*3.6. PCA Analysis and Spearman's Correlation Coe*ffi*cient (r2) Values Extracted from XLSTAT Software of All the Studied Attributes of Maize Plants Foliar-Applied with* α*-Toc*

PCA and correlations coefficients among studied attributes revealed a significant positive correlation of total-Toc contents in leaf, root, and stem with morphological and growth attributes, levels of antioxidants, and uptake of mineral nutrients (K, Ca, Mg, N, and P) in all studied tissues of maize. A positive correlation of leaf and stem Toc was found with leaf area (0.768 \*\*\* and 0.664 \*\*) and fresh weights (0.921 \*\*\* and 0.661 \*\*\*), respectively, that depicts the role of Toc in the improved growth under drought stress. Positive correlation was also recorded of shoot dry weight with Toc levels in studied plant tissues such as in leaf (0.578 \*\*) and root (0.643 \*\*\*), respectively. Significantly positive correlation was found of Toc levels in the root with LRWC (0.721 \*\*\*). CAT, POD, and SOD activities in different plant parts like leaf (0.966 \*\*\*, 0.961 \*\*\* and 0.936 \*\*\*) and stem (0.863 \*\*\*, 0.872 \*\*\* and 0.859 \*\*\*), respectively, were also positively correlated with plant Toc levels. Tocopherol contents were also positively correlated with potassium and calcium contents in leaf (0.553 \*\* and 0.606 \*\*, 0.569 \*\* and 0.633 \*\*\*), root (0.555 \*\* and 0.675 \*\*\*, 0.674 \*\* and 0.461 \*), and stem (0.470 \* and 0.673 \*\*\*, 0.749 \*\*\* and 0.437 \*), respectively. Furthermore, a positive correlation was also recorded between nitrogen and phosphorus contents with Toc levels in studied plant tissues such as in leaf (0.610 \*\* and 0.613 \*\*, 0.539 \*\*, and 0.683 \*\*\*), root (0.669 \*\*\*, 0.494 \* and 0.488 \* and 0.729 \*\*\*, 0.430 \* and 0.620 \*\*), and stem (0.626 \*\*\* and 0.601 \*\*, 0.536 \*\*, and 0.688 \*\*\*), respectively. Figure 3 shows the PCA analysis of varying studied attributes that confirmed correlation studies. Of the extracted components, F1 has a major contribution (67.43%) that has divided the studied attributes in different groups. Of them, the major group encircled has parameters that are positively correlated include Pr L, RFW, RDW, N R, S L, SDW, K L, Ca L, P S, P L, K S, and LRWC, and L A, Ca R, Ca S, Mg L, Pr R, P R, N R, and N L contributed maximally in determining the variance. The F2 component has less variance (17.70%). Both components have a total variance of 80.13% (Figure 3; Table 6).

**Figure 3.** Principle component analysis of tocopherol levels in different plant tissues of maize with studied growth and physio-biochemical attributes, and nutrient accumulation.


**Table 6.** Spearman correlation coefficient values (*r*2) of Toc levels in different plant parts of maize with growth, biochemical attributes, and nutrient uptake.

#### **4. Discussion**

The exogenous application of water-soluble antioxidants have been widely investigated to improve stress tolerance, but plant growth modulations by foliar application of lipophilic antioxidants like α-Toc has been little studied, probably due to limited information regarding their application, absorption, and translocation within the plant. Kumar et al. [60] and Ali et al. [61] reported that the exogenously applied α-Toc can partly alleviate the deleterious impacts of heat and water stress in wheat. In another study, it was found that the exogenous application of α-Toc effectively decreased the adverse effects of salt stress in flax cultivars [44]. In most of the earlier studies, the α-Toc was applied at adult growth stages. However, the seedling stage (among other growth stages) is considered important due to its involvement in better seed yield by establishing better crop stand [14]. In view of the available information in literature, the present experiment was planned with the objective to study the involvement of α-Toc in the improvement of water stress tolerance in relation to the growth modulations of maize depending upon tissue specific partitioning of macro-nutrients and antioxidants in relation with its own translocation/synthesis in specific terms. For this purpose, the response of selected maize genotypes (Agaiti-2002 and EV-1098) was examined under water stress at an early growth stage with and without foliar spray of α-Toc.

#### *4.1. Tocopherol Content in Di*ff*erent Plant Parts*

Foliar spray of α-Toc significantly increased the leaf tocopherol levels under non-stressed and stressed conditions, which pointed out the existence of an appropriate mechanism for the uptake of α-Toc in the leaves of maize. The increments in root tocopherol contents exhibited a similar pattern, as did the leaves, after foliar application, which suggests an efficient basipetal translocation of α-Toc in maize. Our findings are in agreement with Kumar et al. [60], who reported an elevation in the endogenous levels of α-Toc in heat-stressed wheat plants after its exogenous application. Furthermore, it has been reported that the exogenous application of these organic compounds, along with altering the cellular metabolic activities, also controls the plant's own metabolism. In the present study, the improvement in the internal levels of α-Toc by its exogenous application might also be due to its involvement in regulating plant metabolism [34,36,40].

#### *4.2. Growth, Water Relations, and Photosynthetic Pigments*

Seedling growth of maize plants was adversely affected in plants grown without foliar application of α-Toc under water stress, which is in line with the findings that drought-caused growth reduction is a clear phenomenon in crop plants [8,14]. Similarly, in the present study, a drought-induced decrease was recorded in root and shoot lengths, root and shoot fresh and dry weights, leaf area, and number of leaves of both maize genotypes. Growth is dependent on physiological factors, including the content of plant photosynthetic pigments and water relations that directly influences the leaf photosynthetic rate by affecting the capacity of light capturing and assimilation process [61,62]. Different plant species and even cultivars in the same species have different potentials to tolerate the adverse conditions regarding these attributes [63].

In the present study, water-stress-induced reduction in biomass is associated with reduced photosynthetic pigment along with disturbed plant water relations, and this reduction was less in cv. Agaiti-2002, showing its better tolerance to drought [34]. The foliar spray of α-Toc substantially elevated the plant's endogenous levels and resulted in significant growth improvement under stressed and non-stressed conditions. Increments in plant biomass production is positively associated with the improvement in plant water relations and biosynthesis of biosynthetic pigments such as chlorophyll and carotenoids under the influence of α-Toc foliar application. The increment in plant water status might probably be due to impact of α-Toc on H-ATPase system showing its role in cellular osmotic adjustment, due to a necessary part of cellular membranes. This involvement of alpha tocopherol in cellular osmotic adjustment confers its role in maintaining the cellular water relations under stressful conditions. Similar might be in present study where foliar application of alpha tocopherol improved the leaf relative water content of water stressed maize plants. This improvement in plant water relations further confers its role in improving the leaf net photosynthetic efficiency because plant better water content is necessary to regulate stomatal regulation for better photosynthesis [62]. Furthermore, it is found that α-Toc, being a part of cellular membranes, plays a significant role in decreasing the degradation of photosynthetic pigments in a stressful environment [64]. Tocopherols also protect D1 protein [65] and chloroplastic membranes from damaging effects when grown under stressful conditions.

In the present study, foliar-applied α-Toc under drought stress further enhanced its internal levels in parallel with the improvement in leaf photosynthetic pigments, which might be due to the significant role of alpha tocopherol in reducing the adverse effects on leaf photosynthetic pigments, resulting in improved photosynthetic efficiency along with better plant water relations that resulted in better plant biomass production. In an earlier study, it was found by Sakr and El-Metwally [43] and El-Quesni [66] in wheat and *Hibiscus rosa sinensis*, respectively, that exogenous application of α-Toc enhanced plant biomass production, which might be due to the role of α-Toc in the accumulation of total carbohydrates and protein biosynthesis, confirming its role in photosynthesis and assimilation [67]; this can be correlated with present findings, where higher biomass production was associated with α-Toc levels in different parts that improved plant water relations and net photosynthesis as a result of better net assimilation with improved biomass production. Furthermore, this study reveals the increased plant dry weights due to foliar application of α-Toc, which points toward the improved photosynthetic activity and assimilation with the establishment of new binding sites [68] after its exogenous application.

Furthermore, in the present study, both maize cultivars maintained an optimum level of their carotenoid contents even under drought and α-Toc supplementation, which further enhanced the plant carotenoid contents, especially in leaf and root. These observations point out that α-Toc-induced improvement in the growth of maize plants might be due to an improvement in the contents of accessory pigments as additional support to different photosynthetic attributes. In an earlier study, it was found that, in different wheat cultivars [43] and *Vicia faba* [69], foliar-applied α-Toc improved the leaf carotenoid concentration in association with its enhanced growth. Without α-Toc application, a decrease in leaf water contents was found in maize plants, which is a well-known phenomenon in all plants. α-Toc foliar application significantly increased the leaf water content of water-stressed maize plants, showing its protective role in drought-stressed plants, which might be due to its role in the management of cellular turgor potential through imparting its role in cellular osmotic adjustment by enhancing biosynthesis of osmolytes [7], resulting in better growth by providing an environment for increased cell division and provide an environment for better photosynthesis.

#### *4.3. Lipid Peroxidation and Antioxidative Defence Mechanism*

An increase in the levels of ROS under stressful environment is a general phenomenon due to O2 excitation to form singlet oxygen or its conversion to hydroxyl radicals (OH−), hydrogen peroxide (H2O2), or superoxide (O<sup>−</sup>2) due to the transfer of excited electrons, respectively [34,70], with restricted e<sup>−</sup> transfer at different steps in photosynthesis and respiration under reduced metabolic activities. These overly produced ROS directly affect different cellular membranes through lipid peroxidation. As a defense for the protection of the cellular membranes and other components from the deleterious and damaging effects of overproduced ROS, plants have evolved well-developed mechanisms for the antioxidation of ROS, i.e., comprised of non-enzymatic (AsA, phenolics, carotenoids, flavonoids, tocopherol, etc.) and enzymatic (SOD, POD, CAT, APX) components [14,34,71]. This antioxidative system works well in combination. In the present study, the α-Toc-treated plants suffered significantly lower oxidative damage, especially in root and leaf, as depicted by the lower MDA contents in these plant parts relative to untreated ones (as reported earlier for wheat) [60]. Drought stress significantly increased oxidative stress in maize plants; this is obvious

from increased levels of MDA, a product of lipid peroxidation. Damage to biological membranes due to oxidative stress is a general phenomenon that generally increases in specific environments [14,45]. In an earlier study, significantly lower oxidative stress was recorded in α-Toc applied plants as obvious from lower membrane permeability which is in line with its role in quenching lipid peroxyl radicals, responsible for propagating lipid peroxidation [69,72,73]. It was reported that during early growth stages, α-Toc played a significant role in counteracting the adverse effects of membrane lipid peroxidation. Furthermore, being lipophilic, α-Toc has a significant role in membrane stabilization [74] and also protects them from ROS [75]. Furthermore, α-Toc directly scavenges singlet oxygen [76], giving rise an intermediate tocopherol quinone, which again yields α-Toc in chloroplasts, thereby conferring the recycling for oxidized tocopherols [77]. Reports exist that α-Tocopherol is also an excellent quencher and scavenger of singlet oxygen by controlling the lifetime of ROS. By resonance energy transfer, one α-Toc molecule can neutralize up to 120 molecules of singlet oxygen [78]. The activities of antioxidants such as SOD, POD, and CAT were found to be higher in leaves and roots of maize plant after α-Toc treatment, which suggested their antioxidative role to be stimulated in the presence of α-Toc.

Higher activity rates of these enzymes were found in leaves and roots where more accumulation of α-Toc was found in comparison with stem, showing the supportive role of α-Toc in the activities of antioxidative enzymes. Furthermore, the higher levels of non-enzymatic antioxidant in root and leaf as compared to stem (such as AsA, phenolics, and flavonoids) are also associated with high content of α-Toc in these plant parts. These findings show that α-Toc application after its translocation to the studied plant parts played a significant role in increasing the activities of antioxidative enzymes and the levels of non-enzymatic antioxidant compounds and thus played an imperative role in protecting cellular membranes by boosting the plant's own mechanism. It was found by Fahrenholtz et al. [79] that α-Toc acts as an antioxidative defense mechanism in plants. It was also found that α-Toc minimizes the oxidative changes in the cellular membrane in a significant way with other antioxidants [80–82].

#### *4.4. Uptake of Mineral Nutrients*

Drought-induced growth reduction can also be attributed to disturbances in the uptake of mineral nutrients along with other physiological attributes. It is well known that disturbance or reductions in the leaf uptake of mineral nutrient in plants is probably due to nutrient availability, partitioning, and transport, which is negatively affected under drought conditions. Plant mineral nutrients status played a major role in determining drought tolerance [83]. In the present study, the PCA analysis and the correlations studied suggest that an improvement in the levels of α-Toc contents in different plant parts induced by its foliar application increased the uptake of mineral nutrients (K, Ca, N, and P). Mineral nutrients effectively decrease the harsh effects of water stress by various mechanisms [22]. For example, it has been found that better uptake of mineral nutrients like Ca2<sup>+</sup>, N, and K<sup>+</sup> reduces the deleterious effects of over produced ROS by increasing the concentration of antioxidants like CAT, POD, and SOD [22]. It has been reported that P, K, and Mg improve root growth, which results in improved water intake conferring the drought tolerance. It can be interpreted that optimum nutrient levels maintained after α-Toc application confer drought tolerance induction in maize plants in parallel with improved growth. This is more likely because leaf water contents were significantly improved by foliar spray of α-Toc. The supportive role of α-Toc after its application in the absorption of nutrients from the soil in stressful environment has been found extensively [44,78], and it is reported that α-Toc induced increase uptake of nutrients due to α-Toc being an antioxidant, along with membrane permeability. Furthermore, previous studies found that α-Toc induced an increase in growth, water relation, and nutrient uptake associated with improved stem and leaf anatomy, which further improved translocation to different plant parts. Therefore, the studies confirm that, as in the present study, α-Toc application might improve the uptake and translocation of different nutrients from the soil solution to the roots and then to different plant parts, resulting in better assimilation and growth.

#### **5. Conclusions**

It can be concluded that endogenous levels of α-Toc have an important role in enhancing water stress tolerance of maize cultivars, and its foliar application is found to be effective in reducing water-stress-induced adversative effects on growth by modulating different metabolic activities. Our results confirmed that α-Tocopherol application resulted in membrane protection through increased activities of antioxidative enzymes (CAT, POD, and SOD) and the content of non-enzymatic antioxidants with improved water relations. The correlations and PCA analysis revealed that the increase in α-Tocopherol contents in different plant parts after its foliar application increased the uptake of mineral nutrients (K+, Ca2+, N, and P). Optimum water content and nutrients, along with better antioxidant potential, ultimately resulted in drought tolerance in both maize cultivars that increased growth. In relation to translocation-dependent effects, it was found that α-Toc followed basipetal translocation, concentrating mainly in the roots rather than the shoot after its foliar application. Therefore, analysis of the impact of foliar application of α-Toc on seed yield and nutritional quality of arable crops under stressful environment should be the subject of future studies.

**Author Contributions:** Conceptualization, Q.A., M.R., S.A., M.N.A., H.A.E.-S., and F.A.A.-M.; Data curation, Q.A., M.T.J., M.Z.H., and R.P.; Formal analysis, Q.A., M.T.J., N.H., and R.P.; Funding acquisition, Q.A., M.N.A., H.A.E.-S., and F.A.A.-M.; Investigation, N.H. and R.P.; Methodology, M.T.J., M.Z.H., and R.P.; Resources, M.R., S.A., M.N.A., H.A.E.-S., and F.A.A.-M.; Software, M.R., S.A., M.N.A., H.A.E.-S., and F.A.A.-M.; Supervision, Q.A. and M.T.J.; Validation, M.T.J., M.Z.H., and N.H.; Visualization, M.Z.H. and N.H.; Writing—original draft, Q.A. and F.A.A.-M.; Writing—review and editing, M.R., S.A., M.N.A., and H.A.E.-S. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP-2020/19), King Saud University, Riyadh, Saudi Arabia.

**Acknowledgments:** The authors are grateful to the Higher Education Commission (HEC) Islamabad, Pakistan for its support.

**Conflicts of Interest:** The authors declare that there are no conflicts of interest regarding the publication of this paper.

#### **Abbreviations**



#### **References**


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