinity Depends on the Duration of Salinity Stress*

Studies of the effects of salinity on halophytes on a wide time scale (in short-time and long timescale) are significant to fully understand the adaptation strategy and plant interaction with the environment. The response of *T. pannonicum* to salinity stress was two stages to enable more efficient and comprehensive adaptiveness (Figure 3a–c). The H2O2 concentration peaked at 48 h (long-term salt stress), which corresponds with the increased activity of APX and POD, the key enzymes responsible for H2O2 scavenging during salinity stress in plants. A significant result is that the activity of both investigated peroxidases is synergistic (Figure 3a). It seems reasonable to cope with NaCl stress "with redoubled strength". It is also evidence of an early- and late-cellular stress response, which was for the first time demonstrated for *T. pannonicum*. Similar onset/reinforcement of antioxidant systems were also observed for groundnut [12] and soybean [51] under salt stress. For halophyte *S. aralocaspica*, adaptation to the external salinity changes for a period of 24–48 h has been reported [52]. Hernández et al., 2000 [53] noticed that the induction of antioxidant defence is at least one component of the tolerance mechanism of *Pisum sativum* L. to long-term salt stress. According to Fraire-Velazquez and Emmanuel [17], the observed initial, fast response to salinity stress is temporary and thus separated clearly from the pathological consequences of exposure to the same level of salinity over a longer period, which is catastrophic for non-adapted plants.

The highest concentrations of H2O2 (162.1 μM) and proline (166.08 μg/mL) were recorded at 48 h and 5 days after salinity treatment, respectively. Huang et al., 2013 [20] observed the same dependencies in *H. tuberosus* during the initial 72-h period. Compared with enzyme activity, the increase in the concentration of salinity stress indicators was a part of the late stress response (Figure 3b,c) therefore these compounds may be more responsible for the long-term adaptation process of the plant to the extreme environment than antioxidant enzymes, as in the study of Naliwajski and Skłodowska 2021 [54]. The action of H2O2 and proline is also synergistic to intensify the defense mechanisms against NaCl stress. Studies by Huang et al. 2013 [20] demonstrated that the gene expression of key enzymes in the proline biosynthetic pathway changed significantly in roots of *H. tuberosus* after 4 h treatment, which may be responsible for the increase in the concentration of proline observed after 5 h of salinity treatment in our study (Figure 3b).

#### *4.4. Salt-Tolerance and Salt-Adaptation of T. pannonicum*

We indicated that germination success determines the future plant biomass (FMT) and biomass of shoots (FMS) (Table S4). Bayuelo-Jiménez et al., 2002 [15] observed that faster germination allowed emerging seedlings to obtain a higher biomass. The strong negative correlation observed in our study for RL and MGT (Table S4) corresponds with the findings by Robin et al., 2016 [55]. Salinity induced a reduction in the surface area of the root and changes in the main root in wheat [55], which may explain the extended mean germination time of *T. pannonicum* seeds under salinity. Although *T. pannonicum* is a halophyte, saline conditions at the germination stage will be defined as the further adaptation success of this plant. Low soil salinity under the first step of plant growth allowed this plant to obtain a better biomass and overcome interspecific competition in the environment. A PCA analysis indicated a correlation between the activity of antioxidant enzymes and proline concentrations in the investigated organs under NaCl stress, which can be essential for *T. pannonicum* adaptation (Figure 4b, Table S5). The early cellular stress response observed after 1 h and 5 h of NaCl exposure (Figures 3a–c and 4c) can help the plant to restore its performance more quickly and is essential to stress neutralization and further plant survival [17]. Then, the changes in enzyme activities affecting the concentrations of salinity stress indicators (H2O2 and proline) were part of the late cellular stress response observed 24 h after salinity stress (Figure 3a–c). The first line of defense against ROS caused by oxidative stress comprises antioxidant enzymes, so they are activated more quickly than the production/accumulation of proline [56]. The growth response to salinity stress can be divided into two steps: a fast reaction to the increase of the external osmotic pressure, and a slower response as a result of the accumulation of Na+ in plant organs, which was documented by Munns and Tester 2008 [57]. The results of our studies strongly indicate that the levels of stress indicators and the activity of antioxidant enzymes contribute to the tolerance and adaptation of *T. pannonicum* to salinity stress.

The high salt tolerance was noticed for the germination and late growth of *T. pannonicum* results from its natural growth in very stressful and variable habitats. *T. pannonicum* is part of inland and coastal plant communities [8,9,23]. The ecological habitats of sea aster are extremely unfavorable: inland saline meadows flooded in the spring after snowmelt and dried in the summer, or coastal seashores with sea tides. This species in anthropogenic areas can be also exposed to salty waste from the soda or potassium industry [58]. These difficult growth conditions increase the adaptability of *T. pannonicum.* Investigated salt-adaptation traits, such as a high total biomass under salinity, the high and organ-specific activities of APX, POD, and CAT, and the highest efficiency of the antioxidant system in the root with a leaves-specific accumulation of proline, are examples of plant answers for the high variability of the habitats where they grow.

#### **5. Conclusions**

*T. pannonicum* is a member of a diverse group of halophytes that seems to be a promising cash crop to desalinize and reclaim degraded land. However, some basic physiological, biochemical, and molecular mechanisms in the adaptive processes of *T. pannonicum,* such as salt tolerance, are still not well recognized. Our study and recent genetic and omic experiments have indicated that salt tolerance is a complex mechanism that depends on the growth phase, organs, and duration of salinity exposure. The antioxidant system of *T. pannonicum* was very active at 800 mM NaCl and APX. POD, and CAT activity were greatest for the roots. The demonstrated different responses of the organs to NaCl application have significant potential to further proteomic and metabolomic analyses of halophyte adaptions. The time-dependent regulation of adaptive processes involved in the tolerance of high and extended salinity, but also shorter episodes of salinity, also requires a deeper explanation. A better understanding of the adaptabilities of *T. pannonicum* to high salinity could be helpful in the restoration of the fragmented aster population and studies of these plants' application as energy crops for cultivation on saline lands or as cash crop vegetables.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/life13020462/s1, Table S1: Analysis of variance (mean squares) for the germination and the growth parameters of *T. pannonicum* in four salt concentrations, Table S2: Analysis of variance (mean squares) for the activity of antioxidant enzymes (APX and POD) and salinity stress indicators

(H2O2 and proline) of *T. pannonicum* in four salt concentrations, Table S3: Analysis of variance (mean squares) for the activity of antioxidant enzymes (APX and POD) and salinity stress indicators (H2O2 and proline) of *T. pannonicum* in eight different times of measurements, Table S4: Correlation coefficient matrix between germination and growth parameters under NaCl stress, Table S5: Correlation coefficient matrix between antioxidant enzymes and salinity stress indicators under NaCl stress, Table S6: Correlation coefficient matrix between antioxidant enzymes and salinity stress indicators under the different times of the duration of salinity stress.

**Author Contributions:** Conducted experiments and data interpretation, wrote the manuscript, A.L.; supported biochemical analysis, A.C.; supported statistical analysis, A.R.D.; supported timedependent experiments, S.C.-P.; supported data interpretation, A.P.; reviewed & edited the manuscript, A.C., A.R.D., S.C.-P. and A.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by funds for the science from Nicolaus Copernicus University in Toru ´n, Poland, and IDUB Emerging Field Ecology & Biodiversity.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets generated during the current study are available from the corresponding author upon reasonable request.

**Acknowledgments:** Thanks are extended to Katarzyna Roszek (Department of Biochemistry, NCU) for technical and equipment support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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