*2.6. H2O2 Concentration*

Increasing the B toxicity enhanced the H2O2 content effectively. The H2O2 content was highly affected by 30 mg kg−<sup>1</sup> B in soil. The effective treatment in term of reducing the H2O2 content was 100 μM SA, which decreased the H2O2 content up to 84% (Table 3, Figure 2).

#### *2.7. Proline Content*

The toxic effects of B significantly increased the proline contents. The B treatment of 30 mg kg−<sup>1</sup> increased the proline contents up to 43.75% as compared to the control. Priming with SA remained productive in reducing the B toxic effects. Only 100 and 150 μM SA effectively mitigated the toxic effects in plants grown in pots containing 15 mg kg−<sup>1</sup> B. However, under a high boron toxicity, only 150 μM SA significantly reduced the toxic effects by increasing the proline content in comparison with the control, as shown in Table 3, Figure 2.

#### *2.8. Glycine Betaine*

An outstanding improvement was noted in the leaf GB contents of plants grown under B stress. Exogenous applications of SA further increased the GB content in the leaves of plants experiencing B toxicity stress. All the treatments of SA affected the GB level, however 100 μM SA increased the GB contents up to 100% under 30 mg kg−<sup>1</sup> of B treatment as compared to the control, as indicated in Table 3, Figure 2.

#### *2.9. Potassium Content*

An increase in the K contents was observed in response to the B toxicity. Applications of SA reduced the K contents and a maximum reduction of up to 27.8% was noted in the plants primed with 100 μM of SA. The K uptake and accumulation exhibited quite similar patterns in the plant root and shoot (Figures 2 and 4a,b).

#### *2.10. Calcium Content*

Boron toxicity significantly influenced the Ca accumulation in the root of the maize cultivar Gohar-19. The Ca content was reduced at a lower SA treatment level as compared to the control. With increasing the SA concentration up to 100 and 150 μM, higher increments in the Ca contents relative to the control were recorded. A total of 100 μM SA treatment was found to be effective in reducing toxic effects by lowering the Ca content up to 28.7% in the 30 mg kg−<sup>1</sup> B group. The Ca

accumulation in the plant leaves was reduced due to the B toxicity even at high concentrations of SA applications. No significant effects of SA on Ca contents were observed (Figures 2 and 4a,b).

#### *2.11. Nitrate Concentration*

An increase in the nitrate contents was observed with an increasing B level. Various SA concentrations (0, 50, 100 and 150 μM) reduced the nitrate content. The application of 100 μM of SA reduced the nitrate content up to 20% in the most destructive B treatment of 30 mg kg−<sup>1</sup> (Figures 2 and 4a,b).

The relationship between B toxicity and the morpho-physiological attributes of maize under SA application is illustrated in Figure 2.

A Pearson correlation analysis was conducted to quantify the interactive effects of B toxicity and SA application on plant growth and biomass, chlorophyll contents, lipid peroxidation and the antioxidant and nutrient uptake of maize (Figure 2). B toxicity was negatively correlated with plant growth and biomass, photosynthetic pigments, oxidative stress and antioxidative response. Chlorophyll contents were positively correlated with plant biomass accumulation. Positive correlations were also identified among growth attributes and K, Ca and nitrate contents.

#### *2.12. Principal Component Analysis*

The combinatorial effect of B toxicity and SA application was evaluated on important attributes of maize plants by the synthesis of the score and loading plots of PCA, as presented in Figure 4. All the three applied B treatments with and without SA were successfully dispersed by the first two principal components (Figure 1a). The maximum variance among all the components was based on extracted components—i.e., PC1 (Dim1) and PC2 (Dim2), where component Dim1 contributed 69.9% while the contribution of Dim2 was 18.9% (Figure 1b).

#### **3. Discussion**

The only non-metallic element of group 13 of the periodic Table is B, which exhibits a trivalent oxidation state. Naturally, B is present in the form of borate, boric acid and borosilicate mineral. In the Earth crust, the B level varies between 1–500 mg kg−<sup>1</sup> and 2–100 mg kg-1 as per the geographical region and soil composition status [32].

B has a considerable importance due to its supportive role in plant development and growth. It helps in the processes of cell division, the formation of cell wall and the elongation of cells [33]. However, B causes toxic effects at very high or very low levels. B toxicity mostly co-exists together with some other abiotic stresses—e.g., salt and drought stress [34]. A high B toxicity reduces the plant growth and other attributes.

EI-Shazoly [25] conducted a study to describe the SA effects on B toxicity stress in wheat. The results of such a study were in agreement with those of the present study. The SA application also enhanced the root and shoot length, supporting the findings of the previous works [35]. It has been reported that a high level of B causes abnormal cell division in the root meristematic zone [35], hypodermis formation and suberin deposition [36], thus limiting plant growth and development. The excess of B also causes cytotoxic effects during mitosis, which in turn reduces the root and shoot biomass [37,38]. In the present study, 100 μM of SA significantly enhanced the plant biomass by mitigating the B toxicity. Sarafi et al. [39] reported that the B toxicity reduces the plant dry weight up to 48%, the number of leaves and the root dry weight in the pepper plant (*Capsicum annuum*). In this study, the applications of melatonin (MEL) and resveratrol (RES) were studied, where a treatment of 100 μM of RES and 1 μM of MEL effectively reversed the reductions in fresh and dry weights under B toxic effects, respectively. Eser and Aydemir [22] reported that kinetin application prevented the B-induced reductions in the plant fresh and dry weight of wheat plants under B stress. Moreover, the high B content (50 mg kg<sup>−</sup>1) in soil reduced the shoot fresh and dry weight of tomato plants [40]. It has been particularized that B toxicity causes the down-regulation of the photosystem biochemical

components and the inhibition of the electron transport rate [41], thus lowering the activity of carbon fixation enzymes [41,42]. High levels of B can also cause the root growth inhibition, accompanied by a decrease in plant dry weight [43]. The reduction in root growth may be due to the intense lignification of cell wall [44]. However, it has been reported that lignification is not mainly responsible for root growth inhibition, but is rather a defensive attribute for reducing B uptake [36].

The high B level (30 mg kg<sup>−</sup>1) reduced the photosynthetic pigments biosynthesis. However, SA application reversed these negative effects, and the most effective treatment was 100 μM of SA. The findings of present study are in line with those of EI-Shazoly [25]. Plant growth and development are considerably dependent on photosynthetic pigments. It has been reported that the inhibition of plant growth by B stress is associated with reduced photosynthetic pigments. Indeed, the present study indicated that the biosynthesis of chlorophyll and carotenoid was negatively affected by B toxicity stress. Our results depicted a negative relationship between the biosynthesis of photosynthetic pigments and the increasing applied B stress regimes. This decline in photosynthetic pigments might be owing to H2O2 accumulation, which damages the photosynthetic reaction centers. Papadakis et al. [45] reported that one of the possible reasons for the reduction in photosynthetic activity in plants grown under excess of B was the structural damage of thylakoids. In general, SA, being a versatile molecule, interacts with other hormones to promote the induction of enzymes and antioxidants to alleviate the toxic effects of stress [46].

Regarding the mineral contents, Kaya and Ashraf [46] described that B toxicity significantly reduces the N, K and Ca contents in tomato. However, nitric oxide application induced the level of minerals and minimized the B toxicity effects. EI-Shazoly [25] described that a low level of boron (3 mg kg−<sup>1</sup> soil) does not affect the K content in wheat plants, however a high level could decrease it. Moreover, the Ca level was reduced due to the B toxicity (3 mg kg−<sup>1</sup> soil), but increased upon SA and thiamin application.

High levels of B increased the anthocyanin contents in sweet basil (*Ocimum basilicum* L.) plants, indicating possible stress responses or poor nutrient mobilization from the plant root [47]. We also found that B stress elevated the anthocyanin levels in the root and shoot of maize cultivar Gohar-19. The application of SA at higher levels reduced the stress level. Additionally, the reduction in anthocyanin content in plants treated with SA predicts a reduction in stress severity.

Ascorbic acid is an important antioxidant and scavenger of ROS [48–50]. The ASA content was significantly affected by B stress in the present study. Abiotic stresses result in a higher accumulation of ASA than that of other stress. Increased ROS scavenging enzymatic and nonenzymatic activity by excessive B concentrations has already been reported in barley, chickpea, tomato and grapes [51–54].

In general, plants up-regulate the synthesis of different osmolytes in cytosol and other organelles to cope with the deleterious effects of environmental stresses. Proline and GB are considered to be key osmolytes for the osmotic adjustment. Proline, being a secondary metabolite, plays a key role in stress tolerance as an antioxidant and osmoprotectant [55]. Stress-related genes are activated by GB to detoxify ROS and protect photosynthetic machinery under stressed conditions [56]. In the current study, SA applications triggered the accumulation of proline and GB to cope with the B toxicity effects through scavenging ROS and the activation of the antioxidant defense systems.

The PCA results depicted that the application of salicylic acid had a significant ameliorative effect for B toxicity on the studied parameters of maize plants. The same effects of SA have been reported in salt-stressed sunflower plants [57]. Overall, the applied B stress exerted hazardous effects on the growth and ecophysiological attributes of maize. These results were in accordance with the findings of previous reports which have reported decreases in the growth traits of various plant species grown under environmental stress conditions [58–66]. Based on the findings of the current study, we conclude that SA applications improved the growth of B-stressed maize plants at the seedling stage through increasing the biosynthesis of photosynthetic pigments, osmolytes and antioxidants. The high level of B deteriorates photosystem II centers, as the low levels of chlorophyll and carotenoids are linked with biomass reduction caused by B toxicity. High levels of osmoprotectants such as proline may act as signaling molecules for scavenging ROS, thus stabilizing the membrane structures as well as cascading the stress-tolerant gene expression. Further studies at the molecular level may elaborate the comprehensive understanding lying behind these modulations of SA against B toxicity in maize. The induction of B toxicity tolerance in maize plants after SA application is also associated with antioxidant defense system improvement.

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