*2.3. Plant Biomass*

Different plant samples (roots, shoots, and seeds) were dried until they attained a constant weight following the method previously described [40]. Plants were separated at 80 days after sowing (DAS) in root and shoots for various physiological and biochemical analyses.

#### *2.4. Chlorophyll Content*

Chlorophyll extraction was carried out from leaf discs of plants following the method previously described [41], and chlorophyll a, b, and total chlorophyll were computed from the extinction values following the equation of [42].

#### *2.5. Total Protein, Relative Water Content, and Harvest Index*

Total protein was estimated following the method previously described by Bates et al. [43]. Leaf relative water content (RWC) was calculated by the method described by Grieve & Grattan [44]. For chlorophyll a and b extraction, leaf discs of plants were mixed with 5 mL of 80% acetone overnight. After centrifugation, the supernatant was used for absorbance reading at 645 nm (chl a) and 663 nm (chl b) using a spectrophotometer (Hitachi-U2001, Tokyo, Japan). Relative water content (RWC) was measured following Cavell [45], where selected leaves were rehydrated by soaking in deionized water for 24 h. Fully turgid leaves were weighed and, subsequently, oven-dried for 48 h at 80 ◦C. Here, FW is fresh weight, DW is dry weight, and TW is turgid weight. Plant yield efficiency in terms of the harvest index (HI) was computed according to Mehraban et al. [46]. The amount of aboveground biomass production invested into harvestable organs was calculated as follows:

HI = (Seed dry weight/Aboveground plant biomass at harvest) × 100

#### *2.6. Proline and Glycine Betaine Content*

Proline content was analyzed following absorbance of toluene soluble brick-red colored complex at 520 nm [47]. The concentration of proline was estimated by referring to a standard curve drawn from known concentrations of proline. GB was determined following the absorbance of the betaine−peridotite complex with iodide in an acidic medium at 360 nm as per the method of Dubois et al. [48]. Reference standards of GB were prepared as 50–200 μg mL−<sup>1</sup> for sample estimation.

#### *2.7. Catalase and Superoxide Dismutase Estimation*

CAT and SOD were measured using 0.2 g fresh leaf samples. The obtained mixture (0.05 M Tris–HCl buffer (pH = 7.5) and samples) was centrifuged at 13,000 rpm for 20 min at 4 ◦C.

After centrifugation, the supernatant was used to estimate CAT according to a modified method of Kar and Mishra [49], and SOD was assayed by the method described by Beauchamp and Fridovich [50].

### *2.8. Nutrient Analysis and Zinc Content*

Nutrient extraction was done according to Carvalho et al. ([51], modified). Two g of flour for each sample was placed in labeled crucibles and ashed for 3 h in a furnace at 550 ◦C. Samples were digested with 2–2.5 mL of concentrated HNO3,then placed into the furnace at 550 ◦C for 1h. After that, 10 mL of diluted HNO3 (HNO3:H2O 1:2 dilution ratio) was added to the sample and placed for 5 min in a sand bath. The mixture was filtered through Whatman paper for purification. The atomic absorption spectroscopy (AAS) (Varian AAS FS 240 Model, Varian Inc., Palo Alto, CA, USA) method was used to analyze the mineral concentration. Five replicates were done per sample.

#### *2.9. Statistical Analysis*

Each parameter was investigated in its separate independent experiment. Analysis using variance (ANOVA) was performed, and subsequent comparison of the means was done using Duncan's multiple range test at *p* = 0.05. Treatment mean ± SE (*n* = 12) are for growth and yield attributes and (*n* = 4) for the other tested characteristics.

#### **3. Results**

#### *3.1. Plant Growth, Yield, and Yield-Related Traits*

Drought stress significantly affected (*p* < 0.05) growth parameters, yield, and yield components (Table 2). Zn application and AMF inoculation significantly enhanced plant growth and yield components under well-watered conditions and drought stress. Zn application and/or AMF inoculation enhanced all growth parameters and yield attributes. For example, RDW increased by 25, 30, and 46%, respectively, for these three treatments, compared to the control treatment. For 1000-grain weight, the increase was 9, 0.4, and 3% for the same three treatments (Table 2). Drought stress significantly decreased plant growth and grain yield attributes. The combination of Zn application and AMF inoculation alleviated the adverse effect of drought stress on all parameters except for grain weight per spike, which decreased by 45.9%. The decrease in 1000-grain weight was noticeably smaller after the application of Zn and AMF compared to drought stress only (Table 2). Under this constraint, Zn significantly enhanced 1000-grain weight. However, AMF or Zn did not affect grain number and grain weight per spike under drought stress (Table 2).

**Table 2.** Application effect of Zinc fertilizer and mycorrhizal inoculation on yield attributes of bread wheat.


Grown under water-stress conditions, SDW = Shoot dry weight, SL = shoot length, RDW = root dry weight, RL = root length. Values in columns followed by different letters are significantly different at *p* ≤ 0.05. Means ± standard deviation.

#### *3.2. Chlorophyll Content*

There was significant variability of chlorophyll content due to Zn fertilization and AMF inoculation. Chlorophyll compounds were increased by Zn and AMF inoculation and their combination under both control and drought conditions. Chl a content increased by 69, 68, and 75%, Chl b content by 84, 87, and 90%, and Chl a+b content by 73, 74, and 80%, respectively, after the application of Zn and AMF inoculation and their combination compared with the control. However, there were nonsignificant effects on Chl a/b content under drought stress after Zn and AMF treatments. Overall, the highest chlorophyll content was observed in the plants treated with combined Zn and AMF under both control and drought-stress conditions (Figure 1).

**Figure 1.** Individual and combined application of Zn and/or AMF effects on chlorophyll a, chlorophyll b, and chlorophyll (a+b) of a bread wheat cultivar under control (well-watered) and droughtstress conditions. Bars with different letters are significantly different at *p* ≤ 0.05.

#### *3.3. Protein, Relative Water Content, and Harvest Index*

Protein, relative water content, and HI were significantly(*p* ≤ 0.05) affected by drought stress. However, AMF inoculation and/or Zn and their combination improved protein content by about 15%. The highest level was 15.35% in plants that received combined Zn and AMF treatment under drought stress (Figure 2). Under well-watered conditions, Zn application, AMF inoculation, and their combination enhanced RWC by 14.10, 16.23, and 23.90%, respectively (Figure 2), although it decreased by 20.35, 20.15 and 21.66%, respectively, under drought stress. Under drought stress, Zn application and/or AMF inoculation enhanced HI by 45.91, 84.80 and 28.82%, respectively, compared to control conditions (Figure 2).

**Figure 2.** Zn application and/or AMF inoculation effect on protein content, relative water content (RWC), and harvest index (HI) of bread wheat cultivar under control (well-watered) and droughtstress conditions. Bars with different letters are significantly different at (*p* < 0.05).

#### *3.4. Accumulation of Glycine Betaine and Proline Content under Drought Stress*

Treatment effects were significant for GB and proline. Under drought stress, the application of Zn and/or AMF inoculation increased GB compared to control conditions by 31.71, 10.36, and 70.70%, respectively. However, the level of GB was higher in the control under the same conditions (1.69 μmol g−1). Regarding proline content, results showed significant variability (*p* < 0.05) under both control and drought conditions. AMF inoculation improved proline content more than Zn application. Generally, drought stress decreased proline content compared to control conditions (Figure 3).

**Figure 3.** Zn application and/or AMF inoculation effect on glycine betaine and proline content in the bread wheat under control (well-watered) and drought-stress conditions. Bars with different letters are significantly different at *p* ≤ 0.05.

#### *3.5. Activities of Antioxidant Enzymes*

The antioxidant defense was enhanced significantly (*p* < 0.05) under drought stress mostly for peroxide dismutase activity, and the increase was outworn by 50% for all the treatments compared to the well-watered conditions. AMF inoculation and Zn application improved SOD and CAT activity by 58 and 56%, respectively, under drought stress (Figure 4). Under well-watered conditions, Zn and/or AMF did not significantly a meliorate the enzymatic reaction (Figure 4).

**Figure 4.** Zn application and/or AMF inoculation effects on catalase (CAT) and peroxide dismutase (SOD) in the bread wheat under control (well-watered) and drought-stress conditions. Bars with different letters are significantly different at *p* ≤ 0.05.

#### *3.6. Nutrient Composition of Wheat Flour*

Macro and/or microelements in wheat flour showed significant variability due to the combination of Zn application and AMF inoculation under drought stress (Figure 5). However, treatment effects were nonsignificant under control conditions for micronutrients. Drought stress significantly increased Na and Cu, compared to the control, by 21.68 and 36.13%, respectively. The microelements Fe, Mn, Zn, and Cu in the flour had very low concentrations (0.003–0.089%). On the contrary, macro elements were significantly affected by drought stress. Zn and/or AMF inoculation improved K, Ca, and P. For example, Zn combined with AMF increased K and P by 51.61 and 75%, respectively, under drought stress (Figure 5).

**Figure 5.** Zn application and/or AMF inoculation effect on macronutrient and micronutrient content in bread wheat cultivar under control (well-watered) and drought-stress conditions. Bars with different letters are significantly different at *p* ≤ 0.05.

#### **4. Discussion**

The main objective of this study was to analyze the potential of Zn fertilizer and AMF for improving wheat performance under drought stress. Drought significantly affects wheat yield worldwide [52,53]. AMF improved water assimilation in many plants under drought stress as fungus mycelia can penetrate the soil and increase water absorption and transportation from roots to other plant parts as a tolerance mechanism to drought stress [54]. Fertilization using several nutrient sources increased plant vigor against environmental stress [55]. Zn fertilization and its co-application with AMF were evaluated by studying variability in different physiochemical mechanisms as described in a previous study [30]. AM fungus and/or Zn application positively affected morphological traits, increasing plant growth and yield attributes, as was reported previously [3]. Therefore, the efficiency of Zn and AMF application is confirmed in this study. Drought stress decreased plant dry weight and length. This was confirmed in another study [56]. Osmotic variability due to variations in osmotic potential caused a significant decrease in the fresh weight of plants due to a decrease in cellular division, consequently causing a decrease in total plant weight [57]. Zn combined with AMF treatment effectively improved plant growth under drought stress by sustaining higher water content in cells, thus ameliorating drought stress. All parameters were alleviated by Zn application and AMF inoculation, except for grain weight per spike, which decreased by 45.9% under drought stress. Zn improved chlorophyll synthesis, as it acts as a catalyst and cofactor of various enzymes [58]. This finding was confirmed in this study. Cell membranes, which cause improvement in the photosynthetic process, were protected by the application of Zn and AMF [59]. Similar findings were observed in rice and wheat plants. Zn increased all studied photosynthetic pigments [60].

Protein content was significantly enhanced only under stress conditions, and the effect was increased with Zn treatment. The potential effect of Zn on soluble protein in wheat under drought stress was previously reported [61]. Also, amino acid synthesis, which helps in protecting plants from drought stress, is related to Zn application [62]. Faced with drought stress, plant tolerance can be improved via drought escape by early flowering time in drier environments, avoidance by transpiration regulation, development of extensive root systems, trait flexibility, maintenance of water management in tissues, antioxidant scavenging, and secretion of plant growth substances by plant growth regulators and osmotic regulation [63]. Under drought stress, plants used stomatal closure to reduce the transpiration rate, causing an increase in leaf temperature. However, compared to the control, under the same conditions, Zn and/or AMF increased RWC and HI. These findings confirmed that Zn, at an optimum dose, maintained water status, stomatal conductance, and osmotic adjustment in many plants, such as chickpea, under drought stress [64]. For osmotic homeostasis regulation under stress conditions, proline as an osmolyte played an important role in protecting plants against drought [65]. The compatible solute accumulation leads to improved turgor potential and water content of plants, which contributes to enhanced plant growth performance under stress conditions. AMF was also reported to stimulate compatible solute and protein content under stress conditions [66]. The results of this study confirmed previous findings [67], mentioning that Zn and AMF acted synergistically to enhance proline and total protein content.

Drought stress reduces the assimilation of nutrients and inhibits the activities of important enzymes that are involved in the synthetic processes of energy for plant growth. For that, plants have an antioxidant defense against stress conditions, having different antioxidant reactions protecting plants under water deficit [68].This defense reaction was expressed by different enzymes which convert these harmful oxygen species to reduce their negative effect on plant growth [69]. In our study, drought stress increased levels of CAT and SOD compared to the control (well-watered). AMF inoculation or Zn application enhanced the activity of these antioxidant enzymes under drought-stress conditions, being more pronounced when applied together. This finding was confirmed by many reports mentioning enhancement in the enzymatic antioxidant defense system due to AMF and/or

Zn application in wheat under drought stress [70]. Zn reduced oxidative damage under stress conditions, which confers stress tolerance to plants [71]. The increase in antioxidant enzyme activity is assessed through decreased malondialdehyde content and H2O2 content as noted in many crops, for example, in sunflower (*Helianthus annuus*), chickpea (*Cicer arietinum*) [72], lentil (*Lens culinaris*) [73], and wheat leaves.

Moreover, nonenzymatic molecules, such as the accumulation of GB in wheat leaves, decreased the impact of drought stress as an antioxidant defense. Zn and AMF increased the activity of GB under drought stress, being more pronounced when applied together (Figure 3). It was reported [74] that enzymatic antioxidant defense systems were enhanced in wheat due to Zn application under drought-stress conditions.

In addition, as a strategy to tolerate stress, plants balance the concentrations of macro and micro elements. The results showed that drought stress significantly increased Na and Cu. However, Fe, Mn, Zn, and Cu were present in the flour at very low concentrations (0.003–0.089%). Many reports confirmed this finding explaining that a different nutrient supply as Zn and biofertilizer (AMF) can increase plant growth under water stress, depending on the severity of the drought, the concentration of the elements in the soil, and other conditions [75]. Application of Zn, AMF inoculation, and their combination increased K, Ca, Mg, and P. For example, Zn combined with AMF increased K and P by 51.61 and 75%, respectively, under drought stress.

#### **5. Conclusions**

This study investigated the effect of Zn application and AM fertilization. Wheat growth, yield, the antioxidant mechanism (enzymes, osmoprotectors, and osmolytes), and nutrient balance were improved. Root proliferation was significantly enhanced due to Zn and AMF fertilization under stress conditions. Zn fertilizer combined with AMF had larger impacts on measured traits. As a work perspective, deep research is needed to be done under field conditions to confirm these results on the effects of Zn and AMF and to be recommended to improve wheat production under drought stress. Moreover, extensive work on molecular studies as the contribution of differentially expressing endogenous genes encoding antioxidant enzymes should be established.

**Author Contributions:** N.A.—writing manuscript, methodology, data analysis; A.V.B. methodology and assistance; C.S.—methodology; M.L.—data analysis, writing-review, visualization, and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the National Research Foundation through the South African Research Chairs Initiative (UID 84647).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy and ethical reasons.

**Acknowledgments:** This research was funded by the National Research Foundation through the South African Research Chairs Initiative.

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

#### **References**


**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
