**1. Introduction**

Among different environmental adversities, water shortage is of major focus, which has hampered the production of global agricultural systems [1,2]. At a global level, about 45% of all land is prevailed by drought [3]. On the other hand, an estimated increase in the world population will be about 2.5 billion in the next 25 years, which will exert huge pressure on agriculture to fulfill world food demand and on the available freshwater resources. From the last two decades, Pakistan has also faced the problem of agricultural productivity to fulfill the food demand of the sixth largest population in the world. With an agriculture-based economy, Pakistan is predominantly categorized as arid country lying within the geographic coordinates of 23.38◦–30.25◦ N latitude and 61.78◦–74.30◦ E longitude, with a total land area of 796,096 km<sup>2</sup> [4]. The interannual rainfall variability makes the arid region (covering 75% land area of Pakistan) more susceptible to drought risks. Approximately 34.15 Mha of land area is in agriculture use, and uncultivated land is 23.60 Mha. About 25% of the cultivated land is rainfed, which plays a vital role in the country's economy [5]. Due to the major contribution of the agriculture sector in Pakistan's economy, Pakistan is more susceptible to drought risks [6]. In recent decades, unexpected and rapid changes in climate have severely affected socioeconomic and environmental conditions in Pakistan [6]. The major cause of drought stress is a decrease in soil water contents in combination with evaporation due to over-changing atmospheric conditions [7]. Shortage of water induces drastic changes in plants' physio-biochemical and molecular properties that ultimately affects all growth stages of a plant's life cycle, including the final yield [8,9]. At present (and in the near future), the maintenance of crop productivity for a large population under limited water supply is a challenge for the researchers working in the agriculture sector.

To survive under water deficit conditions, plants have manipulated metabolic defensive systems/mechanisms, which are species- and genotype-specific [10–12]. Disturbance in plant water status is the important effect of water shortage that triggers various other metabolic processes to survive under water stress [10,11,13,14]. It results in reduced growth and final grain yield due to perturbations in photosynthesis by disturbances in the biosynthesis of photosynthetic pigments and impaired nutrient uptake [15,16]. Water deficit conditions cause sub-optimal plant photosynthetic efficiency due to limited CO2 diffusion into the leaves due to less stomatal opening or reduced Rubisco activity [17,18]. To cope with a stressful environment, the plant mineral uptake mechanism plays a significant role in improving resistance [19,20]. Generally, under water deficit conditions, mineral uptake and transport reduces due to a decrease in the nutrient diffusion rate [16,21]. Among different nutrients, potassium (K+), nitrogen (N), calcium (Ca2<sup>+</sup>), phosphorus (P), and magnesium (Mg2<sup>+</sup>) have prime importance due to their vital functions in plant physio-biochemical processes [14,20,22].

The stress tolerance in crop plants that results in better yield is growth-stage and species-specific [23,24]. The seedling stage is of prime importance in potentially contributing to better seed yield. Uniform crop stand leads to better yield, which depends on better seedling growth [25,26]. Furthermore, it was found that at early seedling stages, crop cultivars with better antioxidative potential are more drought tolerant than cultivars with less antioxidative activity [27] because the disturbances in different physiological mechanisms results in another secondary stress (oxidative stress) by excessive production of reactive oxygen species (ROS).

Stress-induced oxidative stress due to production of ROS (O2 <sup>−</sup>, H2O2, OH<sup>−</sup>, and O\*) is a common phenomenon in all organisms [28]. Over-production of ROS damages membrane lipids [28], thereby increasing malondialdehyde (MDA) accumulation due to limited activity of antioxidative defense mechanisms [29]. Under stressful environments, the levels of MDA are parallel with antioxidant enzyme activities, which are the indices to assess the status of the extent of damage due to the overproduction of ROS [30]. Other than the levels of antioxidant enzyme to counteract ROS damage, plants also have non-enzymatic antioxidative defense mechanisms such as the production of ascorbic acid, phenolic acid, carotenoids, tocopherols, etc. [31]. Furthermore, it is well known that the antioxidative defensive phenomenon is inter-species, cultivar, and growth-stage-specific. However, most of the higher yielding genotypes are not drought tolerant when considering stress tolerance mechanisms [32].

Furthermore, some high-yield crop cultivars are deficit with regard to such anti-stress mechanisms [14,28,33]. For the induction of drought tolerance, different approaches have been adopted, including the exogenous application of secondary growth metabolic compounds [34–37]. Exogenous

application such as the foliar spray of different secondary metabolites of which the plant is in deficit is considered as an effective means among others for stress tolerance induction [38,39]. It is well known that foliar application of such compounds is translocatable to different plant parts. Furthermore, after their translocation to different plant parts, they play a potential role in the induction of drought tolerance. Along with modulating metabolic activities, plants also control their own metabolisms [34,40]. Among different secondary metabolic compounds, the tocopherols are lipophilic in nature and scavenge ROS, with the ability to recycle themselves and, as a result, reduce lipid peroxidation. Tocopherols belong to a family of eight members including α, β, γ, and δ tocopherols, along with their respective precursors (tocotrienols) that have high antioxidative activity and protect plants from stress through different metabolic processes [41]. Among these, α-Toc is largely known as vitamin E, with large antioxidant potential in comparison with other family members, but the production of α-Toc to reduce oxidative damage is cultivar-specific [42]. However, α-Toc exogenous application was found to be helpful for stress tolerance induction. For example, in wheat, exogenous application of α-Toc improved salt-stress tolerance [43]. In flax, genotypes foliar-applied tocopherol significantly improved salt stress tolerance [44]. Most of the studies presented are regarding salt tolerance induction and the application of α-Toc on adult-stage plants, and there is a lack of knowledge regarding its exogenous use at other growth stages. However, the discovery of the proper plant stage for better drought-stress induction through exogenous use of this compound is of prime importance [45].

Furthermore, there are missing gaps in understanding the proper physiological mechanism for the induction of stress tolerance at different growth stages by the exogenous use of organic compounds like that of α-Toc, also considering its translocation to specific plant parts. Therefore, the current work was aimed to quantify to which extent the foliar applied α-Toc could modulate growth in water-stressed maize plants and when it should be applied in the early growth stage. The goal of the study was to draw parallels among tissue-specific alterations in endogenous tocopherol levels, antioxidative defense mechanisms, and nutrient mobility patterns after α-Toc foliar application in maize plants grown in a drought-stressed rhizosphere. The research outcomes are helpful for optimizing strategies for growing maize with limited irrigation and in semi-arid and arid regions for better growth and production.

Maize (*Zea mays* L.) is the third most commonly produced cereal, after wheat and rice. It has a potential to grow in a wide range of environmental conditions and has gained great economic priority due to its potential nutritional quality all over the world, including in Pakistan [46]. In Pakistan, 1.016 million hectares are under maize cultivation, and 35% of the total cultivated area is rainfed, which is now facing problems in getting better production under dry environmental spells; this situation has further become more severe due to the present change in environmental conditions. Maize kernels are not only good and cheap source of carbohydrates but are also a rich source of carotenoids, proteins, and edible oil. However, due to changes in rainfall patterns along with the shortage of fresh water for irrigation, its production is under threat, along with that of other crops.

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

The present experiment was arranged in the research area of the Department of Botany, Government College University Faisalabad, Pakistan, (latitude 30◦30 N, longitude 73◦10 E, and altitude 213 m) under natural environmental conditions during August–September 2018. To avoid disturbances due to rain, the experimental area was covered with a polyethylene sheet. The design of the experiment was completely randomized in factorial arrangement, with three replications of each treatment. The experiment consisted of two drought levels (control and 70% field capacity), two highly yielding maize genotypes (EV-1098 and Agaiti-2002), and two levels of α-Toc (0 mmol and 50 mmol) in solution form applied as foliar spray with three replications of each treatment. The 70% field capacity used in the present study was selected following some earlier studies [47,48]. These two maize cultivars selected for study are used frequently in breeding programs to produce high-yielding hybrid genotypes. The experimental unit was comprised a total 24 equal-size plastic pots (28 cm × 30 cm), each filled with 10 kg soil. The soil was fully irrigated with canal water before seed sowing. When the soil

was at field capacity, seeds of both maize genotypes were hand sown. Before sowing, the soil was prepared well by hand digging. The seeds of both maize genotypes were purchased from Maize and Millet Research Institute, Yousafwala Sahiwal, Pakistan. Ten healthy seeds were sown in each pot. After five days of the completion of seed germination, five seedlings per pot were maintained by thinning. The water stress treatment was started just after the thinning of the seedlings by controlling the irrigation of half of the pots at 70% field capacity, and the other half of the pots were treated as control plants and irrigated to maintain 100% field capacity. Average mean daily length was 13/11 h, mean minimum and maximum day/night temperatures were 38 ± 3/30 ± 3 ◦C and 25 ± 2.5/20 ± 2.5 ◦C, respectively, the mean relative humidity during whole experiment (at daytime) was 50%. During the whole experimental period the averaged photosynthetically available radiation (PAR) measured at noon was varied from 794 μmolm−<sup>2</sup> s−<sup>1</sup> to 1154 μmolm−<sup>2</sup> s<sup>−</sup>1. Soil moisture content was maintained on daily basis and using a tensiometer, (Irrometer, Model RT-12 inch Riverside, CA, USA). Ten days after thinning, the seedlings were supplied exogenously as foliar spray with 0 mmol and 50 mmol solution of α-toc. Foliar spray of α-Toc solution was done in evening before sunset for the maximum absorption of the solution in leaf. The spray of α-Toc solution was made only once during the whole experimental period. An aliquot of 50 mL solution of each of α-Toc level was applied manually per replicate as foliar spray that costs only \$0.015 USD for six plants and \$65 USD per acre. The solution was prepared by dissolving the required measured quantity in minimal amount of ethanol, and then the final volume was maintained with distilled water. The 0 mmol treatment without α-Toc was considered as control treatment. Before foliar spray, 0.1% of Tween-20 was added as the surfactant to the finally prepared solution for the maximum absorption of the solution. The data for varying attributes was calculated after 15 days of α-Toc foliar spray. Fresh leaf material was taken in liquid nitrogen and stored at −80 ◦C for different biochemical studies.

#### *2.1. Soil Analysis*

The soil used was sandy loam with a saturation percentage of 47.5, average pH, and the ECe of the soil solution was 7.63 ds.m−<sup>1</sup> and 0.045 ds.m<sup>−</sup>1, respectively, organic matter (1.21%), with the available P (0.051 mg kg<sup>−</sup>1), K (30 mg kg<sup>−</sup>1), and total N (6.1 mg kg<sup>−</sup>1). The soil solution had soluble CO3 <sup>2</sup><sup>−</sup> (traces), HCO3 <sup>−</sup> (5.01 meq L<sup>−</sup>1), Cl<sup>−</sup> (8.49 meq L<sup>−</sup>1), SO4 <sup>−</sup><sup>2</sup> (2.01 meq L<sup>−</sup>1), Na (3.01 meq L<sup>−</sup>1), Ca<sup>2</sup>++Mg2<sup>+</sup> (13.91 meq L<sup>−</sup>1), and SAR (0.079 meq L<sup>−</sup>1).

#### *2.2. Estimation of Di*ff*erent Growth Parameters*

Two plants per replicate were uprooted and washed with distilled water for the estimation of different growth attributes. After calculating root and shoot lengths, number of leaves, leaf area, and fresh masses of roots and shoots, the same plants was then oven-dried using an electric oven at 70 ◦C for 48 h, and their dry masses were calculated.

#### *2.3. Estimation of Leaf Photosynthetic Pigments*

For the estimation of leaf chlorophyll (Chl.) *a*, *b*, total Chl, and Chl *a*/*b*, we followed the method described by Arnon [49]. The content of carotenoids (Car) was estimated following Kirk and Allen [50]. The extraction of the pigments was done using 80% acetone. Briefly, fresh leaf material (0.1 g) was chopped and put in 10 mL acetone for overnight at 4 ◦C and the absorbance of the extract was read at 663, 645, and 480 nm using a spectrophotometer (Hitachi U-2001, Tokyo, Japan). The quantities were computed using the specific formulae:

$$\text{Chl. } a = \text{[12.7 (OD 663) - 2.69 (OD 645)]} \times \text{V/1000} \times \text{W} \tag{1}$$

$$\text{Chl. } b = \text{[22.9 (OD 645) - 4.68 (OD 663)]} \times \text{V/1000} \times \text{W} \tag{2}$$

$$\text{Total Chl.} = \text{[20.2 (\Delta A \& 45) - 8.02 (\Delta A \& 63)]} \times \text{v/w} \times 1/1000 \tag{3}$$

$$\text{A caroterooid } (\text{\(\mu\text{g/g FW}\)} = \Delta \text{A} 480 + (0.114 \times \Delta \text{A} \, 663) - (0.638 \times \Delta \text{A} \, 645) \tag{4}$$

$$\text{Car} = \text{A Car/Em} \, 100\% \times 100 \,\tag{5}$$

$$\text{Emission} = \text{Em } 100\% = 2500\tag{6}$$

$$
\Delta \mathbf{A} = \text{absorbrane at respective wavelength} \tag{7}
$$

$$\mathbf{V} = \text{volume of the extract (mL)}\tag{8}$$

$$\mathcal{W} = \text{weight of the fresh leaf tissue (g)}\tag{9}$$

#### *2.4. Leaf Relative Water Content (LRWC)*

For the estimation of LRWC, the second one from top was used. In first step, after excising the leaf, the fresh weight was measured and tagged with a specific mark. Then, the leaf was soaked in dH2O for 4 h. Then, the leaf was taken out of the water, it absorbed the extra surface water, and we measured its weight again and termed the result the turgid weight. The same leaf was then oven-dried at 75 ◦C for 48 h and again weighed and termed this the dry weight of leaf. Then LRWC was estimated using the formula from the obtained data

$$\text{LRWC} \left( \% \right) = \frac{\text{Fresh weight of leaf -- dry weight of leaf}}{\text{Turing weight of leaf -- dry weight of leaf}} \times 100 \tag{10}$$

#### *2.5. Leaf Relative Membrane Permeability*

We followed the method described by Yang et al. [51] to find out the leaf relative membrane permeability (LRMP). The known amount (0.5 g) of excised leaf was cut into small pieces (approximately 1 cm) and put in test tubes having 20 mL of deionized dH2O. After vortexing well for 5 s, the EC of the assayed material was measured and termed as EC0. The test tubes containing leaf were then kept at 4 ◦C for 24 h, and the EC1 was measured. These test tubes containing leaf material were then autoclaved for 30 min at 120 ◦C and assayed the EC2. The LRMP was measured using the following equation:

$$\text{RMP } (\%) \, = \frac{\text{EC1 } - \text{EC0}}{\text{EC2 } - \text{EC0}} \times 100 \tag{11}$$

#### *2.6. Estimation of Leaf Malondialdehyde Content*

Content of malondialdehyde (MDA) was measured using the method given by Cakmak and Horst [52] as the measure of lipid peroxidation. The trichloroacetic acid (TCA) method was used for the estimation of MDA content. One gram of freshly taken leaf material was ground in TCA (10% solution). The supernatant (0.5 mL) was obtained from the homogenized material and mixed with 3 mL of thiobarbituric acid (TBA), prepared in 20% TCA. Test tubes having the triturate were kept at 95 ◦C for 50 min and then cooled immediately in chilled water. After centrifugation (10,000× *g*) of mixture for 10 min, the absorbance of colored part was read at 600 nm and 532 nm. The content of MDA was calculated using the following formula:

$$\text{MDA (mmol)} = \text{A (A532 nm} - \text{A 600 nm)} / 1.56 \times 105 \tag{12}$$

Absorption coefficient for the calculation of MDA is 156 mmol−<sup>1</sup> cm<sup>−</sup>1.

#### *2.7. Extraction of Antioxidant Enzymes and Total Soluble Proteins from Di*ff*erent Plant Parts*

For the extraction of antioxidant enzymes and total soluble proteins (TSP) from each plant part (root, stem, leaf), fresh material was ground (0.5 g) in chilled (10 mL) 50-mM phosphate buffer (pH 7.8). The mixture was then centrifuged at 10,000× *g* for 20 min at 4 ◦C. The supernatant so obtained was then used for the estimation of total soluble proteins (TSP) and estimation of antioxidative enzymes activities.

#### 2.7.1. Estimation of Total Soluble Proteins in Different Plant Parts

TSP in the buffer extracts was estimated following the method of Bradford [53]. The absorbance of the triturate was measured at 595 nm, and the quantities of the TSP in samples were computed using a series of protein standards (200–1400 mg/kg) prepared from analytical-grade bovine serum albumin (BSA).
