Table 1 shows the contents of fatty acids in the leaves of 90 days old plant under salineinduced stress. The contents of myristic acid (C14:0), palmitic acid(C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidic acid (C20:0), and behenic acid (C22:0) were affected differently for the different treatments. The contents of C14:0, C16:0, and C18:0 were not significantly altered for different treatments compared to S0. With increasing salinity, the content of unsaturated fatty acids (C16:1, C18:1, C18:2, and C18:3) was observed to decrease for control (S1, S2, S3), biochar (BS1, BS2, BS3), and AMF (AS1, AS2, AS3) treatments. Compared to S0, the contents of C16:1, C18:1, C18:2, C18:3 was significantly (*p* < 0.05) decreased by 44.02%, 33.49%, 25.99%, and 0.53% in S3 treatment, respectively. Nevertheless, the contents of C20:0 (52.82%) and C22:0 (87.77%) were increased significantly (*p* < 0.05) improved for the same

treatment. Also, relative to S3 treatment, BS3 treatment positively impacted the contents of C16:1, C18:2, C20:0, and C22:0while negatively affecting C18:1 and C18:3 (Table 1). For AS3 treatment, the contents of C16:1, C18:1, C20:0, and C22:0 were significantly decreased while C18:2 and C18:3 were only slightly increased. The combined application of biochar and AMF (ABS3) demonstrated the most significant positive effect on the contents of all fatty compared to S0 while C22:0 was negatively affected relative to S3. Specifically, the contents of C16:1, C18:1, C18:2, C18:3 and C20:0 were increased by 161.36%, 101.3%, 65.07%, 12.17%, 18.23% relative to S3 or 46.31%, 33.89%, 22.17%, 11.58%, 88.31%, and 56.46% relative to S0.

#### *2.2. Influence of AMF and Biochar on Soil Nutrient Content*

The following acronyms will be used as defined in the materials and method section (Table 2): S0, S1, S2 and S3 (0, 50, 100, and 150 Mm NaCl treatment, respectively); BS0, BS1, BS2 and BS3 (biochar + 0, 50, 100, and 150 Mm NaCl treatment, respectively); AS0, AS1, AS2 and AS3 (AMF + 0, 50, 100, and 150 Mm NaCl treatment, respectively); ABS0, ABS1, ABS2 and ABS3 (biochar + AMF + 0, 50, 100, and 150 Mm NaCl treatment, respectively). Figure 1 gives a summary of the selected physicochemical properties of the soil after the growth of maize. After maize growth, the soil pH was decreased in the S0, S1, BS0, AS0, ABS0, ABS1 treatments by 0.42, 0.03, 0.48, 0.38, 0.44, 0.38 units while S2, S3, and A3 had an increase of 0.06, 0.20, and 0.05 units compared to the original soil (pH 8.25), respectively (Figure 1A). Compared to the S0 treatment, only the BS0 and ABS0 treatments experienced a slight decrease in pH. For different treatments, the content of potassium was either increased or decreased with an increment in the concentration of NaCl (Figure 1B). For instance, under no amendment, the content of K was decreased by 3.83%, 23.76%, and 23.8% when the concentration of NaCl was 50 (S1), 100 (S2) and 150 mM (S3), respectively. This decrement was also observed for AS0, AS1, AS2, AS3, and ABS3 with values lowered by 24.68%, 20.72%, 8.82%, 11.64%, and 21.37%, respectively. This observation shows that increase in the concentration of Na+ ions has a negative impact on available K+ ions needed for plant growth, which has been sighted as a major concern for saline soils [17]. Nevertheless, amending the alkaline soil with biochar (BS0, BS1, BS2, BS3) and biochar + AMF (ABS0, ABS1, and ABS2) significantly (*p* < 0.05) improved the content of K; with the largest increment recorded for BS3 (32.49%), ABS0 (24.62%), ABS1 and (23.31%) relative to S0. When compared with the saline-treated control (S1, S2, S3), biochar (BS1, BS2, BS3), AMF (A1, A3, except A1), and biochar + AMF (ABS1, ABS2, ABS3) treatments significantly (*p* < 0.05) improved K in soil. Thus, biochar amendment with/without AMF can effectively mitigate K+ ion loss in high pH saline soils. Phosphorus and nitrogen are important nutrient requirements for plant growth which can become deficient when the soil health and fertility are threatened. For the control (S0, S1, S2, and S3) and AMF (AS0, AS1, AS2, and AS3) treatments, the content of P was decreased as the concentration of NaCl was increased (Figure 1C). Relative to the S0 treatment, the content of P was increased by 22.58% for B0 but was decreased by 38.45% and 30.41% for A0 and AB0 treatments, respectively. Also, relative to the saline (S1, S2, S3) treatments, biochar (BS1, BS2, BS3), AMF (AS1, AS2, AS3), and biochar + AMF (ABS1, ABS2, ABS3) amendments showed contrasting effects on available P. For instance, the available P was increased by 21.43%, 12.78%, 11.97%, 4.23%, 7.43%, 46.43%, 18.94% for BS1, BS2, BS3, ABS1, ABS2, ABS3, and AS1, respectively, but decreased for AS2 and AS3 treatments. From this result, it is evident that salinity negatively impacts available P and the individual or co-application of biochar with AMF can mitigate this negative effect.

*Plants* **2021**, *10*, 2490

**Table 1.** Fatty acid composition in lipids of the maize plant.

Data are presented as the means±

standard deviation of three replicates and mean values followed by different letters are

significantly

 different at *p* < 0.05 according to Duncan multiple range test.

**Table 2.** Treatment arrangement.

From Figure 1, it can be observed that the content of N significantly decreases in the control treatment as the saline content was increased from 0–150 mM; except for S1 treatment (Figure 1D). For instance, compared to the S0 treatment, N was increased by 26.4 mg kg−1 and decreased by 47.3 and 74.9 mg kg−1 when the soil was treated with 50 (S1), 100 (S2), and 150 Mm (S3) of NaCl, respectively. The application of biochar alone (BS0, BS1, BS2, BS3) and in combination with AMF (ABS0, ABS1, ABS2, ABS3) significantly improved the content of N under increasing salt stress. For no salt treatments (BS0, AS0, and ABS0), N content was improved by up to 74.25%, 16.61%, and 90.89% for BS0, AS0, and ABS0 amendments relative to S0 treatment, respectively. Under increasing salt stress, N content was improved progressively when biochar and/or AMF were applied. Specifically, for BS1, BS2, and BS3 treatments, the amount of N (relative to S1, S2, and S3) increased by 37.3%, 88.91%, and 105.6% (or 50.25, 57, and 50.57% relative to S0), respectively. Similarly, the recorded increment in the content of N for ABS1, ABS2, and ABS3 was 50.88%, 95.79%, and 102.44% (or 65.11, 62.71, and 48.29% relative to S0), respectively. Additionally, applying AMF alone did not mitigate N loss (−18.05%) when 50 mM NaCl was added but showed a positive effect at saline concentrations of 100 (8.25%) and 150 mM (26.08%). Thus, amending alkaline soils with biochar, biochar + AMF, and AMF alone can mitigate the negative effect of Na+ ions on N content, with biochar and biochar + AMF exhibiting the most significant effects at 100 and 150 mM of Na+ ions.

**Figure 1.** Influence of *Arbuscular mycorrhizal* fungi (AMF) and biochar on (**A**) soil pH, (**B**) exchangeable K, (**C**) available P, and (**D**) total N after harvesting. Data present the mean ± standard deviation of three replicates and mean values followed by different letters are significantly different at *p* < 0.05 according to Duncan multiple range test. S0, S1, S2, and S3 (0, 50, 100, and 150 Mm NaCl treatment, respectively); BS0, BS1, BS2 and BS3 (biochar + 0, 50, 100, and 150 Mm NaCl treatment, respectively); AS0, AS1, AS2 and AS3 (AMF + 0, 50, 100, and 150 Mm NaCl treatment, respectively); ABS0, ABS1, ABS2 and ABS3 (biochar + AMF + 0, 50, 100, and 150 Mm NaCl treatment, respectively).

#### *2.3. The Effects of Salinity, Biochar, and AMF on Plant Growth Parameters*

Plant growth parameters were evaluated based on the maize height after 45 and 90 days of growth, total fresh weight, and the total number of leaves after 90 days (Figures 2 and 3). The plant height after 45 days of growth was 2.73%, 16.02%, and 4.92% shorter compared to S0 when the saline concentration was 50 (S1), 100 (S2), and 150 mM (S3), respectively (Figure 2A). After 90 days of growth for the same treatments (Figure 2B), the plant height was 6.22%, 17.84%, and 17.3% shorter, respectively. When the soil was amended with biochar and/or AMF without NaCl, the plant height was increased by 23.67%, 28.67%, 2.11% after 45 days and by 21.08%, 18.65%, 7.3% after 90 days for BS0, ABS0, and AS0 compared to S0, respectively. Under increasing saline stress, biochar enhanced plant growth by 19.68% (BS1), 24.19% (BS2), 14.38% (BS3) after 45 days and by 24.21% (BS1), 26.97% (BS2), 13.4% (BS3) after 90 days when compared to S1, S2, and S3, respectively. This significant (*p* < 0.05) increment in the plant height was also recorded when the soil was amended with biochar + AMF (ABS1, ABS2, and ABS3). Comparatively, the biochar treatments demonstrated a significant effect in improving the plant's height compared to single AMF treatment; with biochar + AMF being a better option.

**Figure 2.** Impact of AMF and biochar on the height of corn plants under different levels of salinity after 45 (**A**) and 90 (**B**) days of growth. Data present the mean ± standard deviation of three replicates and mean values followed by different letters are significantly different at *p* < 0.05 according to Duncan multiple range test. The significance of acronyms is the same as in Figure 1.

**Figure 3.** Impact of AMF and biochar on plant fresh weight (**A**), number of healthy leaves (**B**), and dry weight (**C**) under different concentrations of salinity. Data present the mean ± standard deviation of three replicates and mean values followed by different letters are significantly different at *p* < 0.05 according to Duncan multiple range test. The significance of acronyms is the same as in Figure 1.

Figure 3 shows the difference in the total plant fresh weight (Figure 3A), the number of leaves (Figure 3B), and the total plant dry weight (Figure 3C). As shown, increasing the concentration of NaCl negatively affects the total plant fresh weight (Figure 3A). Increasing the NaCl concentration by 50 (S1), 100 (S2), and 150 mM (S3) significantly decreased the plant's fresh weight by 31.62%, 39.28%, and 32.28% relative to S0, respectively. When no

salt was applied to the soil, biochar and AMF amendments improved the plant's fresh weight by 50.21%, 54.28%, and 34.4% for BS0, ABS0, and AS0, respectively. Compared to the saline treatments (S1, S2, and S3), biochar amendments improved the plant's fresh weight by 74.84%, 62.36%, 71.14% for BS1, BS2, BS3 and 108.5%, 107.7%, 67.48% for ABS1, ABS2, and ABS3, respectively. For the treatments containing only AMF, the plant fresh weight was increased for AS1 (51.49%) and AS2 (24.76%) but was decreased as the salt content was increased to 150 mM for AS3 (−14.91%). Compared to the S0 treatment, all the treatments with biochar induced a significant improvement in plant fresh weight and demonstrates the individual ability of biochar or when combined with AMF to alleviate saline-related stress on plant growth.

The mean number of healthy leaves decreased with increasing salt stress for the control treatment (S1, S2, S3) (Figure 3B). Amending the soil with biochar (BS0, BS1, BS3) or combined with AMF (ABS0, ABS1, ABS2) or with AMF alone (AS0, AS1, AS2) mitigated the adverse effect of increasing saline concentration on the number of healthy leaves. Nevertheless, at a higher saline concentration (150 mM), the different amendments had fewer leaves when compared to S0 but higher when compared to the S3 treatment.

The total plant dry weight (Figure 3C) was negatively affected by increasing NaCl concentration. This observation corroborates the negative impact of saline stress on plant water content observed in Figure 3A. The plant dry weight was 4.03 g for S0 treatment but was decreased by 35.06%, 35.21%, and 36.95% in the S1, S2, and S3 treatments, respectively. The different amendments demonstrated positive impacts on the plant dry weight, both in the absence and presence of saline stress. For instance, biochar (BS0), AMF (AS0), and biochar + AMF (ABS0) increased the plant dry weight by up to 75.38%, 50.74%, and 90.31% when compared to S0, respectively. Under increased NaCl concentration, biochar, AMF (except AS3), and biochar + AMF treatments significantly improved the plant dry weight. For example, at the highest saline concentration and when compared to S0, the plant dry weight was 76.4% (BS3) and 63.6% (ABS3) larger than the corresponding untreated S3 sample.

#### *2.4. The Effects of Salinity, Biochar, and AMF on the Nutrient Uptake Ability of the Maize Plant during Growth*

Figure 4 shows the nutrient uptake ability of maize plants under different saline concentrations before and after amending the soil with biochar and/or AMF. It can be observed that increasing the saline concentration from 0 to 150 mM (S0 to S3) induced a negative effect on the nutrient uptake ability of the plant. Specifically, %N was decreased from 3.481 to 1.694, %P from 0.265 to 0.189, and %K from 1.562 to 0.937. This corroborates the observed negative effect of increasing salt stress on maize growth observed above (Figures 2 and 3). Under no salt stress, the biochar and AMF treatments (AS0, ABS0, and BS0) enhanced the nutrient uptake ability of the plant compared to S0 treatment. Under increasing saline stress, all treatments containing biochar exhibited a superior effect on promoting nutrient uptake compared to single AMF treatments, with a combined application of biochar + AMF showing the best effect relative to S0 and saline controls (S1, S2, and S3). From this result, it can be inferred that the application of biochar and/or AMF to alkaline and saline soils can enhance plant growth by suppressing salt-related stress thereby improving soil fertility, improving nutrient availability, and promoting the uptake of essential nutrients required for growth.

#### *2.5. Influence of AMF and Biochar on Photosynthetic Pigments in Plant*

The effect of increasing saline concentration and the mitigating effects of different amendments on photosynthetic pigments were evaluated and the results are shown in Figure 5. Compared to S0, the content of chlorophyll a was decreased by 27.11%, 51.56%, and 65.78% (S3) as the salt content was increased in S1, S2, and S3, respectively (Figure 5A). Similarly, the contents of chlorophyll b were decreased by 26.8%, 30.93%, and 51.55% (Figure 5B) while carotenoid was decreased by 16.08%, 44.48%, and 44.41% (Figure 5C) in S1, S2, and S3, respectively. From this result, it is evident that under increasing salt stress,

plant growth becomes inhibited as the required nutrients are made less available which results in a reduced photosynthetic rate.

**Figure 4.** Effect of biochar and/or AMF on percentage of N, P, and K uptake by the maize plant under salinity stress. Data present the mean ± standard deviation of three replicates and mean values followed by different letters are significantly different at *p* < 0.05 according to Duncan multiple range test. The significance of acronyms is the same as in Figure 1.

Amending the alkaline soil with biochar alone or combined with AMF significantly improved the plant's photosynthetic ability as chlorophyll a, chlorophyll b, and carotenoids were significantly (*p* < 0.05) improved. The contents of chlorophyll a, chlorophyll b, and carotenoids were increased by 52.89%, 15.46%, 25.78% for B0 and 68%, 52.58%, 89.46% for AB0 compared to S0, respectively. Similarly, the A0 treatment improved the contents of chlorophyll a and carotenoids by 34.22% and 50.34%, respectively, while decreasing that of chlorophyll b by 6.19%. Under increasing saline stress, all amendments containing biochar showed positive effects on improving the photosynthetic ability of the maize plant while the single AMF treatment demonstrated negative effects on chlorophyll b at 100 (AS2) and 150 mM NaCl (AS3). Of all treatments, the combined application of biochar + AMF induced the most significant effect on improving the contents of photosynthetic pigments under salt stress. Relative to the S0 treatment, the content of chlorophyll a was 61.33%, 78.67%, 86.22%; chlorophyll b was 38.14%, 19.59%, 19.59%; and carotenoid was 130.6%, 95.89%, 71.18% higher in ABS1, ABS2, ABS3 treatments than in the S1, S2, S3 treatments, respectively. This observation is in agreement with the superior ability of biochar + AMF treatments in significantly enhancing nutrient uptake by the plant (Figure 4) under increased saline stress relative to other treatments. Therefore, amending alkaline and saline soils with AMF, biochar, or biochar + AMF can play an important role in mitigating stress-related adverse effects in these soils that inhibit plant growth.

#### *2.6. Impact of AMF and Biochar on Antioxidant Enzyme Activities*

The activities of SOD and POD in the leaves of the maize plant were significantly (*p* < 0.05) affected after 90 days of growth under different saline conditions (Figure 6). Compared to S0, increasing the saline concentration reduced the activity of SOD by 25.41%,

32.26%, and 34.04% in S1, S2, and S3 treatments, respectively (Figure 6A). For similar treatments, the content of POD was increased by 15.41% for S1butdecreased by 38.44% for S2 and 44.09% for S3. After amending the soil with biochar (BS0), AMF (AS0), and biochar + AMF (ABS0), the activity of SOD was increased by 1.55%, 10.01%, and 15.18%, respectively. Also, while the BS0 (7.14%) and ABS0 (21.04%) treatments enhanced the activity of POD, the AS0 treatment induced a 4.41% decrease in its activity compared to S0.

**Figure 5.** Effect of biochar and AMF on photosynthetic pigments: (**A**) Chlorophyll a; (**B**) Chlorophyll b; (**C**) Carotenoid. Data present the mean ± standard deviation of three replicates and mean values followed by different letters are significantly different at *p* < 0.05 according to Duncan multiple range test. The significance of acronyms is the same as in Figure 1.

**Figure 6.** Impact of AMF and biochar on the activity of SOD (**A**) and POD (**B**) in maize under different levels of salinity. Data present the mean ± standard deviation of three replicates and mean values followed by different letters are significantly different at *p* < 0.05 according to Duncan multiple range test. The significance of acronyms is the same as in Figure 1.

Under different saline conditions, the amendments showed diverse effects on the activities of SOD and POD. Relative to S0 treatment, the content of SOD in biochar amendments was lower by 21.52% (BS1) or insignificantly higher by 0.08% (BS2) and 0.17% (BS3) when compared to S1, S2, and S3, respectively. Similarly, an insignificant increase was observed for AS1 (1.69%) and AS2 (3.42%) and a decrease for AS3 (7.91%). The application of biochar mitigated the adverse effect of saline stress on the activity of POD (BS1, BS2, BS3) while AMF only had a positive effect at a lower saline concentration (AS1). Of all treatments, the combined application of biochar and AMF showed the most significant (*p* < 0.05) effect on the activities of SOD and POD under saline stress. Compared to S0, the activity of SOD was 28.74%, 40.63%, and 47.5% higher for ABS1, ABS2, ABS3 than for S1, S2, S3, respectively. Similarly, the activity of POD was19.86%, 61.03%, and 77.21% higher in ABS1, ABS2, ABS3 treatments than for S1, S2, S3, respectively. This result shows that biochar + AMF had the most significant effect on the activities of SOD and POD under increasing saline stress, making this combination the best for improving antioxidant activities in alkaline and saline soils.

#### **3. Discussion**

The application of biochar to alkaline soils have demonstrated both positive and negative effects on the availability of nutrients such as K, P, and N. Chen et al. [18] showed that biochar nanoparticles can reduce P retention in alkaline soils by up to 23% and 18% and the authors associated this effect to increased leaching of P associated to Fe/Al oxides in soils. Contrarily, Cui et al. [13] observed that the co-application of biochar and effective microorganisms enhanced the growth of *Sesbania cannabina* in coastal saline-alkali soil. Our study shows that the application of biochar without/without *Arbuscular mycorrhizal* fungi (AMF) (*Glomus mosseae*) had a positive effect on the available nutrients (N, K, and P) in alkaline soil (Figure 1). Although the overall effect of biochar and/or AMF on soil pH was not significant, a significant increase was observed in the nutrient uptake ability of maize plants (Figure 4) and the measured growth parameters (Figures 2 and 3).

Salt-affected soils are prone to nutrient deficiency and excess Na+ ions in soils affect the availability of plant nutrients either by direct competition or indirectly by increasing the osmotic pressure of the soil solution and retarding mass uptake of nutrients by the roots [19,20]. For saline soils, nutrients such as N, P, and K are in low quantities mostly due to low levels and rapid loss of organic matter [21]. This observation agrees with our results (Figure 1) given that the contents of N, P, and K were respectively reduced by 26.75%, 28.94%, and 23.8% when the NaCl concentration was increased to 150 mM for the control (S3). Under similar conditions, the individual application of biochar and combined application with AMF demonstrated the most significant effect in alleviating the adverse effects of saline stress on nutrient availability. Specifically, when compared to S0, the contents of N, P, and K were higher by 77.32%, 8.51%, and 56.3% in BS3 compared to S3 treatment, while in biochar + AMF (ABS3) treatment, it was 75.04%, 32.99%, 2.44% higher, respectively (Figure 1).

Nutrient deficiency and low use efficiency have negative impacts on the photosynthetic potentials of plants and their subsequent resistance to stress [22]. When plants are under stress, they may suffer chlorophyll degradation which results in a reduction of the plant's photosynthetic ability [23]. From Figure 5, it can be observed that increasing salinity reduced the plant's photosynthetic ability. The saline-related stress significantly reduced the contents of photosynthetic pigments, with the reduction increasing with NaCl concentrations and most severe for chlorophylls a and b. Amending the soils with biochar and/or AMF alleviated the negative effects of increasing salinity on maize photosynthetic ability, with the biochar treatments being more effective than single AMF treatments. The colonization of plant roots by AMF has been reported to be beneficial for plant growth in low pH soils, and this effect was significantly improved when AMF was co-applied with biochar [15]. Thus, biochar application to soils played a modifying role in improving the physicochemical characteristics for effective colonization by AMF and it can be inferred that

by improving soil fertility and nutrient uptake, biochar and/or AMF provided a conducive environment for plant growth.

The secretion of antioxidant enzymes is one key mechanism by which plants mitigate the adverse effects of biotic and abiotic stress [24,25]. By scavenging ROS, antioxidants can mitigate the adverse effects of oxidative stress induced by high saline concentrations thereby promoting plant growth [14]. As observed by Farhangi-Abriz and Torabian [10], an increase in the concentration of NaCl causes an increase in antioxidant enzymes such as CAT, SOD, and POD in the leaves and roots of bean seedlings. In our study, increasing the saline concentration decreased the contents of both SOD and POD (Figure 6) while co-application with biochar and/or AMF showed diverse effects. Nevertheless, applying biochar alone or combined with AMF mostly demonstrated an enhancing effect on the activities of SOD and POD; with the combined application of biochar + AMF showing the most significant (*p* < 0.05) increase. Our result may differ from that of Farhangi-Abriz and Torabian [10] primarily due to the difference in the plant types and growth stages considered.

The ability of plants to alter the contents of unsaturated fatty acids is an important mechanism through which they adapt to stress. Through the activities of fatty acid desaturases, plants modify their membranes to provide a suitable environment for the functioning of photosynthetic proteins [26]. Oleic acid (18:1) has been reported to be critical for resistance against pathogens [27] while linolenic acid (C18:3) is an important stress signal [28]. The importance of C18:1 and C18:2 levels have been documented in several studies and it was reported that they play important roles in the regulation of fungal development, seed colonization, and mycotoxin production by *Aspergillus* spp. [29,30]. When plants are subjected to saline stress, the content of C18:3 may decrease to indicate damages caused by the stress [26]. Under drought stress, *Brassica napus* leaves showed decreased levels of C18:3 and C18:2 [31]. Pál et al. [32] studied the effect of cadmium contamination on the content of membrane lipids of maize plants. They found that while the content of C16:0 in the leaves decreased that of C18:2 and C18:3 increased with increasing Cd levels. Also, they observed that for the roots, the levels of C18:0 and C18:1 decreased while those of C18:2 and C18:3 increased. We observed that saturated fatty acids (C14:0, C16:0, and C18:0) that play an important role in controlling the hydrophobicity of membrane proteins [33] were not significantly altered under saline stress and in the presence of biochar. Nevertheless, the levels of saturated acids with longer chains (C20:0 and C22:0) were significantly improved under increasing saline stress and in the presence of biochar. Conversely, increasing salinity negatively affected unsaturated fatty acid concentrations (C16:1, C18:1, C18:2) while the application of biochar demonstrated a mitigating effect and increased the levels of unsaturated fatty acids. The combined application of biochar and AMF showed the most significant effect in increasing the concentrations of unsaturated fatty acids. Thus, by improving the levels of membrane unsaturation, the co-application of biochar and AMF improved the growth of maize in alkaline soil under increasing saline stress. Therefore, we infer that under conditions of stress, plants become better adapted to handle different levels of stress by adjusting the fatty acid unsaturation levels. Also, soil amendments that can improve the content of fatty acid unsaturation may enhance plants' adaptability to stress.

#### **4. Materials and Methods**

#### *4.1. Experimental Materials*

The agricultural soil used in this study was a loess soil collected from Lanzhou, China, with a bulk density of 1.25 g cm−3 and sampled from the surface 0–20 cm. The soil was air-dried and sieved with a 2 mm mesh sieve. On average, the annual rainfall of the study site was 415 mm and the annual temperature was about 6.2 ◦C. For soil characterization, five soil samples were collected and properly mixed to make a representative composite soil sample that had TN content of 1.74 ± 0.01 g kg−1, available phosphorus 25.1 ± 0.19 mg kg−1, soil organic carbon of 9.08 ± 0.09 g kg−1, and soil pH (in solution)

8.25 ± 0.11. Soil pH was determined using a METTLER TOLEDO Desktop pH meter after the soil sample was equilibrated in distilled water (1:5). Soil organic carbon was estimated by the Wet Oxidation method [34]. TN and phosphorus were determined by the Kjeldahl method [35] and Olsen method [36], respectively. The exchangeable K was evaluated by the Ammonium Acetate extraction method [37]. The soil test was performed in the laboratory of the College of Resources and Environmental Sciences at Gansu Agricultural University, Lanzhou, China. For this study, a pot experiment was conducted in greenhouse conditions (air temperature 30 ◦C; relative humidity 50%) from July to October 2020 at the Dingxi experimental station of Gansu Academy of Agricultural Sciences (Tangjiao Town, Dingxi city, 35◦350 N, 104◦360 E, 1970 m a.s.l), Gansu, China.

The biochar was prepared from corn straws collected from a cornfield in Tangjiao Town, Dingxi city, and transported to the College of Resources and Environmental Sciences of Gansu Agricultural University, Lanzhou, China. The straws were cut into 10–15 cm pieces and pyrolyzed at a temperature ranging from 350 to 500 ◦C for 1 h under an oxygendeficient condition in an Oven-Electric Furnace (Heraeus MR 170, Meinerzhagen, Germany). After pyrolysis and cooling, the biochar produced was ground to pass a 250 μm mesh sieve and characterized. Total Organic Carbon (TOC) of biochar samples was measured after oxidization with potassium dichromate following Nelson and Sommers [38]. The total K contents were determined with a flame photometer (Jenway Flame Photometer, Bibby Scientific Ltd-Stone-Staffs-St15 0SA–UK.), P was estimated by a spectrophotometer as described by Sparks [39], and TN was determined by the micro-Kjeldahl method [40]. The %TN, %P, %K, and %TOC of biochar were 0.43 ± 0.01, 0.17 ± 0.004, 0.46 ± 0.003, and 41.2 ± 0.41, respectively. The seeds of corn (*Zea mays* L.) were collected from Gansu Provincial Key Laboratory of Arid Land Crop Science and kept at 4 ◦C for 24 h and later washed with running distilled water for 30 min before planting. Round plastic pots (21 cm in diameter \* 16 cm in height) were used in this experiment. At the harvest period, the plant nutrient uptake (N, P, and K) were estimated following Hashem et al. [15]. To determine the contents of N and K in maize shoot, dried and ground samples were digested with H2SO4-H2O2 at 260–270 ◦C. N contents were determined by an Auto-analyzer 3 digital colorimeter (AA3, Bran + Luebbe, Hamburg, Germany) and K contents were estimated using Flame Photometry (FP6400, Shanghai Precision Scientific Instrument, Shanghai, China). The phosphorus was extracted by nitric-perchloric acid digestion and measured using the Vanado-molybdophosphoric colorimetric method. Standard curve of each mineral (10–100 μg−1 mL) used as reference.

#### *4.2. Arbuscular Mycorrhizal Fungi (AMF) Inoculum*

The Arbuscular mycorrhizal fungi (AMF) strain used was a single genus of *Glomus mosseae* provided by the Gansu Provincial Key Laboratory of Arid Land Crop Science, Lanzhou, China. It was multiplied with *Zea mays* (L.) as the host plant for 4 months in sterilized soil in the greenhouse of the College of Resources and Environmental Sciences of Gansu Agricultural University, Lanzhou, China. The methods previously described by Gerdemann and Nicolson [41] and Giovannetti and Mosse [42] were used to determine AMF inoculum characteristics. Root mycorrhizal colonization, soil spores content, arbuscules, and extraradical hyphae in the roots samples were observed accordingly. The AMF inoculum consisted of mycorrhizal roots (80% root mycorrhizal colonization), soil containing spores (50–80 per 10 g inoculum), and extraradical hyphae (2.5 m per 1 g soil) mixed with soil.

#### *4.3. Experimental Treatments and Design*

The greenhouse pot experiment was conducted in a completely randomized design with five replications per treatment and increasing salt stress. In total, there were 16 treatments as mentioned in Table 2 The NaCl concentration in the pots was gradually increased from 50 to 150 mM at a rate of 50 mM per 24 h. Frequent irrigations (thrice a week) with saline solution permitted the various salt concentrations in the pots to be maintained at

a constant level. There was a total of 80 pots in the present greenhouse experiment. The biochar (5%) and/or AMF (20 g) were applied and thoroughly mixed with soil in each treated pot. This was followed by the sowing of eight imbibed seeds in each pot. After germination, the seedlings were thinned to four per pot.

#### *4.4. Measurement of Plant Growth Parameters*

The maize plant height was measured just before the final harvest. The height was measured by holding a measuring tape close to the stem of the plant. Plant height was recorded from the ground level to the base of the highest fully expanded leaf. The plant fresh weight (FW) was also determined by weighing the different parts of the harvested plant. The pots were cut away on two sides to permit careful root separation from the soil. For each treatment, plants were removed from the soil and washed with distilled water. After adsorbing residual water using tissue paper, an electronic balance was used to measure plant shoot and roots FW.

#### *4.5. Lipid Extraction and Analysis*

3 g 12-week-old maize leaves were harvested from plants grown on saline soils and transferred into 1.5 mL polypropylene reaction tubes. Fresh leaves were flash-frozen in liquid nitrogen and stored at −80 ◦C. To each sample, 300 μL extraction solvent composed of methanol, chloroform and formic acid (20:10:1, *v*/*v*/*v*) was used. The mixture was then vigorously shaken (using a paint shaker) for 5 min. Briefly, 150 μL of 0.2 M phosphoric acid, 1 M potassium chloride was added, and samples were centrifuged at 13,000× *g* at room temperature for 1 min. Lipids were extracted and followed by separation using two-dimensional thin-layer chromatography (TLC) on silica gel plates (pre-coated silica gel plates, Merck 5626) according to Xu and Siegenthaler [43]. The first developing solvent was acetone/toluene/H2O (91:30:8, by volume) and the second was chloroform/methanol/25% NH3/H2O (65:35:3:2, by volume). The plates were shortly air-dried before being delicately sprayed with 0.01% primuline and viewed under ultraviolet radiation. The transesterification of individual lipids previously separated by TLC was performed with 5% H2SO4 in methanol at 85 ◦C for 1 h. A Hewlett-Packard 5890 gas chromatography system equipped with a hydrogen flame ionization detector and an FFAP capillary column (30 m; i.d. 0.53 mm) was used to separate the fatty acid methyl esters. The column was run isothermally at 190 ◦C, while the detector was kept at 230 ◦C. The internal standard was heptadecanoic acid provided by Sigma Aldrich Chemical Co. (St. Louis, MO, USA). All other used chemicals were purchased from Merck (Darmstadt, Germany) and were of analytical purity.

#### *4.6. Antioxidant Enzyme Activities*

For protein extraction, young fresh leaves were collected from 6- and 12-week-old maize plants. The leaves (1 g) were immediately frozen in liquid nitrogen, lyophilized, and homogenized in 2-mL of 0.1 mM potassium phosphate (pH 7.8). The samples were centrifuged for 15 min at 12,000× *g* (4 ◦C). The supernatants were collected into tubes and stored at −20 ◦C until needed for enzymatic activity assays. The activity of superoxide dismutase (SOD) was measured spectrophotometrically by determining the inhibition of blue diformazan formation in the presence of riboflavin/nitroblue tetrazolium (NBT) and light [44]. The modified assay solution was prepared with 1-mL of 50 mM sodium phosphate (pH 7.8), 0.1 mM EDTA, 0.3 mM riboflavin, and 30 L of leaf extract. After 5 min at room temperature, the solution was mixed with NBT to obtain a final concentration of 0.03 mM NBT. The reaction mixture was then illuminated for 3 min with a fluorescent light (75 W, 20 cm above the mixture) and absorbance was determined at 560 nm. The reaction mixture without extract was used to calculate the control rate. NBT absorption was insignificant. The activity of SOD is presented in min−1 mg−1 protein with one unit described as 50% inhibition of blue diformazan formation. Also, the activity of Peroxidase (POD) was determined by measuring the increase rate in absorbance at 470 nm with o-

dianisidine as the substrate [45]. The assay solution was 1 mL of 0.01 M sodium phosphate (pH 6.0) containing 1.3 mM H2O2, 1 mM o-dianisidine and 5 μL of extract. The activity was expressed as ΔOD470 nm min−1 mg−1 protein.

#### *4.7. Photosynthetic Pigments*

Fresh leaves from 12-week-old maize plants were sampled for photosynthetic pigments assessment [46,47]. The leaves were finely cut into small sections (~0.1 g) and ground to a powder in 80% acetone (10 mL) and then centrifuged for 5 min at 10,000 rpm. After collecting the supernatant, the procedure was repeated until the residue was colorless. The absorbance of the solution was recorded at 480, 645 nm, and 663 nm. 80% acetone was included as the blank solution. The photosynthetic pigments of leaves were determined by estimating the contents of chlorophylls (Equations (1)–(3)) and carotenoids content by (Equation (3)).

$$\text{Chlorophyll a (mg g}^{-1}\text{ FW)} = \text{(0.0127 }^\circ\text{ A}\_{663}\text{)} - \text{(0.00269 }^\circ\text{ A}\_{645}\text{)} \text{ \* V/W} \tag{1}$$

$$\text{Chlorophyll b (mg g}^{-1}\text{ FW)} = \text{(0.0229 }^\circ\text{ A}\_{645}) - \text{(0.00468 }^\circ\text{ A}\_{663}) \text{ \* V/W} \tag{2}$$

$$\text{Caortenoids (mg g}^{-1}\text{ FW)} = \left[\text{A}\_{480} + \text{(0.114 }^\circ\text{A}\_{663}) - \text{(0.638 }\cdot\text{A}\_{645})\right] \text{\* V/W} \tag{3}$$

where A663, A645, A480 are absorbance at 663, 645, and 480 nm, respectively, and V is the total volume of sample solution and W is the sample weight.

#### *4.8. Statistical Analysis*

Data were analyzed by one-way analysis of variance (ANOVA) using Genstat statistical software (ver.12). Significant differences among treatments were computed by Duncan's multiple range tests (*p* < 0.05).

#### **5. Conclusions**

Maize cultivated on alkaline soils suffer from nutrient deficiency and this situation may become worse if such soils are affected by salinization. The use of biochar or AMF alone in amending such soils can mitigate the negative impacts of nutrient deficiency and low levels of salinization but become inefficient under high saline concentrations. Our study demonstrates that the combined application of biochar and AMF can effectively alleviate the adverse effects of saline stress on plant growth by (a) improving soil fertility, (b) increasing antioxidant enzymes activities and (c) increasing the levels of unsaturated fatty acid. Nevertheless, biochar application in alkaline and saline soils has shown contrasting results in different studies and this study adds to the limited literature on this. It is suggested that (1) broader studies be conducted in the greenhouse with different plants and different alkaline soils to have a larger picture of the effect of this amendment on alkaline soils under saline stress, (2) researchers should perform small scale field experiments with actual alkaline-saline soils to compare the mechanisms of different amendments in improving soil fertility, and (3) researchers should evaluate the difference in plant-growth improvement mechanisms of these amendments between actual alkaline-saline soils and alkaline soils which are salinized in laboratory studies.

**Author Contributions:** Conceptualization, N.I.N. and C.L.; methodology, N.I.N.; software, N.I.N.; validation, N.I.N., Q.S. and J.N.N.; formal analysis, N.I.N.; writing—original draft preparation, N.I.N.; writing—review and editing, Q.S., F.U.H., A.M., N.I.N. and J.N.N.; supervision, C.L.; funding acquisition, C.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was financially supported by the National Natural Science Foundation of China (41661049).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank Richard Dormatey, Cheikh Ndiaye, Babakar Thioye, and S. Ndour for their assistance during the preparation of this manuscript.

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

#### **References**

### *Article* **Roles of Exogenous** α**-Lipoic Acid and Cysteine in Mitigation of Drought Stress and Restoration of Grain Quality in Wheat**

**Amr Elkelish 1 , Mohamed M. El-Mogy 2,\* , Gniewko Niedbała 3 , Magdalena Piekutowska 4 , Mohamed A. M. Atia 5 , Maha M. A. Hamada 6, Mostafa Shahin 6 , Soumya Mukherjee 7 , Ahmed Abou El-Yazied 8 , Mohamed Shebl 9, Mohammad Shah Jahan 10,11 , Ali Osman 12, Hany G. Abd El-Gawad 8 , Hatem Ashour 13, Reham Farag 13 , Samy Selim 14 and Mohamed F. M. Ibrahim 13,\***

**Abstract:** Cysteine (Cys) and α-lipoic acid (ALA) are naturally occurring antioxidants (sulfurcontaining compounds) that can protect plants against a wide spectrum of environmental stresses. However, up to now, there are no conclusive data on their integrative roles in mitigation of drought stress in wheat plants. Here, we studied the influence of ALA at 0.02 mM (grain dipping precultivation treatment) and Cys (25 and 50 ppm as a foliar application) under well watered and deficit irrigation (100% and 70% of recommended dose). The results showed that deficit irrigation markedly caused obvious cellular oxidative damage as indicated by elevating the malondialdehyde (MDA) and hydrogen peroxide content (H2O2). Moreover, water stressed plants exhibited multiple changes in physiological metabolism, which affected the quantitative and qualitative variables of grain yield. The enzymatic antioxidants, including superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POX) were improved by Cys application. SOD and APX had the same response when treated with ALA, but CAT and POX did not. Moreover, both studied molecules stimulated chlorophyll (Chl) and osmolytes' biosynthesis. In contrast, the Chl a/b ratio was decreased, while flavonoids were not affected by either of the examined molecules. Interestingly, all above-mentioned changes were associated with an improvement in the scavenging

**Citation:** Elkelish, A.; El-Mogy, M.M.; Niedbała, G.; Piekutowska, M.; Atia, M.A.M.; Hamada, M.M.A.; Shahin, M.; Mukherjee, S.; El-Yazied, A.A.; Shebl, M.; et al. Roles of Exogenous α-Lipoic Acid and Cysteine in Mitigation of Drought Stress and Restoration of Grain Quality in Wheat. *Plants* **2021**, *10*, 2318. https://doi.org/10.3390/ plants10112318

Academic Editors: M. Iftikhar Hussain, Adele Muscolo and Mukhtar Ahmed

Received: 11 September 2021 Accepted: 15 October 2021 Published: 28 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

capacity of reactive oxygen species (ROS), leaf relative water content (RWC), grain number, total grain yield, weight of 1000 kernels, gluten index, falling number, and alveographic parameters (P, W, and P/L values). Furthermore, heatmap plot analysis revealed several significant correlations between different studied parameters, which may explore the importance of applied Cys and ALA as effective compounds in wheat cultivation under water deficit conditions.

**Keywords:** wheat; water stress; antioxidant capacity; grain quality; alveographic parameters; alpha-lipoic acid; cysteine

#### **1. Introduction**

Wheat (*Triticum aestivum* L.) is an extremely important cereal crop for human nutrition and animal feed worldwide. Though wheat cultivation is pervasive in a wide spectrum of ecosystems, many parts of the world (particularly in the arid and semiarid regions) suffer from drought stress as a major constraint hindering its expansion from meeting the growing demands day by day for the population [1,2].

Frequent climate change scenarios and rareness of fresh water are two major limiting constraints to sustainable agriculture and preserving food security worldwide. These two major problems may be exacerbated in the semiarid regions where the annual precipitation (200 to 750 mm/year) is not sufficient to meet the needs for farming throughout the year [3,4]. In plants, drought stress or water deficit can trigger a wide spectrum of successive events at morphological, biochemical, and molecular levels that may contribute to the adaptation or tolerance processes to such conditions [5–8]. Among these responses is the excessive release of reactive oxygen species (ROS) that induce oxidative damage to plant cell components, i.e., protein, lipids and, nucleic acids [9–11], and the development of several efficient non-enzymatic and enzymatic antioxidant systems [1,12,13]. Plant resilience to drought stress is mostly attained by accumulating compatible solutes, ion homeostasis, and redox management [5,14–16]. Additionally, water shortage can trigger a cascade of interconnected responses that coordinate the physiological metabolisms of plants, such as altering the signaling pathways, gene expression, hormonal homeostasis, and photosynthetic machinery, leading eventually to affecting crop productivity [8,17–21].

Sulfur-containing molecules and a number of non-protein and protein thiols have been evidenced to play a fundamental role in plant tolerance to various abiotic stresses [22–24]. These molecules can work together, representing a crucial network of responses that enable plants to cope with environmental stresses [24]. Therefore, special attention should be given to reveal a complete picture in this respect.

Alpha-lipoic acid (1,2-dithiolane-3-pentanoic acid; ALA) is a dithiol short-chain fatty acid that is ubiquitous both in prokaryotic or eukaryotic organisms [25]. It has received great attention as a dietary supplement for humans because of its antioxidant and therapeutic properties [26,27]. This antioxidant capacity depends on its two sulfhydryl moieties [25] which enable it to scavenge free radicals and chelate metals [28]. In cereal crops, a few studies during the seedling stage have reported that exogenous ALA can enhance the photosynthetic performance in maize under drought stress [29], ameliorate lipid peroxidation, and induce the antioxidant systems of maize under osmotic stress [25]. Furthermore, it can regulate the ion homeostasis and osmotic potential in wheat under saline conditions [30]. In addition, it has been found that there is a close connection between the endogenous ALA and the cellular redox status of wheat seedlings grown under excess copper [31].

Cysteine (Cys) is an essential thiol-containing amino acid that comprises amino group (NH2), carboxylic acid (COOH) and sulfhydryl group (SH) as reactive centers. This distinct structure enables Cys to be a potent antioxidant and efficient scavenger for reactive oxygen species (ROS). This is due to the presence of the thiol side chain, which smoothly oxidizes, protecting against the oxidative damage induced by biotic and abiotic stresses [32,33]. Moreover, Cys is involved in synthesizing a wide array of vital and defensive molecules

such as protein, glutathione, phytoalexins, thionins, glucosinolates, metallothioneins, and phytochelatins [23,32,34–36]. Additionally, Cys is implicated in different sulfur metabolism pathways and the synthesis of methionine [33]. These responses can regulate plant growth and development via the importance of methionine and/or S-adenosylmethionine (SAM) as precursors for some vital phytohormones, i.e., ethylene and polyamines [37,38]. Several lines of evidence have suggested that exogenous Cys can alleviate the deleterious effects of heavy metals in plants through its roles in metal sequestration and detoxification [35,36]. Similarly, applied Cys has been shown to delay the senescence of postharvest green leafy vegetables by decreasing the rate of Chl degradation, cellular respiration, and ethylene biosynthesis [39]. Up to now, there are no conclusive data to elucidate the role of ALA and/or Cys in wheat plants subjected to water deficit. The current research explores novel approaches to learn more about the combined effect of biomolecules on the diverse parameters of wheat plants under water deficit.

#### **2. Materials and Methods**

#### *2.1. Plant Material, Treatments, and Experimental Design*

A field experiment was conducted during the period from November to April in 2018/2019 and 2019/2020 at a private farm at Beheira Governorate, Egypt (latitude: 30.8481 N; longitude: 30.34 E; mean altitude: 60 m above sea level). Healthy and uniform wheat grains (*Triticum aestivum* L, Cv. Giza 168) were divided into two groups. The first group of grains was soaked in distilled water for 4 h and then sown in the experimental soil, whereas the second group was soaked for 4 h in a solution containing 0.02 mM α-lipoic acid (ALA, Cayman Chemical Company, Michigan, USA). The experimental design was Split-Split-Plot arranged into a randomized complete block design (RCBD) with three replicates. Irrigation with two application levels of 70% and 100% of the recommended dose for wheat plants was set up in the main plots, while ALA-treated and non-treated grains were distributed in the sub plots. Cys foliar applications (Cys, Alpha Chemika, Mumbai, India) at 0, 25 and 50 ppm plus 0.05% (*v*/*v*) Tween 20 as a non-ionic surfactant were randomly distributed in the experimental units. The experimental unit was 15 m2 (3 × 5 m) with sowing space 12 cm. Applied Cys was repeated three times during the growing season, starting from the tillering to stem extension stage at 25, 45, and 65 days after sowing. At the anthesis stage (85 days after sowing), leaves were collected for different biochemical estimations, whereas the total yield (ton·ha−1), various physical/chemical properties and alveographic parameters of grains were determined at the end of each season (158 days after sowing).

#### *2.2. Irrigation Requirements*

A local weather station registered meteorological data. The mean daily temperature data, wind speed at the height of 2 m, precipitation, daily solar radiation, and relative humidity were used in developing the Reference Evapotranspiration (ETo) in mm/day. The ETo was created according to the calculation procedure given in FAO paper n. 56 [40]. Estimation of irrigation requirement (IR) in m3/ha is derived from crop evapotranspiration (ETc), which can be calculated by multiplying the ETo with the crop coefficient (Kc). It has been taken into account that the sprinkler irrigation efficiency is 75% when calculating the IR. Based on the quantities of IR calculated in Table 1, the irrigation quantities in the main experimental plots were determined with two application levels of 70% and 100% from IR. The period considered in this study was calculated ETo for two consecutive seasons (2018/2019 and 2019/2020). The ETo equation can be expressed as:

$$\text{ET}\_0 = \frac{0.408\,\Delta\left(R\_\text{fl} - G\right) + \gamma \left(\frac{900}{(T + 273)}\right) \text{UL}\_2(\text{e}\_s - \text{e}\_d)}{\Delta + (1 + 0.34\,\text{l}\Omega\_2)}\tag{1}$$

where Δ is the slope of the vapor pressure curve (kPa· ◦C−1), Rn the surface net radiation (MJ·m−2·day−1), G soil heat flux density (MJ·m−2·day−1), *γ* psychometric constant

(kPa· ◦C−1), T mean daily air temperature (◦C), U2 wind speed (m·s−1), *es* saturated vapor pressure, *ea* actual vapor pressure.

Kc = Crop Coefficient, ETo = Reference Evapotranspiration (mm), IR = Irrigation Requirements (m3/ha). (2)

**Table 1.** Details of irrigation requirements of two consecutive seasons (2018/2019 and 2019/2020) for wheat based on the reference evapotranspiration calculated by FAO 56 Penman–Monteith equation. The irrigation system efficiency of 75% is considered in the calculations.

#### *2.3. Agricultural Management*

Wheat grains were sown using 178.57 kg ha−1 at the 10th and 8th of November in the first and second season, respectively. The recommended doses of mineral fertilizers (NPK) were applied as follows: P (36 kg ha−1) was supplemented during the soil preparation as calcium super phosphate 15.5% P2O5, while N (187.5 kg ha−1) was added as ammonium nitrate (33.5% N) into seven equal portions, at 10, 20, 30, 40, 50, 60 and 70 days after sowing. Additionally, K (79.2 kg ha−1) was supplemented as potassium sulfate (48% K2O) in two equal portions at sowing and heading stages.

#### *2.4. Determination of Chlorophyll (Chl)*

The leaf content of Chl a and Chl b was determined as described by Costache et al. [41] with some modification, small pieces of fresh leaves (0.5 g) were submerged into 10 mL pure acetone for 24 h/4 ◦C in small dark bottles to avoid pigment degradation. The homogenate was centrifuged at 4000 rpm for 15 min then the absorbance was measured at 645 and 663 nm, respectively. The concentration was calculated using the following equations:

$$\text{Chl a (mg/g FWHM)} = 11.75 \text{ A}\_{662} - 2.350 \text{ A}\_{645} \times (\text{V/1000} \times \text{W}) \tag{3}$$

$$\text{Chl} \,\text{b} \,\text{(mg/g FWHM)} = 18.61 \,\text{A}\_{645} - 3.960 \,\text{A}\_{662} \times \text{(V/1000 \times W)}\tag{4}$$

where, A is the absorbance at 645 and 663 nm, V is the final volume of Chl extract in pure acetone and W is the fresh weight of tissue extract. Additionally, Chl a+b and Chl a/b ratio were calculated.

#### *2.5. Determination of Osmolytes and Leaf Water Status*

Proline concentration was determined with ninhydrin reagent as described by Bates et al. [42]. Total soluble sugars were estimated by phenol-sulfuric acid method as described by Chow and Landhäusser [43]. Leaf relative water content was determined according to Ünyayar et al. [44]. Leaf discs from 10 leaves were weighed (FW) and placed immediately in distilled water for 2 h at 25 ◦C and then their turgid weights (TW) were recorded. The samples were then dried in an oven at 110 ◦C for 24 h (DW). Relative water content (RWC) was calculated by using the following formula: RWC = (FW − DW)/(TW − DW) × 100.

#### *2.6. Determination of Oxidative Damage and Scavenging Capacity*

The level of lipid peroxidation was measured by the determination of malondialdehyde (MDA) as described by Heath and Packer [45]. One gram of fresh leaves was

homogenized in 10 mL diluted trichloroacetic acid (TCA; 0.1% *w*/*v*). The homogenate was centrifuged at 4500 rpm for 15 min. The reaction mixture contained 1 mL from the supernatant and 4 mL 0.5% (*w*/*v*) thiobarbituric acid (TBA) dissolved in 20% (*w*/*v*) TCA. The mixture was heated in boiling water for 30 min then the mixture was cooled at room temperature and centrifuged at 4500 rpm for 15 min. The absorbance of the supernatant was measured at 535 nm and corrected for non-specific turbidity at 600 nm using a spectrophotometer (UV-1601PC; Shimadzu, Tokyo, Japan). The MDA concentration (nmol·g−1 FW) was calculated using Δ OD (A532-A600) and the extinction coefficient (ε = 155 mm−1 cm−1).

Hydrogen peroxide (H2O2) concentration was determined according to Velikova et al. [46] with some modifications. Leaf samples of 0.5 g were homogenized in 3 mL of 1% (*w*/*v*) tri-chloroacetic acid (TCA). The homogenate was centrifuged at 10,000 rpm and 4 ◦C for 10 min. Subsequently, 0.75 mL of the supernatant was added to 0.75 mL of 10 mM K-phosphate buffer (pH 7.0) and 1.5 mL of 1M KI. H2O2 concentration was evaluated by comparing its absorbance at 390 nm to a standard calibration curve. The concentration of H2O2 was calculated from a standard curve plotted in the range from 0 to 15 nmol mL−1.

The scavenging capacity of free radicals was estimated by the reduction of the reaction color method of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) with sample extract as described by Huang et al. [47]. One gram of fresh leaves was homogenized in 20 mL of 70% methanol (*v*/*v*) for 15 h and kept in dark until assay. A final concentration (0.15 mM) of DPPH solution (3.9 mL) was mixed with sample solution (0.1 mL). The mixture was kept in the dark at ambient temperature. The absorbance of the mixtures was recorded at 515 nm for exactly 30 min. Blank was made from 3.9 mL of DPPH and 0.1 mL methanol and measured absorbance at T0. The scavenging of DPPH was calculated according to the following equation:

$$\% \text{ DPPH scavenging} = [(\text{Abs } \text{T}\_0 - \text{Abs } \text{T}\_{30})/\text{Abs } \text{T}\_0] \times 100\tag{5}$$

where Abs (T0) = absorbance of DPPH at 0 time, T30 absorbance at 30 min.

#### *2.7. Determination of Non-Enzymatic Antioxidants*

Carotenoids were quantified using the acetone and petroleum ether method as described by de Carvalho et al. [48] using the following formula:

$$\text{Caroteroids (mg/g FW)} = \text{A}\_{450} \times \text{V (mL)} \times 10 / \text{[A}^{1\%} \text{}\_{1\text{cm}} \times \text{W (g)]} \tag{6}$$

where A450 = absorbance at 450 nm, V = total extract volume; W = sample weight; A1% 1cm = 2592 (β-carotene coefficient in petroleum ether). The specific wavelengths for all estimated leaf pigments were determined using UV visible spectrophotometer (UV-1601PC; Shimadzu, Tokyo, Japan).

Aluminum chloride colorimetric method was used for flavonoids determination [49]. Each plant was extracted (0.5 mL of 1:10 g mL−1) in methanol and was separately mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. It was kept at room temperature for 30 min; the reaction mixture's absorbance was measured at 415 nm. The calibration curve was obtained by preparing quercetin solutions at concentrations of 12.5 to 100 μg/mL.

The extraction of fresh leaves in cold MeOH 85% was used to determine total soluble phenols according to the method of Folin–Denis as described by Shahidi et al. [50]. One milliliter of crude extract was mixed with 0.5 mL of Folin–Denis reagent and were well mixed in dry test tube, the tube was thoroughly shaken for 3 min, 1.0 mL of saturated Na2CO3 solution was added and well mixed then 3 mL of distilled water was added. After one hour, phenolic compounds were determined by reading the developed blue color at 725 nm by using a spectrophotometer. Ascorbic acid (AsA) was determined using 2, 6-Dichloroindophenol titrimetric method [51].

#### *2.8. Determination of Antioxidant Enzymes Activity*

To prepare the extraction of enzyme and soluble proteins, fresh leaves (0.5 g) were homogenized in 4 mL 0.1 M sodium phosphate buffer (pH 7.0) containing 1% (*w*/*v*) polyvinylpyrrolidone (PVP) and 0.1 mM EDTA, centrifuged at 10,000× *g* for 20 min at 4 ◦C and then the supernatant was used for assays. Soluble proteins were evaluated by the method of Bradford [52]. All studied enzyme activities and protein concentration in the crude enzyme extract were measured using a spectrophotometer (UV-1601PC; Shimadzu, Tokyo, Japan) as follows.

Superoxide dismutase (SOD) assay was based on the method described by Beyer et al. [53]. The reaction mixture with a total volume of 3 mL contained 100 μL crude enzyme, 50 mM phosphate buffer (pH 7.8), 75 μM NBT, 13 mM L-methionine, 0.1 mM EDTA and 0.5 mM riboflavin. The addition of riboflavin initiated the reaction then the reaction mixture was illuminated for 20 min with a 20 W fluorescent lamp. One enzyme activity unit was defined as the amount of enzyme required to result in a 50% inhibition in the rate of nitro blue tetrazolium (NBT) reduction at 560 nm.

Catalase (CAT) activity was measured by monitoring the decrease in absorbance at 240 nm as described by Cakmak et al. [54]. The reaction mixture with a total volume of 3 mL contained 15 mM H2O2 in 50 mM phosphate buffer (pH = 7). The reaction was initiated by adding 50 μL crude enzyme. The activity was calculated from the extinction coefficient (ε = 40 mm−1 cm−1) for H2O2. One unit of enzyme activity was defined as the decomposition of 1 μmol of H2O2 per minute.

The activity of ascorbate peroxidase (APX) was determined according to Nakano and Asada [55]. The decrease of absorbance at 290 nm was monitored for 3 min. The reaction mixture with a total volume of 3 mL included 100 μL crude enzyme, 50 mM phosphate buffer (pH 7), 0.1 mM EDTA, 0.5 mM ascorbic acid, and 0.1 mM H2O2. The addition of H2O2 initiated the reaction. One enzyme activity unit was defined as the amount of enzyme required for oxidation of 1 μmol of ascorbate per minute. The rate of ascorbate oxidation was calculated using the extinction coefficient (ε = 2.8 mm−1 cm−1).

Peroxidase (POX) activity was quantified by the method of Dias and Costa [56] with some minor modifications. The assay mixture (100 mL) contained 10 mL of 1% (*v*/*v*) guaiacol, 10 mL of 0.3% H2O2 and 80 mL of 50 mM phosphate buffer (pH = 6.6). The volume of 100 μL of the crude enzyme was added to 2.9 mL of the assay mixture to start the reaction. The absorbance was recorded every 30 s for 3 min at 470 nm.

#### *2.9. Properties of Wheat Grains*

#### 2.9.1. Physical/Chemical Characteristics

Moisture was determined according to AACC standard method No 44-15.02 [57]. Weight per 1000 kernels and hectoliter weight were determined according to AACC standard method No 55-10.01 [57]. Wet gluten and gluten index were determined for wheat meal. Ground whole grain was performed by laboratory mill (3100 with 0.8-mm screen). Wet gluten was washed from whole-grain wheat meal by an automatic gluten washing apparatus (Glutomatic system 2200, Perten Instruments AB, Huddinge, Sweden). Then, it was centrifuged on a specially constructed sieve under standardized conditions. The weight of wet gluten was forced through the sieve then total wet gluten was measured according to AACC standard method No 38-12.01 [57]. The total wet gluten was expressed as percent of sample, and the gluten index was expressed as percentage of wet gluten remaining on the sieve after centrifuging. Hagberg falling number was determined according to AACC standard method No 56-81.03 [57] using FN 1500 (Perten Instruments AB, Hagersten, Sweden). This method is based on the ability of α-amylase to liquefy a starch gel. The activity of the enzyme is measured by falling number (FN), defined as time in sec required to stir and allow stirrer to fall a measured distance through a hot aqueous flour or meal gel undergoing liquefaction. α-Amylase activity is associated with kernel sprouting, and both of these are inversely correlated with FN. Protein (N×5.7) content was analyzed according to AOAC standard method No 920.87 [58].

#### 2.9.2. Alveograph Test

Alveographic properties were evaluated using Chopin Alveograph (Chopin, Villeneuve La Garenne cedex—France) according to AACC standard method No 54-30.02 [57]. Alveograph test was carried out to determine resistance of dough to extension (maximum overpressure, *P*; this is average of maximum ordinates, measured in mm), extensibility (average abscissa at rupture, *L*; abscissa at rupture of each curve is measured in mm on zero line, from origin of curve to point corresponding vertically with clear drop due to rupture of bubble; average of abscissae at rupture points of curves, expressed to nearest unit, represents length), curve configuration ratio, *P*/*L*, and Deformation energy of dough, *W*. Average curve is drawn based on average of ordinates up to *L*, expressed in 10−4 J.

#### *2.10. Statistical Analysis and Data Visualization*

A combined analysis of variance over the two seasons was carried out (Table 2). The significance of difference among the studied varieties was tested by analyzing variance (ANOVA) test as outlined by Snedecor and Cochran [59]. Mean comparisons for variables were made among genotypes using least significant differences (LSD at 5%) tests. Moreover, silhouette analysis was performed to evaluate the quality of the irrigation, seed soaking, and foliar application treatments measurements clustering by testing the cluster distances within and between each cluster [60]. Additionally, we performed a multidimensional preference analysis to disclose the interrelationships amongst parameters in addition to the similarity classification of in terms of dependent and independent variables [61]. Finally, hierarchical clustering based on the correlation analysis was conducted and two-dimensional heatmap plotting was constructed. Boxplot of the parameter's classes and "seed soaking and foliar application" levels are represented graphically, showing distribution of the data, and the X inside the box represents the mean. Colored Mosaic plot was displayed to summarizing the high dimensional data levels. The heatmap, Mosaic plot and Boxplot were drawn with R software.

**Table 2.** Analysis of variance (mean square) of leaf chlorophyll concentration.

ns, \*\*, \*\*\* not significant or significant at *p* ≤ 0.01, *p* ≤ 0.001, analysis of variance.

#### **3. Results**

#### *3.1. Changes in Chlorophyll Composition*

Analysis of variance (Table 2) shows that all leaf chlorophyll compositions including Chl a, Chl b, Chl a+b, and Chl a/b ratio were dramatically and significantly affected by irrigation level (I), ALA as seed soaking (S) and Cys as foliar application (F) treatments. However, the interaction between the irrigation level and seed soaking (I × S) was significant in respect to the Chl a, Chl a+b and Chl a/ b ratios. Furthermore, the Chl a/b ratio was significantly affected by the interaction treatments of I × F and S × F. Generally, deficit irrigation caused an obvious and significant (*p* ≤ 0.05) decrease in Chl a, Chl b and Chl a+b, whereas the Chl a/b ratio was significantly increased particularly in the plants not treated with ALA (Figure 1). This trend indicates the positive effect of ALA as a seed soaking treatment on maintaining the content of Chl b under water shortage. The highest significant increase in Chl a, Chl b and Chl a+b was obtained by the treatment of 0.02 mM ALA + 50 ppm Cys under well watered conditions. In contrast, under limited water supply,

the treatments of 0.02 mM ALA and/or 50 ppm Cys significantly reduced the Chl a/b ratio compared to the untreated plants.

**Figure 1.** Effect of irrigation level, seed soaking by α-lipoic acid (ALA; 0 and 0.02 mM) and foliar application by cysteine (Cys; 0, 25 and 50 ppm) on the chlorophyll concentration of wheat plants. (**A**) chlorophyll a, (**B**) chlorophyll b, (**C**) total chlorophyll, (**D**) chlorophyll a/b ratio. For each parameter, the mean values ± SD followed by a different letter are significantly different according to Tukey's range test (*p* ≤ 0.05).

#### *3.2. Oxidative Stress and Scavenging Capacity*

To assess the efficiency of plants to scavenge the cytotoxic molecules which lead to the oxidative damage, antioxidant activity by DPPH, and lipid peroxidation as indicated by malondialdehyde (MDA) and H2O2 were determined (Table 3, Figure 2). The results showed that all above-mentioned variables were significantly affected by irrigation level (I), ALA as seed soaking (S) and Cys as foliar application (F) treatments. The interaction treatment of I × S was significant with H2O2, while the interaction treatment of I × F was significant with MDA and H2O2. The general tendency was that deficit irrigation resulted in a significant increase (*p* ≤ 0.05) in the antioxidant activity, MDA and H2O2 compared to the well watered plants. Despite being under water deficit, the treatment of 0.02 mM ALA individually reduced the concentration of MDA and H2O2, and the increase of antioxidant activity by DPPH did not reach the level of significance compared to ALA-untreated plants. The foliar applications of Cys (25/50 ppm) either individually or combined with ALA led to improve significantly (*p* ≤ 0.05) the antioxidant activity by DPPH in parallel with reducing the concentration of MDA and H2O2 under water deficit conditions.

**Table 3.** Analysis of variance (mean square) of ROS and scavenging capacity.

ns, \*, \*\*\* not significant or significant at *p* ≤ 0.05, *p* ≤ 0.001, analysis of variance.

**Figure 2.** Effect of irrigation level, seed soaking by α-lipoic acid (ALA; 0 and 0.02 mM) and foliar application by cysteine (Cys; 0, 25 and 50 ppm) on the oxidative stress and scavenging capacity of wheat plants. (**A**) total antioxidant activity, (**B**) Lipid peroxidation (MDA), (**C**) Hydrogen peroxide content. For each parameter, the mean values ± SD followed by a different letter are significantly different according to Tukey's range test (*p* ≤ 0.05).

#### *3.3. Non-Enzymatic Antioxidants*

Non-enzymatic antioxidants including carotenoids, total soluble phenols and ascorbic acid were significantly influenced by the irrigation level (I), ALA as seed soaking (S) and Cys as foliar application (F) treatments, whereas flavonoids were significantly affected by reducing the irrigation level (Table 4). The interactive treatments revealed that flavonoids and total soluble phenols were affected by I × S. Ascorbic acid was also significantly changed by I × F interaction treatment. Generally, carotenoids were significantly (*p* ≤ 0.05) decreased under water deficit. Conversely, flavonoids, total soluble phenols and ascorbic acid were increased with reducing the irrigation level (Figure 3). The highest significant (*p* ≤ 0.05) increase in carotenoids was obtained by the treatment of 0.02 mM ALA + 50 ppm Cys under well watered conditions. On the other hand, the treatment of 0.02 mM ALA + 50 ppm Cys achieved the maximum significant (*p* ≤ 0.05) increase in flavonoids, total soluble phenols and ascorbic acid compared to the untreated plants under water shortage.

#### *3.4. Changes in the Activities of Antioxidant Enzymes*

The activities of antioxidant enzymes including SOD, CAT, POX, and APX were significantly affected by the irrigation level (I), ALA as seed soaking (S) and Cys as foliar application (F) treatments (Table 5). Moreover, POX was affected by all the interactions except the irrigation × seed soaking × Cys interaction. All antioxidant enzymes were increased under water deficit condition compared to well watered condition (Figure 4). Under water deficit conditions, SOD and APX were significantly (*p* ≤ 0.05) increased at higher ALA concentration (0.02 mM) compared to the untreated plants with ALA (0 mM). Conversely, the activity of CAT was significantly decreased at higher ALA concentration, whereas POX did not show any significant changes. All antioxidant enzymes were increased by increasing the concentrations of Cys except POX at higher ALA under well water condition. Under water shortage, the treatment of 50 ppm Cys without ALA and all Cys treatments with ALA recorded the maximum significant (*p* ≤ 0.05) activities in SOD. Additionally, the highest activity of CAT was achieved by the treatment of 50 ppm Cys without ALA under water deficit conditions, whereas the highest POX value was recorded at zero ALA concentration and 25 ppm Cys. Moreover, the treatment of 0.02 ALA + 50 ppm Cys achieved the highest significant (*p* ≤ 0.05) activity regarding to APX.

**Table 4.** Analysis of variance (mean square) of non-enzymatic antioxidants.

ns, \*, \*\*, \*\*\* not significant or significant at *p* ≤ 0.05, *p* ≤ 0.01, *p* ≤ 0.001, analysis of variance.

**Figure 3.** Effect of irrigation level, seed soaking by α-lipoic acid (ALA; 0 and 0.02 mM) and foliar application by cysteine (Cys; 0, 25 and 50 ppm) on the concentration of non-enzymatic antioxidants in wheat plants. (**A**) Carotenoids, (**B**) Flavonoids, (**C**) Total soluble phenols, (**D**) Ascorbic acid. For each parameter, the mean values ± SD followed by a different letter are significantly different according to Tukey's range test (*p* ≤ 0.05).

**Table 5.** Analysis of variance (mean square) of antioxidant enzymes.

ns, \*, \*\*, \*\*\* not significant or significant at *p* ≤ 0.05, *p* ≤ 0.01, *p* ≤ 0.001, analysis of variance.

#### *3.5. Changes in Osmolytes and Leaf Relative Water Content*

Proline, total soluble sugars, and leaf relative water content (RWC) were significantly affected by the irrigation level (I), ALA as seed soaking (S) and Cys as foliar application (F) treatments (Table 6). The interaction treatments revealed that there was a significant relationship between the irrigation level and the seed soaking in ALA in respect to all traits (proline, total soluble sugars and RWC), as well as the interaction treatment of irrigation x foliar applications by Cys demonstrated a significant effect in proline. Generally, plants subjected to water deficit exhibited an obvious and significant (*p* ≤ 0.05) increase in both investigated osmolytes (proline and sugars). However, an opposite trend was observed in respect to RWC (Figure 5). The foliar applications of Cys at both examined concentrations (25/50 ppm) were showed to display a significant (*p* ≤ 0.05) increase in proline in ALA-treated plants under well watered conditions. In this respect, the treatment

of 50 ppm Cys + 0.02 mM ALA recorded the highest significant values under water deficit conditions. In addition, total soluble sugars were significantly (*p* ≤ 0.05) improved by increasing the concentration of Cys either in ALA-treated or non-treated plants under both investigated levels of irrigation. Leaf relative water content (RWC) demonstrated remarkable and significant (*p* ≤ 0.05) improvement in the plants that exposed to both investigated concentrations of Cys under water deficit conditions.

**Table 6.** Analysis of variance (mean square) of osmolytes and leaf water status.

**Figure 5.** Effect of irrigation level, seed soaking by α-lipoic acid (ALA; 0 and 0.02 mM) and foliar application by cysteine (Cys; 0, 25 and 50 ppm) on the concentration of osmolytes and leaf relative water content (RWC) of wheat plants. (**A**) Proline, (**B**) Total soluble sugars, (**C**) RWC. For each parameter, the mean values ± SD followed by a different letter are significantly different according to Tukey's range test (*p* ≤ 0.05).

#### *3.6. Grain Yield and Its Components*

Analysis of variance (Table 7) shows that number of spikes per m2, number of grains per plant and total grain yield (ton/ha) were significantly affected by the irrigation level (I), ALA as seed soaking (S) and Cys as foliar application (F) treatments. The interaction treatments demonstrated that number of spikes and total grain yield were affected by the interaction between the irrigation level and seed soaking treatment by ALA. Additionally, total grain yield was affected by the interaction between the irrigation level and Cys treatments. The highest significant (*p* ≤ 0.05) number of spikes/m2 was obtained by the treatment of ALA 0.02 + Cys 50 under well watered conditions (Figure 6A). Under limited water supply, plants that were treated by Cys (25 or 50 ppm) displayed a significant (*p* ≤ 0.05) increase in the number of spikes compared to the untreated plants regardless of the ALA treatments. Additionally, the highest number of grains per plant was achieved by the treatment of ALA 0.02 + Cys 50 under well watered conditions (Figure 6B). A similar trend was observed under water deficit conditions. Enhancement of the number of spikes and number of grains by ALA and Cys treatments was eventually reflected on the total grain yield (ton/ha) (Figure 6C).

**Table 7.** Analysis of variance (mean square) of grain yield and its components.

ns, \*, \*\*, \*\*\* not significant or significant at *p* ≤ 0.05, *p* ≤ 0.01, *p* ≤ 0.001, analysis of variance.

**Figure 6.** Effect of irrigation level, seed soaking by α-lipoic acid (ALA; 0 and 0.02 mM) and foliar application by cysteine (Cys; 0, 25 and 50 ppm) on grain yield and its components of wheat plants. (**A**) number of spikes, (**B**) number of grains, (**C**) Grain yield. For each parameter, the mean values ± SD followed by a different letter are significantly different according to Tukey's range test (*p* ≤ 0.05).

#### *3.7. The Physical/Chemical Properties of Grains*

Quality assessment of wheat grains was performed by evaluating some simple physical/chemical parameters (Table 8). All studied parameters including moisture content, wet gluten, gluten index, falling number, weight of 1000 kernels and hectoliter weight were significantly affected by reducing the irrigation level. Moreover, ALA as a seed soaking treatment significantly affected gluten index, whereas applied Cys significantly affected moisture content, gluten index, falling number and weight of 1000 kernels. No significant effects were detected between all possible interaction treatments except irrigation x Cys treatments in respect to the moisture content and falling number.

The highest significant (*p* ≤ 0.05) moisture content (Figure 7A) was obtained by the treatment of 50 ppm Cys under well watered conditions compared to the control. The wet gluten (Figure 7B) revealed an obvious and significant (*p* ≤ 0.05) increase under water deficit conditions especially with increasing the concentration of Cys up to 50 ppm. The highest significant (*p* ≤ 0.05) value in the gluten index (Figure 7C) was achieved by the treatment of 0.02 mM ALA + 25 ppm Cys under deficit irrigation conditions. Falling number displayed a significant (*p* ≤ 0.05) decrease in all treatments of Cys at both examined concentrations compared to the Cys-untreated plants (Figure 7D). This increase was more pronounced under deficit irrigation compared to the well watered conditions. The weight of 1000 kernels (Figure 7E) was significantly (*p* ≤ 0.05) increased in parallel with rising the concentration of Cys up to 50 ppm under both investigated levels of irrigation, but the treatment of 0.02 mM ALA did not show any significant results. Furthermore, no significant changes were observed in the hectoliter weight by all investigated treatments under the same level of irrigation (Figure 7F). However, the treatment of 0.0 mM ALA + 50 ppm Cys under well watered condition revealed a significant increase compared to the treatments of

zero ALA + zero Cys, zero ALA + 25 ppm Cys and 0.02 mM ALA + 50 ppm Cys under water limited supply.

**Table 8.** Analysis of variance (mean square) of physical and chemical properties of grains.

ns, \*, \*\*, \*\*\* not significant or significant at *p* ≤ 0.05, *p* ≤ 0.01, *p* ≤ 0.001, analysis of variance.

**Figure 7.** Effect of irrigation level, seed soaking by α-lipoic acid (ALA; 0 and 0.02 mM) and foliar application by cysteine (Cys; 0, 25 and 50 ppm) on the physical/chemical properties of wheat grains. (**A**) Moisture content, (**B**) Wet gluten, (**C**) Gluten index, (**D**) Falling number, (**E**) Weight per 1000 kernels, (**F**) Hectoliter weight. For each parameter, the mean values ± SD followed by a different letter are significantly different according to Tukey's range test (*p* ≤ 0.05).

#### *3.8. Alveographic Parameters of Wheat Grains*

All alveographic parameters of wheat grains were significantly affected by irrigation, seed soaking, and foliar application treatments interaction except seed soaking of *L* (Table 9, Figure 8). All alveographic parameters of wheat grains were significantly increased by water deficit, high concentration of ALA (except *L*), and the highest Cys concentration (except *L*). *L* values were decreased with increasing Cys concentration.

**Table 9.** Analysis of variance (mean square) of alveographic parameters of wheat grains.

ns, \*\*, \*\*\* not significant or significant at *p* ≤ 0.01, *p* ≤ 0.001, analysis of variance.

**Figure 8.** Effect of irrigation level, seed soaking by α-lipoic acid (ALA; 0 and 0.02 mM) and foliar application by cysteine (Cys; 0, 25 and 50 ppm) on the alveographic parameters of wheat grains. (**A**) P, (**B**) L, (**C**) W, (**D**) P/L. For each parameter, the mean values ± SD followed by a different letter are significantly different according to Tukey's range test (*p* ≤ 0.05).

#### *3.9. Correlation between Irrigation, Seed Soaking and Foliar Application Parameters*

To assess the cluster quality of treatments based on the irrigation, seed soaking, and foliar application treatments measurements by testing the cluster distances within and between each cluster, the silhouette analysis plot was calculated and generated based on the Euclidean distance metric (Figure 9). The results revealed that all the parameters except P exhibited positive values indicating that clustering of treatments based on the irrigation and seed soaking and foliar application showed mostly similar behavior with different levels of effect, and the clustering configuration may have few clusters. Meanwhile, two-dimensional heatmap plotting based on all parameters clustered the APX, P, and RWC (%) in a separate cluster, indicating that these parameters almost have the similar power to elucidate the water deficit effects when compared to the well watered condition. While the second cluster comprised two distinctive sub-clusters, the first comprised MDA, sugars, grain yield, weight per 1000 kernels, SOD, Chl a+b, wet gluten (%), and falling number parameters/traits. At the same time, the second sub-cluster comprised Chl a, Chl

a/b, proline, ascorbic acid, Chl b, protein (%), POX, H2O2, CAT, carotenoids, moisture (%), P/L, and flavonoids parameters/traits (Figure 10). Boxplot analysis revealed that the average of the parameter-classes leaf pigments, grain yield, and physical/chemical properties of wheat grain classes decreased significantly under the water deficit compared to well watered condition. The classes of non-enzymatic antioxidants, ROS and scavenging capacity, antioxidant enzymes, osmolytes, leaf water status, and alveographic parameters of wheat grains increased the water deficit significantly compared to well watered condition (Supplementary Table S1).

**Figure 9.** Plot of silhouette analysis values for clustering of all parameters based on "seed soaking and foliar application" treatments variables. On the y-axis each cluster is ordered by decreasing silhouette value. The silhouette value can range between −1 and 1.

**Figure 10.** Two-dimensional heatmap visualization shows the interaction between the irrigation treatments and both the 31 measured parameters included in the study and the six "seed soaking and foliar application" types.

> Meanwhile, Boxplot representation of the "seed soaking and foliar application" treatments on both water deficit and well watered conditions revealed that the average of all treatments except treatments ALA 0.02 - Cys 25 and ALA 0.02 - Cys 50 showed minor variations under the water deficit compared well watered condition (Supplementary Table S1). Moreover, multidimensional preference analysis was performed to summarize the interrelationships amongst treatments, parameters, and classes. The plot shows a high level of consistency and interrelationships between each parameter and treatment type. Ultimately, to summarize and visualize such high-dimensional data levels, a Mosaic plot representing a contingency matrix of the water deficit and well watered treatments versus the 31 parameters and their classes included in the study was developed (Figure 11). The plot further confirmed that the six "seed soaking and foliar application" treatments applied on both water deficit and well watered plant groups exhibited different levels of effectiveness (moderate to minor) to alleviate the negative effect of water deficit when compared to well watered condition.

**Figure 11.** Mosaic plot representing a contingency table of the water deficit and well watered treatments variants versus the 31 parameters included in the study. The vertical size of the cells is proportional to the number of variants found in the respective parameter; the horizontal size of the cells is proportional to the effect level of the six "seed soaking and foliar application" types for each parameter under each parameter-class. The colors of the variants (seed soaking and foliar application) are indicated at the bottom of the Mosaic plot in colored squares similar to the respective bars in the Mosaic plot. Variants were not found at all possible locations of each parameter-class, which causes the reduction of several bars to dashed lines drawn as place holders and indicating that at the particular location no variant has been found in the parameter-class.

#### **4. Discussion**

In the present study, exposing wheat plants to deficit irrigation manifestly altered the leaf chlorophyll composition. In this respect, it can be observed that Chl a, Chl b, Chl a+b contents were significantly decreased in the water stressed plants compared to the well watered ones (Figure 1A–C). This reduction could be attributed to increase the activity of chlorophyllase [62], as well as water deficiency induced oxidative damage (Figure 2B,C) which can collapse the membranes and chloroplast structure where the leaf pigments are localized [63,64]. In contrast, the Chl a/b ratio (Figure 1D) in ALA/Cys-untreated plants was significantly increased under water deficit conditions compared to the well watered plants. It is well known that Chl a is the major cofactor for the photochemical reactions in the plastid because it is required for the assembly of pigment–protein complexes, while Chl b can also act as one of the accessory pigments in light-harvesting chlorophyll complexes (LHCs) [65,66]. In this study, increasing the Chl a/b ratio may act as a protective and adaptive behavior to maintain the function of photosynthetic apparatus under water stress conditions.

On the other hand, applied ALA and/or Cys were shown to enhance the content of photosynthetic pigments (Chl a, Chl b and Chl a+b) under well watered and water stressed conditions. However, an opposite trend was observed in Chl a/b ratio (Figure 1). Sulfur-containing biomolecules (ALA and Cys) are considered very potent antioxidants that can protect plants against abiotic stresses [24,30]. In this context, exogenous ALA was shown to stimulate photosystem II and gene expression of carbon fixation enzymes (Rubisco and PEP carboxylase) in maize seedlings exposed to drought stress [29]. These responses were associated with a simultaneous down-regulation in the chlorophyllase gene (Chlase) [29]. Moreover, Cys has been reported to improve Chl a and Chl b content in oat plants subjected to drought stress [67]. This effect could be attributed to enhancing the uptake of N in the plants, which is essential for Chl biosynthesis [68]. In contrast, the Chl a/b ratio (Figure 1D) differentially responded to ALA treatments under water deficit conditions. It was significantly decreased in the ALA-treated plants compared to the ALA-untreated plants. This effect implies that ALA as an efficient antioxidant has a more positive impact on Chl b than Chl a.

In the present study, water shortage led to an increase of H2O2 and the rate of lipid peroxidation as indicated by malondialdehyde (MDA) (Figure 2B,C). The excessive generation of reactive oxygen species (ROS) under drought stress conditions has been evidenced in several plant species [69–72]. This increase of toxic molecules should be restricted by enhancing the overall antioxidant activities and scavenging capacity of plants (Figure 2A). In this context, the treatments of ALA/Cys revealed beneficial effects as protectant antioxidants by reducing MDA and H2O2 in parallel with enhancing the antioxidant capacity by DPPH (Figure 2A–C). Previous investigations on the antioxidative role of ALA during Pb toxicity revealed ALA-induced accumulation of various antioxidant molecules in wheat plants [73]. Furthermore, ALA can enhance the photosynthetic performance in maize under drought stress [29], ameliorate lipid peroxidation, and induce the antioxidant systems of maize under osmotic stress [25]. Cysteine (Cys) has also been suggested as an important precursor for the synthesis of glutathione in plants [24].

Carotenoids were significantly decreased in the water stressed plants compared to the well watered plants (Figure 3A). This response could be explained by that carotenoids are involved in the biosynthesis of ABA, the major signaling hormone in response to drought stress [74]. In contrast, the treatments of ALA/Cys improved the content of carotenoids under deficit irrigation. This synergistic effect may have occurred to keep the carotenoids that mediate the xanthophyll cycle, leading to dissipating the exceeding light energy and enhancing the photosynthetic capacity under drought stress. In this regard, earlier evidence has confirmed that Cys can hinder energy transfer to prevent photooxidation [75].

On the other hand, flavonoids and total soluble phenols revealed a significant increase in the water stressed plants compared to the well watered ones (Figure 3B,C). This overaccumulation of secondary metabolites can enhance plant tolerance to drought stress and alleviate the induced oxidative damage due to their higher antioxidant capacity [76,77]. In addition to their antioxidant properties, phenolic compounds can be involved in plant tolerance to drought stress as a sink for carbon under stress conditions [78]. Furthermore, flavonoids are widely distributed secondary metabolites that are synthesized through the phenylpropanoid pathway, transforming phenylalanine into 4-coumaroyl-CoA, which finally enters the flavonoid biosynthesis pathway [79]. The carbon skeleton of all secondary metabolites including the compound-mediated phenylpropanoid pathway basically relies on photosynthesis, since several changes in the stressed plants happen in carbon metabolism to achieve the balance between the biomass production and formation of defensive secondary compounds [76]. In this study, flavonoids and total soluble phenols were relatively enhanced by the combined ALA/Cys treatments. This enhancement could be attributed to the increase of the rate of photosynthesis by both compounds [29,80].

Alpha lipoic acid (ALA) is soluble in water and lipid phases, connects the activity of antioxidants in the cell membrane (α-tocopherol) with other antioxidants in the cytoplasm, i.e., ascorbic acid (AsA) and glutathione (GSH), leading to reinforcing the antioxidant

power of plants [31]. Moreover, Cys is an important precursor of GSH biosynthesis in plants [24]. In the current work, the treatments of ALA/Cys increased the concentration of AsA under both investigated levels of irrigation (Figure 3D). These findings indicate that the treatments of ALA/Cys can play a modulatory role in the operation of the GSH-AsA cycle under the circumstances of this study [81].

The activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APX) were observed to be significantly affected by deficit irrigation and ALA/Cys treatments (Table 5; Figure 4). Interestingly, ALA and Cys exhibited differential effects on the activity of antioxidative enzymes, whereas SOD and APX activity were positively upregulated by ALA treatment while CAT and POX activity were decreased. Thus, ALA functions through the activation of the major ROS scavenging system that includes APX and SOD. Reduction of H2O2 content was accompanied by ALA and Cys-mediated elevation in APX, POX, and CAT activity. Further investigations are necessary to reveal the mechanism of ALA and Cys signaling in the modulation of antioxidative enzymes in specific organelles in response to water stress in wheat leaves. Interestingly, Cys treatment during water deficit elevated the activity of all antioxidative enzymes, which was all the higher for POX. Thus, the present work highlights the integrative and synergistic role of ALA and Cys application in the upregulation of the enzymatic antioxidative defense systems. It is further advocated that both ALA and Cys are beneficial to instigate redox management and ROS scavenging activity [25,30,67,73]. Exogenously applied ALA and Cys have been suggested to mitigate the oxidative stress and confer osmotic tolerance to wheat and soybean plants subjected to NaCl stress [30,68].

In the present work, ALA and Cys treatments also increased the proline, total soluble sugars and leaf relative water content (RWC) of drought stressed wheat plants (Table 6, Figure 5). Several lines of evidence demonstrated that to regulate the osmotic potential and enhance water uptake under osmotic stress (drought or salinity), plants usually accumulate considerable concentrations of some organic molecules such as proline and sugars that function as osmotic regulators and enhance plant water relation [64,82,83].

It is noteworthy that the improved osmotic regulation in leaves was also associated with the amelioration of yield attributes (Table 7, Figure 6) including number of spikes/m2, number of grains/plant and grain yield (ton/ha). Several previous studies reported that water deficit has a destructive effect on the yield of wheat plants [84,85]. Generally, water stress can affect many aspects related to the field performance of grain crops such as the photosynthesis and translocation of carbohydrates, which are responsible for the filling of grains [86,87]. In this study, besides the amelioration of drought induced oxidative damage, the treatments of ALA/Cys were evidenced to enhance photosynthetic pigments, accumulation of osmolytes and improving the non-enzymatic and enzymatic antioxidants. These positive effects could explain the increase of grain yield attributes.

ALA and Cys treatments improved moisture percentage and gluten index in grains obtained from water stressed plants. Cys treatment further improved kernel weight (per 1000 kernels) under water deficit conditions. Analysis of alveographic characteristics indicated that ALA and Cys applications were effective in improving flour and dough quality of wheat grains. Total amino acid content, water relation, and growth characteristics are known to be associated with physiochemical properties, as well as grain quality in drought stressed wheat plants [88].

Additionally, Cys and ALA treatments (sulfur-containing molecules) significantly improved grain attributes. In recent years, investigations have revealed the emerging role of ALA (dithiol) as a potential antioxidant in plants [89]. Reduced ALA possesses 2 sulfhydryl moieties which function as potential ROS scavenging sites. ALA is an important sulfurbearing compound that exerts a plethora of effects in plants subjected to abiotic stress [73]. ALA further activates a number of mitochondria-localized metabolic enzymes [90]. Sulfur compounds are important regulators of signaling pathways associated with abiotic stress tolerance in plants [91,92]. In the present work, application of the two sulfur-containing

priming molecules (ALA and Cys) led to several synergistic effects that enhanced the tolerance of wheat plants to water shortage.

Various physiochemical attributes have been improved in the presence of ALA and Cys treatment during water stress in wheat plants. Future investigations are necessary to establish the possible role of ALA and Cys in the modulation of the glutathione-ascorbate cycle in water stressed wheat plants. It is noteworthy that application of the two biomolecules has led to an improvement in the gluten index and quality of grains (Table 8; Figure 7). Thus, using ALA and Cys are effective in the grain filling stage of wheat plants under water stress. This effect may indicate the presence of long distance signaling of ALA and Cys from leaves to grains. Correlation analysis revealed significant interaction among irrigation, seed soaking, and ALA/Cys treatments in wheat plants raised in the absence and presence of drought stress. Thus, ALA and Cys are effective priming molecules and bear agronomic importance in improving wheat yield in the arid cultivable regions. Priming for drought or salinity allows stress tolerance and offers several benefits in many crops [93]. This improvement was extended to the grain processing technology by affecting the alveographic parameters (Tables 8 and 9). Analysis of alveographic characteristics indicated that ALA and Cys application were effective in improving flour and dough quality of wheat grains. In our present work, Cys and ALA treatment (sulfur-containing molecules) significantly improved grain attributes. Various physiochemical attributes of grains have improved in the presence of ALA and Cys treatment during water stress in wheat plants, and alveographic parameters of wheat flour dough also was improved. This improvement extended to the grain processing technology by affecting the alveographic parameters (technological quality parameters). Such parameters are strongly related to the flour yield and flour properties by enhancing the viscoelastic properties of dough [94]. In this context, it has been found that altering water relation and total amino acid content is concomitant with substantial changes in the physiochemical properties and grain quality of drought stressed wheat plants [88].

#### **5. Conclusions**

The present work provides novel insights into the synergistic action of ALA and Cys as effective stress priming molecules in the mitigation of drought stress in wheat plants. Amelioration of drought stress in wheat plants is primarily attained by the enhancement in the function of the antioxidative system and regeneration of osmotic tolerance in leaves. Apart from their antioxidative role, these sulfur-containing compounds also appear beneficial in the improvement of alveographic characteristics of wheat grain which determine various attributes of dough quality. Therefore, applications of these two biomolecules provide both physiological tolerance and restore yield attributes in wheat. Future investigations are necessary to decipher the signaling mechanisms of these two biomolecules in relation to various plant growth regulators in wheat plants. The molecular mechanism of ALA-Cys crosstalk shall also provide new insights to wheat management in arid regions. From our obtained results it can be concluded that applied ALA at 0.02 mM as seed soaking treatment combined by Cys at 50 ppm as a foliar application could be recommended as potent compounds in wheat cultivation under water deficit conditions.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/plants10112318/s1, Table S1: Germination assay of Wheat seed after soaking with different concentration of α-lipoic acid.

**Author Contributions:** Conceptualization, A.E., M.M.E.-M., H.A., R.F., S.S. and M.F.M.I.; methodology, A.E., M.M.E.-M., G.N., M.P., M.A.M.A., M.M.A.H., M.S. (Mostafa Shahin), S.M., A.A.E.-Y., M.S. (Mohamed Shebl), M.S.J., A.O., H.G.A.E.-G., H.A., R.F., S.S. and M.F.M.I.; software, A.E., M.M.E.-M., G.N., M.P., M.A.M.A., M.M.A.H., M.S. (Mostafa Shahin), S.M., A.A.E.-Y., M.S. (Mohamed Shebl), M.S.J., A.O., H.G.A.E.-G., H.A., R.F., S.S. and M.F.M.I.; validation, A.E., M.M.E.-M., G.N., M.P., M.A.M.A., M.M.A.H., M.S. (Mostafa Shahin), S.M., A.A.E.-Y., M.S. (Mohamed Shebl), M.S.J., A.O., H.G.A.E.-G., H.A., R.F., S.S. and M.F.M.I.; formal analysis, A.E., M.M.E.-M., G.N., M.P. and M.F.M.I.; investigation, S.M., A.A.E.-Y., M.S. (Mohamed Shebl), M.S.J., A.O., H.G.A.E.-G., H.A., R.F., S.S.

and M.F.M.I.; resources, A.E., M.M.E.-M., G.N., M.P., M.A.M.A., M.M.A.H., M.S. (Mostafa Shahin), S.M., A.A.E.-Y., M.S. (Mohamed Shebl), M.S.J., A.O., H.G.A.E.-G., H.A., R.F., S.S. and M.F.M.I.; data curation, G.N., M.P., M.A.M.A., M.M.A.H., M.S. (Mostafa Shahin), S.M., A.A.E.-Y., M.S. (Mohamed Shebl), M.S.J., A.O., H.G.A.E.-G., H.A., R.F., S.S. and M.F.M.I.; writing—original draft preparation, A.E., M.M.E.-M., S.M., A.A.E.-Y., M.S. (Mohamed Shebl), M.S.J., A.O., H.G.A.E.-G., R.F., S.S. and M.F.M.I.; writing—review and editing, A.E., M.M.E.-M., G.N., M.P., M.A.M.A., M.M.A.H., M.S. (Mostafa Shahin), S.M., A.A.E.-Y., M.S. (Mohamed Shebl), M.S.J., A.O., H.G.A.E.-G., H.A., R.F., S.S. and M.F.M.I.; visualization, M.A.M.A., M.M.A.H., M.S. (Mostafa Shahin), S.M., A.A.E.-Y., H.A., R.F., S.S. and M.F.M.I.; funding acquisition, A.E., M.M.E.-M., H.G.A.E.-G., H.A., R.F., S.S. and M.F.M.I., All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are grateful to Amr Mossad, Agricultural Engineering Department, Faculty of Agriculture, Ain Shams University, Cairo, Egypt for his great helpful efforts and revising the irrigation requirements of wheat plants at the site of the experiments.

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

#### **References**

**Rasha S. El-Serafy 1 , Abdel-Nasser A. El-Sheshtawy 2,\* , Amira K.G. Atteya 3 , Abdulrahman Al-Hashimi 4 , Arshad Mehmood Abbasi 5,6 and Ibrahim Al-Ashkar 7,\***

**Abstract:** Water shortage is a major problem limiting the expansion of green areas and landscapes. Using seawater as an alternative source of potable water is not a novel idea, but the issue of salt stress needs to be resolved. Salinity has a negative impact on growth and the aesthetic value of ornamental plants. In order to overcome these challenges, *Lathyrus odoratus* seeds were hydro-primed and haloprimed with silicon (Si) and silicon nanoparticles (SiNPs), and exposed to seawater levels. Seawater markedly reduced seed germination and growth of *Lathyrus* seedlings, but halo-priming was shown to significantly alleviate its negative effects. Broadly, SiNPs increased the germination percentage, reduced photosynthetic pigments and carbohydrates decrease, and enhanced water relations, despite having a negative effect on germination speed. Halo-priming significantly increased the proline content and the activities of certain enzymatic (SOD, APX and CAT) and nonenzymatic (phenolic and flavonoids) compounds, that positively influenced oxidative stress (lower MDA and H2O2 accumulation), resulting in seedlings with more salt stress tolerance. Halo-priming with Si or SiNPs enhanced the Si and K+ contents, and K+/Na+ ratio, associated with a reduction in Na+ accumulation. Generally, halo-priming with Si or SiNPs increased *Lathyrus* seedlings salt stress tolerance, which was confirmed using seawater treatments via improving germination percentage, seedlings growth and activation of the antioxidant machinery, which detoxifies reactive oxygen species (ROS).

**Keywords:** *Lathyrus odoratus*; seed priming; seawater; antioxidant; proline; SiNPs

#### **1. Introduction**

Sweet pea *(Lathyrus odoratus*), an annual herb, belongs to the Fabaceae family, and is an important ornamental plant in temperate regions. *Lathyrus* is a climbing plant that reaches up to 2 m in height using tendrils. It is cultivated for its attractive, strongly fragrant, and decorative flowers. It has a range of colors, including pure whites, pinks, oranges, reds, blues, and lavenders. *Lathyrus* is widely used as a bed plant in landscapes and gardens, and is cultivated for the floral industry. As a member of the legume family, these plants are toxic and should not be eaten.

Currently, many countries are attempting to increase the amount of green areas and landscapes in urban environments, due to their vital role in mitigating climate change,

**Citation:** El-Serafy, R.S.;

El-Sheshtawy, A.-N.A.; Atteya, A.K.; Al-Hashimi, A.; Abbasi, A.M.; Al-Ashkar, I. Seed Priming with Silicon as a Potential to Increase Salt Stress Tolerance in *Lathyrus odoratus*. *Plants* **2021**, *10*, 2140. https:// doi.org/10.3390/plants10102140

Academic Editors: Mukhtar Ahmed, M. Iftikhar Hussain and Adele Muscolo

Received: 9 September 2021 Accepted: 6 October 2021 Published: 9 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and the impact of heat [1,2], air pollution [3], and water pollution [4]. In addition, they have a positive effect in terms of reducing respiratory illness, allergies, and improving public health [3,5,6]. Expanding green areas is often challenging as a result of potable water deficiency for irrigation; thus, seeking an alternative to clean water is crucial. Using seawater as an alternative to potable water in agriculture was first attempted after the Second World War. The major challenge for using seawater to irrigate landscapes is the elevated salinity. Leaf necrosis and burns are common symptoms of foliage injury in plants irrigated with low-quality or saline water [7,8]. Moreover, it causes a reduction in shoot and roots growth. Despite this, landscape plants are highly variable in terms of salinity tolerance, which can depend on various factors including climatic conditions, soil or substrate type, irrigation method, plant species, and/or growth stage [9,10]. The impacts of salinity on the growth and appearance of landscapes have attracted much attention. This is in part due to its negative influence on the aesthetics and ornamental appearance. Much effort has been focused on inducing salt tolerance, and seed priming is among the most promising methods for achieving this.

Seed priming is a pre-sowing technique that leaves the seed partially hydrated. This hydration only allows the pre-germinative physiological and biochemical metabolic processes, without radical protrusion [11]. Seed priming can accelerate and homogenize seed emergence, enhance the growth and vigor of seedlings [12]. Halo-priming and hydropriming are types of seed priming. Hydro-priming and halo-priming are defined as soaking seeds in water and salt solutions respectively [13]. Seed priming has been successfully demonstrated to elevate the germination percentage and speed, and enhance seedling vigor under normal and stressed environments [14,15]. Hydro and halo-priming applications enhanced plant growth and performance of wheat under salt stress conditions [16]. Silicon (Si) is a beneficial element commonly used in the halo priming technique, both in bulk size (sodium silicate) and nanoparticle size (SiNPs) [17,18]. Silicon application is an eco-friendly strategy to improve plants' salinity stress response [19]. It boosts plant resistance to salinity and drought stress [20,21], reduces the negative impact of salt stress on chlorophyll content and biomass production [22], boosts adaptive responses, such as phenolic compound production, mineral uptake, and antioxidant activity [23,24]. Seed priming with sodium silicate enhanced germination characteristics and seedling vigor of wheat plants under drought stress [16]. Halo-priming with SiNPs improved seed germination and seedling growth under salinity stress and normal conditions [18,25].

As a result of the importance of sweet pea cultivars as bedding plants in gardens and landscapes, and the scarceness of potable water for landscapes irrigation, the current study aimed to evaluate the effects of seed priming with (Si) and silicon nanoparticle (SiNPs) treatments on plant growth, leaf water status, and the biochemical and physiological traits in *Lathyrus odoratus* under seawater treatments.

#### **2. Results**

#### *2.1. Germination Characteristics*

The results in Figure 1 present the effect of seed priming application on germination characteristics of *Lathyrus* seeds exposed to seawater treatments. Halo-priming application with Si and SiNPs significantly improved the germination percentage (GP) of *Lathyrus* seeds as compared with hydro-priming application. Concerning seawater levels, a gradual decrease was observed in the GP with increasing seawater levels, as the highest salinity level (30%) significantly reduced GP by 21.72%, as compared with unsalted treatments (0%). The highest GP values of 80.02 and 80% were recorded with the treatments Si and SiNPS-primed seeds under non-stressed condition, respectively. Meanwhile, the lowest GP value was obtained by hydro-primed seeds under 30% saline condition (57.48%).

**Figure 1.** Effect of seed priming on germination (%) and mean germination time of *Lathyrus odoratus* seeds irrigated with seawater levels (0, 10, 20, and 30%). Data are mean value ± SE. Bars with different letters are significantly different at *p* ≤ 0.05 level.

Halo-primed seeds with Si had the lowest mean germination time (MGT), which produced 56.03 and 38.32% less than SiNPs and DW, respectively. A significant and growing increase in the MGT value was observed with increased seawater levels, since the highest salinity level (30%) increased MGT by 42.12%, as compared with unsalted treatment (0%). In terms of seed priming and seawater interaction, the lowest MGT was given by Si-primed seeds under non-stressed condition (1.04), while the highest MGT was obtained by SiNPS-primed seeds under 30% saline condition (4.08).

#### *2.2. Plant Growth*

The height of *Lathyrus* seedlings significantly increased following halo-priming application (Si or SiNPs) as compared with hydro-priming application (Figure 2). Seawater treatments negatively impacted *Lathyrus* seedling height, as the lowest height of 16.44 cm was observed with 30% salinity, while unsalted treatment (0%) significantly produced the greatest seedling height (23.5 cm). Regarding the interaction, the tallest seedlings were obtained by SiNPS-primed seeds under non-stressed condition (25.43 cm). However, the shortest seedlings were given by hydro-primed seed under 30% saline condition.

When it comes to the seedling's fresh and dry weights, halo-priming application significantly enhanced seedling fresh and dry weights as compared to hydro-priming application. SiNPs-application significantly produced the highest fresh and dry weights (Figure 2). Seawater treatments led to a significant and gradual decrease in fresh and dry weights with increased seawater levels, as the lowest weights were produced by the highest seawater level (30%). In terms of the interaction, the SiNPS-primed seeds under non-stressed condition significantly produced the heaviest fresh (0.47 g) and dry (0.043 g) weights. However, the lowest fresh (0.17 g) and dry (0.018 g) weights were obtained by hydro-primed seed under 30% saline condition.

**Figure 2.** Effect of seed priming on plant height (cm), fresh weight (g), and dry weight (g) of *Lathyrus odoratus* seedlings irrigated with seawater levels (0, 10, 20, and 30%). Data are mean value ± SE. Bars with different letters are significantly different at *p* ≤ 0.05 level.

#### *2.3. Leaf Water Status*

The water status of *Lathyrus* leaves (leaf water content; LWC) and relative water content (RWC) was significantly enhanced by halo-priming application as compared with the hydro-priming application (Figure 3). Concerning seawater treatments, increasing seawater levels led to a significant and gradual decrease in the LWC and RWC values. Leaf water status was higher with SiNPS-primed seeds under non-stressed condition, which recorded 87.51 and 87.16% for LWC and RWC, respectively. Meanwhile, a lower leaf water status was reported with hydro-primed seeds under 30% saline condition, with respective LWC and RWC of 81.53 and 80.05%.

**Figure 3.** Effect of seed priming on leaf water content (%) and relative water content (%) of *Lathyrus odoratus* leaves irrigated with seawater levels (0, 10, 20, and 30%). Data are mean value ± SE. Bars with different letters are significantly different at *p* ≤ 0.05 level.

#### *2.4. Photosynthetic Pigments*

Leaf photosynthetic pigments (total chlorophyll and carotenoids) were significantly impacted by seed priming and seawater treatments (Figure 4). In this regard, SiNPs produced the highest chlorophyll and carotenoids levels, while the lowest pigment concentrations were obtained by hydro-priming application. Leaf photosynthetic pigments were inversely proportional to seawater treatments, since total chlorophyll and carotenoids content decreased significantly as seawater level increased, reaching its lowest values under 30% salinity (1.36 and 0.367 mg g−1 FW for total chlorophyll and carotenoids, respectively). The treatment of SiNPS-primed seeds under non-stressed conditions significantly produced the highest chlorophyll (2.834 mg g−1 FW) and carotenoids (0.481 mg g−1 FW) concentrations, with the least chlorophyll (1.214 mg g−1 FW) and carotenoids (0.364 mg g−1 FW) concentrations observed with the treatment of hydro-primed seeds under 30% saline conditions.

**Figure 4.** Effect of seed priming on total chlorophyll (mg g−1 FW) and carotenoids (mg g−1 FW) contents of *Lathyrus odoratus* leaves irrigated with seawater levels (0, 10, 20, and 30%). Data are mean value ± SE. Bars with different letters are significantly different at *p* ≤ 0.05 level.

#### *2.5. Biochemical Parameters*

#### 2.5.1. Total Carbohydrates

Seed priming application significantly enhanced total carbohydrate content in *Lathyrus* leaves (Figure 5). Halo-priming application with Si or SiNPs significantly produced higher carbohydrates than hydro-priming application, although there was a non-significant difference between both materials (Si or SiNPs). Total carbohydrates decreased gradually as seawater level increased, reaching its lowest record under 30% salinity, which was roughly 25.35% less than unsalted seedlings (0%). The treatment of SiNPS-primed seeds under non-stressed conditions significantly presented the highest carbohydrate value (27.36%), while the least carbohydrate value was observed with hydro-primed seeds under 30% saline conditions (16.91%).

#### 2.5.2. Proline Content

The results illustrated in Figure 5 show the effect of different seed priming applications on the proline content produced in *Lathyrus* leaves subjected to seawater treatments. Proline content has been decreased by the applications of halo-priming, as the lowest value was noticed with SiNPs-application (3.29 μmol g−1 FW), while it was increased by 20.6% when a hydro-priming application was used. Seawater levels caused remarkable variations in the proline values. A significant and gradual increase was observed with increasing seawater levels, reaching the highest proline content under 30% salinity (5.09 μmol g−1 FW) against the lowest proline content found in unsalted *Lathyrus* leaves (1.98 μmol g−1 FW). The maximum proline content was recorded with the treatment hydro-primed seeds under 30% salinity (5.20 μmol g−1 FW), while the lowest proline content was given by SiNPS-primed seeds under non-stressed conditions (1.78 μmol g−1 FW).

**Figure 5.** Effect of seed priming on carbohydrates (%) and proline (μmol g−1 FW) contents of *Lathyrus odoratus* leaves irrigated with seawater levels (0, 10, 20, and 30%). Data are mean value ± SE. Bars with different letters are significantly different at *p* ≤ 0.05 level.

#### 2.5.3. Total Phenols and Flavonoids Contents

The total phenols and flavonoids content significantly increased after halo-priming applications (with Si or SiNPs) as compared with hydro-priming applications (Figure 6). SiNPs significantly recorded the highest levels in this respect. Increasing seawater levels caused a significant and growing increase, reaching the highest values when plants were irrigated with 30% seawater. The highest levels of phenols (6.544 mg GAE g−1 DW) and flavonoids (4.170 mg CAE g−1 DW) were detected in *Lathyrus* leaves that had been treated with SiNPS-primed seeds under 30% salinity. Meanwhile, the hydro-primed seeds under non-stressed conditions identified the lowest values in this regard (4.670 mg GAE g−1 DW and 2.035 mg CAE g−1 DW for phenols and flavonoids, respectively).

#### 2.5.4. Oxidative Damage Induced

Lipid peroxidation (MDA) and H2O2 content decreased significantly more with halopriming applications than with hydro-priming applications (Figure 7), as SiNPs-application significantly produced the lowest values (34.2% for MDA and 70.9% for H2O2 lower as compared to hydro-priming applications) in this respect. The MDA and H2O2 values gradually increased with increasing seawater levels, with the maximum values being obtained under 30% seawater. The lowest levels of MDA and H2O2 were presented by SiNPS-primed seeds under non-stressed conditions, but hydro-primed seeds under 30% saline conditions significantly caused the maximum values in this respect.

**Figure 6.** Effect of seed priming on total phenols (mg GAE g−1 DW) and flavonoids (mg CAE g−1 DW) contents of *Lathyrus odoratus* leaves irrigated with seawater levels (0, 10, 20, and 30%). Data are mean value ± SE. Bars with different letters are significantly different at *p* ≤ 0.05 level.

**Figure 7.** Effect of seed priming on MDA (mg g−1 FW) and H2O2 (μmol g−1 FW) contents of *Lathyrus odoratus* leaves irrigated with seawater levels (0, 10, 20, and 30%). Data are mean value ± SE. Bars with different letters are significantly different at *p* ≤ 0.05 level.

#### 2.5.5. Antioxidant Enzyme Activities

Applications of halo-priming significantly increased the activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) enzymes in *Lathyrus* leaves relative to hydro-priming applications, as the highest values were produced by the SiNPsapplication (Figure 8). The maximum levels of SOD and APX activities were observed by 30% salinity, while CAT activity decreased with increasing seawater levels. In terms of the interaction effect, the highest levels of SOD and APX were detected by SiNPS-primed seeds under 30% salinity, while the highest CAT value was given by the treatment of SiNPs-primed seeds under non-stressed conditions. On the other hand, the lowest SOD and APX activities were obtained by hydro-primed seeds under non-stressed conditions and by hydro-primed seeds under 30% saline conditions for CAT activity.

**Figure 8.** Effect of seed priming on SOD (U mg−1 protein), CAT (U mg−1 protein), and APX (U mg−1 protein) activities of *Lathyrus odoratus* leaves irrigated with seawater levels (0, 10, 20, and 30%). Data are mean value ± SE. Bars with different letters are significantly different at *p* ≤ 0.05 level.

#### *2.6. Ion Contents*

The results depicted in Figure 9 indicate the content of Si, K+, and Na+ accumulated in *Lathyrus* leaves, and the K+/Na+ ratio in response to priming applications under seawater treatments. Leaf Si content was significantly elevated following halo-priming applications, as compared with the hydro-priming application. Increasing seawater levels caused an increase in Si content reaching its greatest value at 20% salinity, and then decreased after that. Regarding the interaction effect, both Si and SiNPS-primed seeds under 20% saline conditions significantly showed the highest values of Si in *Lathyrus* leaves. The hydropriming application significantly exhibited the highest Na+ content, but SiNPs-application significantly recorded the lowest Na+ value (30.7% less than hydro-priming application). A gradual and significant increase was noticed with increasing salinity levels. Concerning the interaction, the maximum Na+ content was obtained by hydro-primed seeds under 30% salinity. Meanwhile, the Si and SiNPS-primed seeds under non-stressed conditions had the lowest Na+ content.

**Figure 9.** Effect of seed priming on Si (mg Kg−1), Na (mmol g−1 DW), K (mmol g−1 DW) contents, and K/Na ratio (g) of *Lathyrus odoratus* leaves irrigated with seawater levels (0, 10, 20, and 30%). Data are mean value ± SE. Bars with different letters are significantly different at *p* ≤ 0.05 level.

The K+ content and K+/Na+ ratio significantly increased with halo-priming applications as compared with hydro-priming applications, which significantly recorded the lowest K+ value and K+/Na+ ratio. A gradual and significant decrease was observed with increasing seawater concentration. The treatment of SiNPS-primed seeds under nonstressed conditions significantly exhibited the highest K+ value (1.9 mmol g−1 DW) and K+/Na+ ratio (11.23). On the other hand, the treatment of hydro-primed seeds under 30% saline conditions significantly exhibited the lowest K+ (0.70 mmol g−1 DW) and K+/Na+ ratio (0.44). The mechanisms involved in seed priming with Si or SiNPs for gaining salt stress tolerance in *Lathyrus* seedlings are presented in Figure 10.

**Figure 10.** The mechanisms involved in seed primed *Lathyrus* (with Si or SiNPs) for gaining salt stress tolerance.

#### **3. Discussion**

Plant growth and development, as well as productivity, are predicted by seed germination. In the current research study, salt stress decreased GP and prolonged MGT, as well as causing a pronounced inhibition of *Lathyrus* seedling growth. Salinity stress decreased seed germination as a result of limited water absorption, slowed the breakdown of seed storage material, and inhibited the production of storage proteins [26,27]. Basically, salinity decreases cell expansion and division, and inhibits physiological and biochemical processes [28,29], which causes a reduction in the photosynthetic rate, dry matter accumulation, and total carbohydrates content. Furthermore, salt stress reduces chlorophyll synthesis and activates degraded chlorophyllase enzymes [30,31]. In accordance with our results, the negative effects of salt on plant growth features have been previously reported by Al-Yasi et al.; Attia et al., [32,33].

In contrast, Si and SiNPs treatments improved the seed germination of *Lathyrus* seeds. Si was found to be directly linked to the physiological process of seed germination in *Glycyrrhiza uralensis* under saline conditions [29]. Halo-priming with SiNPs exhibited the highest MGT value. Similar results were obtained by Siddiqui and Al-Whaibi [34].

The increase in mean germination time following SiNPs seed priming may be due to the ability of silica to absorb moisture from the surroundings [35]. Furthermore, 1 g of SiO2 nanoparticles (7 nm) has a surface adsorption of 400 m2 [36]. Increasing the seawater levels caused a reduction in the GP and an increase in the MGT. Salts negatively impact the levels of endogenous phytohormones, which essentially inhibit seed germination and plant growth [37].

In our study, halo-priming applications enhanced plant height, shoot FW and DW, leaf water status, leaf pigments, and total carbohydrate under salinity stress, more than

the hydro-priming application. Silicon promotes cell elongation and division, leading to elevated plant height [38,39].

Under salinity conditions, Si enhances photosynthetic activity by decreasing ion toxicity and ROS content, preserving the structure and function of the organelles responsible for photosynthesis [37], maintaining stomatal conductance, transpiration, membrane permeability, net photosynthesis, and chlorophyll levels [29]. Moreover, Si decreases the leaf curve angle and increases leaf flatness, allowing for increasing light interception and more photosynthetic pigments [39], and thus more carbohydrates and dry matter accumulation. Silicon enhances growth performance directly, through blocking Na+ transport, and indirectly, through different physiological processes that alleviate the negative effects of salinity [37]. Increased biomass production is the main indicator of plant resistance [40].

Leaf water status is a good indicator of water relations in plants. Preserving a good water status in plant cells helps to maintain osmotic adjustments and the activity of metabolic processes, and increases plant resistance under salinity stress [41,42]. Under seawater treatments, the LWC and RWC of *Lathyrus* leaves decreased gradually with increasing seawater levels. Salts decrease the osmotic pressure in plant cells, so they have a negative effect on water uptake by plant roots [43]. Halo-priming treatments exhibited better leaf water status. Silicon decreases plant transpiration [29] due to Si accumulation as external layers above cell walls of leaves and stems, leading to thicker leaves and stem cuticle. In addition, Si improves stem hydraulic conductance [39]. Silicon alters the osmotic pressure, which increases plant tolerance under salinity stress conditions [44,45]. Higher water content in Si-plants grown under saline conditions is mainly associated with salt dilution inside the plant, leading to plant growth improvements [44]. Therefore, it can be concluded that Si improves the leaf water status and mitigates the osmotic stress induced by seawater treatments in *Lathyrus* leaves.

Under seawater treatments, proline content gradually increased with increased seawater levels. Proline is normally produced in high amounts under salinity stress [46]. Proline plays a vital role in osmotic adjustment, sub-cellular structure protection, enzyme activities, and can also increase the cellular turgor pressure, which is responsible for cell expansion under salinity conditions [47,48]. Halo-priming treatments presented lower levels of proline. Si decreases proline accumulation under salinity stress, which indicates the role of Si in alleviating damage caused by salts [49]. Total phenols and flavonoids contents of the metabolic *Lathyrus* leaf extract are gradually increased with higher seawater concentrations, because salts stimulate phenolic compound synthesis.

Phenolic compounds, including flavonoids, are among the many sources of antioxidants in plants and are considered to be a response to protect against the oxidative damage caused by salts [50]. The chemical structure of phenols enables them to deactivate singlet oxygen and act as hydrogen donors, allowing them to scavenge ROS [51,52]. Halo-priming treatments demonstrated a significant increase in the total phenolic compound, and SiNPs were superior in this respect which may be due to the nanoscale size of the insoluble SiNPs accumulated in the epidermis. This may allow constitutional phenols to be produced on the large adsorption surfaces of epidermal cells [53,54].

Plants alleviate the oxidative damage that occurs under saline conditions through nonenzymatic (phenolic compounds) generation [55]. These processes play a vital role in protecting plant cells from the oxidative damage [56] that occurs at the cell membrane [28,57], and in ion balance and water status [58].