**3. Results**

### *3.1. Effects of Salt Stress on Plant Growth*

3.1.1. Shoot Length and Lateral Branch Number

The results showed that *T. decumbens* growth response to NaCl was variable (Table 1). The shoot length and lateral branch number were significantly affected by salinity concentrations at *p* ≤ 0.05. The control had the highest shoot length, and this was significantly higher than 100 mM NaCl and 200 NaCl mM concentrations, respectively, but did not differ significantly from the 50 mM NaCl concentration. However, this was not the case with the lateral branch number, where the 50 mM NaCl concentration had the highest number of branches compared to the control.


**Table 1.** Effects of salt stress on growth parameters of *T. decumbens*.

Note. SL: shoot length; BN: branch number; FWS: fresh weight of shoots; DWS: dry weight of shoots; FWSR: fresh weight of stem and roots; DWSR: dry weight of stem and roots; TFW: total fresh weight; TDW: total dry weight. The values (mean ± SE) followed by dissimilar letters in each column are significantly different at *p* ≤ 0.05 (\*).

### 3.1.2. Fresh and Dry Weight of Shoots

Both fresh and dry weights of shoots significantly differed between treatments (Table 1). The highest fresh weight was obtained at 50 mM NaCl concentration. This was significantly higher than all NaCl concentrations, including the control. The lowest fresh weight was obtained at 200 mM NaCl concentration; however, this was not significantly different from the control. As for the dry weight, the highest mean value was again obtained at 50 mM NaCl concentration; this was significantly higher than all other treatments, including the control. The lowest dry weight was obtained at 200 mM NaCl concentration, and this was significantly lower compared to the other treatments, including the control.

### 3.1.3. Fresh and Dry Weight of Stem and Roots

NaCl concentrations positively influenced the fresh and dry weights of the stem and roots. The highest fresh weight of the stem and roots was obtained at 50 mM NaCl concentration. This was significantly higher than the control but did not differ significantly to 100 mM and 200 mM NaCl concentrations, respectively. The highest dry weight of the stem and roots was again recorded at 50 mM concentration. This was significantly higher than the control and 200 mM NaCl but did not differ significantly from the 100 mM NaCl concentration.

### 3.1.4. Total Fresh and Dry Weight

Experimental results also showed that NaCl concentrations significantly (*p* ≤ 0.05) affected the fresh and dry weight of dune spinach (Table 1). Plants exposed to different salt concentrations had fresh weights that were higher than the control. The highest measurement of fresh weight was obtained at 50 mM NaCl concentration, and this was significantly higher than all treatments, including the control. Although plants exposed to the 200 mM NaCl concentration had a higher fresh weight than the control, they did not differ significantly from each other. Conversely, this was not the case in total dry weight, where the 200 mM NaCl concentration recorded the lowest dry weight compared to all treatments, including the control. The highest total dry weight was again obtained at 50 mM NaCl concentration, and this was significantly higher than all other treatments, including the control.

### *3.2. Effect of Salt Stress on the Mineral Content of Dried Leaves of T. decumbens* 3.2.1.Macronutrients

Salinity stress significantly increased macronutrients (N, P, and Na) in the leaves of dune spinach. The highest N, P, and Na were all obtained from the highest salt concentration (200 mM). Conversely, the accumulation of K, Ca, and Mg significantly lowered in comparison to the control (Table 2).


**Table 2.** Effect of salt stress on the concentration of macronutrients in the leaves of *T. decumbens*.

Values (mean ± SE) followed by dissimilar letters in each column are significantly different at *p* ≤ 0.05 (\*); ns = not significant.

### 3.2.2. Micronutrients

Salt stress positively influenced the micronutrients (Mn, Fe, and Cu) in the leaves of dune spinach. The highest Mn, Fe, and Cu were all obtained from the moderate salt concentration (100 mM). However, the highest mean values in Mn and Cu were not significantly (*p* ≤ 0.05) different from all other treatments, including the control. However, the opposite was true for Fe. Conversely, salt stress negatively influenced Zn and B accumulation in the leaves. The control had the highest mean values in both Zn and B, and these were significantly different from all salt treatments (Table 3).


**Table 3.** Effect of salt stress on the concentration of micronutrients in the leaves of *T. decumbens*.

Values (mean ± SE) followed by dissimilar letters in a column are significantly different at *p* ≤ 0.05 (\*); ns = not significant.

### *3.3. Effect of Salt Stress on Chlorophyll Content*

As shown in Figure 1, the total chlorophyll contents were negatively affected by 50 mM, 100 mM, and 200 mM salinity concentrations during the fourth week of growth. However, plants exposed to lower salinity (50 mM NaCl) had the highest SPAD-502 values, which was significantly higher than the other treatments, including the control. During the sixth week, salinity concentrations positively affected the chlorophyll values and were significantly different from one another at *p* ≤ 0.05. The highest SPAD-502 values were obtained at 200 mM NaCl concentration followed by 100 mM, 50 mM, and the control. During the eighth week, chlorophyll values were negatively affected by salinity as all treatments, including the control, had lower chlorophyll values when compared to week 6. However, a higher concentration (200 mM NaCl) had the highest SPAD-502 value, but it was not significantly different from the other treatments except for the control at *p* ≤ 0.05. During the 10th week, salinity stress further reduced the chlorophyll values of all treatments except the control. The control had the highest mean value, which was significantly higher than all salt treatments.

**Figure 1.** The effect of NaCl concentrations on the chlorophyll readings of *T. decumbens* leaves. a–c indicate significant differences in mean values measuredwith Fisher's least significant difference. Bars with different letters in the same week are significantly different at *p* ≤ 0.05.

#### *3.4. Effects of Salt Stress on Phenolic Content and Antioxidant Capacity*

### 3.4.1. Polyphenol Content

The polyphenol content in the leaves of *T. decumbens* varied significantly at *p* ≤ 0.05 when different NaCl concentrations were compared with each other and with the control (Table 4). Plants exposed to the higher NaCl concentration (200 mM) had the highest polyphenol content (2.6 GAE/g DW) compared to all treatments, including the control. This was significantly different from the control and the 50 mM NaCl concentration but did not differ significantly from the moderate NaCl concentration (100 mM).

**Table 4.** Effect of salt stress on the phenolic content and antioxidant capacity of *T. decumbens* leaves.


Values (mean ± SE) followed by dissimilar letters in a column are significantly different at *p* ≤ 0.05 (\*); ns = not significant.

### 3.4.2. ABTS Capacity

Salt stress had a negative influence on ABTS capacity in the leaves of *T. decumbens*. The control had the highest ABTS capacity; however, this was not significantly different from all the treatments (Table 4).

### 3.4.3. FRAP Capacity

The total FRAP capacity in the leaves of *T. decumbens* was significantly influenced by the NaCl concentrations at *p* ≤ 0.05. The lower NaCl concentration (50 mM) had the highest FRAP capacity (14 μM AAE/g DW) compared to the other treatments, including the control. This was significantly higher than the control and 200 mM NaCl concentration but did not differ significantly from the 100 mM NaCl concentration (Table 4).

### **4. Discussion**

There is extensive literature on the reduction of growth caused by salt stress in many plants, which are facilitated by homeostatic transport of Na+ across intra- and inter-cellular cell boundaries predominated by NaCl [46,47]. Nevertheless, the effect of salt stress varies among plants. In the present study, increasing NaCl concentrations led to a significant decrease in plant height. Height reduction as a result of salinity stress has been reported in several plant species and has been mainly associated with the osmotic stress and ion toxicity that causes a reduction in plant growth [48–50]. However, when comparing the number of branches among the treatments, all plants irrigated with NaCl had more branches compared to the control. This might be caused by the natural adaptation of the species to saline environments, which enhances the ability of the species to remediate saline soil and stabilize the coastal dunes. The increase in branch numbers resulted in a higher total fresh weight. These findings agree with the findings of [51], where the halophyte *Ammophila arenaria* showed increased plant biomass in lower to moderate soil salinity. However, the results contradict those reported by [52], where the authors observed that after 12 weeks of cultivating some halophytes, such as *Inula crithmoides* L., *Plantago crassifolia* Forssk. and *Medicago marina* L., the plants whose irrigation water had not been spiked with salt showed better productivity and growth rates. The ability of dune spinach to withstand these varying salt concentrations could be attributed to osmotic, ion, and tissue tolerance. At high salt concentrations, the growth of dune spinach reduced drastically. This has been reported in numerous studies conducted on halophytes, where increasing salinity negatively affected plant growth performance, causing a reduction in biomass, leaf number, and plant height [47,53,54]. Reference [55] also reported that longer salt exposure in the

root zone restricts the flow of water and nutrients into the plant, causing direct injury to plant cells through the accumulation of toxic ions causing a decline in plant growth.

Salt stress has been reported as one of the major environmental factors affecting the nutritional value of many edible plants. In the present study, salt stress increased the uptake of N, P, and Na in the leaves, while K, Ca, and Mg were reduced drastically. The reduction of these elements may be directly linked to excessive Na+ absorption by the roots as reported by [56]. However, sufficient K, Ca, and Mg are required to meet basic metabolic processes such as intracellular K homeostasis, which is essential for optimal functioning of the photosynthetic machinery and maintenance of stomatal opening [57]. These results sugges<sup>t</sup> that dune spinach can transport K, Ca, and Mg to new shoots and leaves under salt stress and maintain a suitable ratio needed for normal metabolism; hence, the chlorophyll content was not affected for 8 weeks. This could be attributed to the water use efficiency and carbon fixing capacity of this species, which uses Crassulacean acid metabolism (CAM) to adapt to harsh conditions. Our results agree with those conducted by [58,59] on *Chenopodium quinoa* (genotype A7) and *Cichorium spinosum* in saline conditions, respectively, where it was reported that higher transport of K and Ca into new shoots and leaves contributed to mitigating ion toxicity in leaf cells.

Moreover, salinity stress also increased the Mn, Fe, and Cu contents in the leaves, while Zn and B were negatively affected. Similar findings were reported on the edible halophyte *Salicornia ramosissima* by [60]. These results indicate that salt stress caused Zn and B deficiency in the leaves of dune spinach, but since they are required in small quantities, visual symptoms of nutrient deficiency did not occur.

It has been reported in the literature that salinity stress damages nutrition and promotes senescence mechanisms in plants, thereby causing a reduction of chlorophyll content in the leaves [49,50]. However, the extent of reduction depends on the salt tolerance of the plant species [61]. Reference [62] reported that salt-tolerant species, such as *Thellungiella halophila*, indicated more or unchanged chlorophyll content when exposed to 0–500 mM NaCl, while salt-sensitive species (glycophyte), such as *Arabidopsis thaliana*, had lower chlorophyll content. In the present study, chlorophyll content was used as a biochemical marker to screen the salt tolerance of dune spinach. SPAD values (chlorophyll content) varied among treatments during the growing weeks, with salt treatments having higher SPAD values on week 6 and 8 when compared to the control (Figure 1). The findings of this study are in agreemen<sup>t</sup> with the results obtained by [63] in M-81E sweet sorghum (salt-tolerant genotype), where the chlorophyll content was not affected by 50 mM NaCl. In another study conducted by [64], spinach cultivar raccoon treated with saline irrigation water maintained SPAD chlorophyll levels but had a reduced photosynthetic rate, stomatal conductance, and transpiration rate.

Under salinity stress, the balance between reactive oxygen species production and activities of an antioxidative enzyme determines whether oxidative damage will occur [65]. To reduce the effects of oxidative stress, plants accumulate metabolites, such as phenolic compounds, which act as reducing agents, hydrogen donors, and singlet oxygen quenchers [49,66]. Moreover, phenolic compounds are of grea<sup>t</sup> interest due to the relevant role they play in the taste and flavor of food products, as well as their health-promoting properties [67,68]. In the present study, the total phenolic content was significantly increased by salinity levels, with more prominent content in plants irrigated with the highest NaCl concentration (200 mM). These findings validate that of [69], where an increase in the total phenolic content, antioxidant activity, and cyanidin-3- glucoside content was found in Khamdoisaket and KDML 105 Thai rice cultivars subjected to salinity stress. A similar trend was also reported by [70] on the effects of salinity on biochemical characteristics of the stock plant (*Matthiola incana* L.), where the phenolic content in severe salt-stressed plants of both cultivars was higher than the control. Reference [71] stated that phenolic compounds may be affected by salinity, but this critically depends on the salt sensitivity of a considered species. The results of the study prove that dune spinach can grow under severe salinity

concentrations and could be considered as an alternative source of nutritional antioxidant in areas with higher and problematic saline soils.

Contrary to the increase in phenolic content, the antioxidant activity (ABTS) in the leaves of *T. decumbens* exposed to various salinity concentrations showed a much weaker antioxidant capacity compared to the control. This contradicts the findings of [16] on edible flowers, where three antioxidant assays (FRAP, DPPH, and ABTS) increased with the application of salinity, with a more pronounced impact at a salinity of 100 mM NaCl. Furthermore, these results also substantiate that of [72] on a traditional Chinese herb, where a much weaker antioxidant capacity was found with increasing NaCl concentrations at 50 mM and 100 mM, respectively, compared to the control. Conversely, the FRAP capacity was positively influenced by salt stress with the strongest antioxidant capacity obtained at 50 mM NaCl. A similar trend was reported earlier by [16] in edible flowers, where the application of salinity enhanced antioxidant activities.
