*2.8. Assay of Antioxidant Enzymes*

The crude enzyme was obtained according to the assay of antioxidant enzyme activities. A fresh flag leaf (2 g) of wheat plants was extracted in 10 mL of 100 mM phosphate buffer (pH 6.8) and kept at 4 ◦C overnight. The extract was centrifuged at 5000 rpm for ten minutes and reserved to assay the activities of enzymes [31].

#### 2.8.1. Peroxidase (POX) Assay

POX activity was assayed according to [32]. A total of 0.2 mL of crude extract was reacted with 5.8 mL of phosphate buffer (50 mM, pH 7.0), 2.0 mL pyrogallol (20 mM), and 2.0 mL hydrogen peroxide (20 mM). The increase in absorbance was determined within 60 s against a reagent without enzyme at 470 nm using a spectrophotometer. The amount of crude enzyme that converts one micromole of hydrogen peroxide in one minute at room temperature equals one unit of enzyme activity [33].

#### 2.8.2. Catalase (CAT) Assay

The CAT activity was assayed [34] by mixing 40 μL of enzyme extract and 9.96 mL phosphate buffer (pH 7.0) containing H2O2 (0.16 mL of 30% H2O2 in 100 mL of 50 mM phosphate buffer). CAT activity was determined by measuring the rate of H2O2 absorbance change in one minute against a buffer blank at 250 nm using a spectrophotometer. One unit of enzyme activity is equivalent to the amount of enzyme that reduced 50% of the hydrogen peroxide in one minute at room temperature.

## *2.9. Lipid Peroxidation*

The level of lipid peroxidation (malondialdehyde; MDA) was measured according to [35]. A total of 200 mg of fresh flag leaf of wheat plants was ground in 10 mL of 5% trichloroacetic acid (TCA) and centrifuged at 15,000 rpm for 10 min. Then, 2.0 mL of the extract was added to 4.0 mL of 0.5% thiobarbituric acid (Mallinckrodt. Inc., Paris, KY 40361, USA) in 20% TCA, heated in a boiling water bath for a half-hour, immediately cooled, and centrifuged at 10,000 rpm for ten minutes. The reading was noted at 532 and 600 nm using a spectrophotometer. By subtracting the absorption value at 600 nm, the absorption coefficient of 155 nmol cm−<sup>1</sup> was used to assess the MDA content as nmol g−<sup>1</sup> FW.

#### *2.10. Protein Profile*

The rapid freeze-dried leaf samples (0.2 g) were extracted with 1 mL of protein buffer and kept in the freezer overnight and then vortexed for 15 s and centrifuged at 5000 rpm at 4 ◦C for 15 min. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS– PAGE) was performed [36]. The molecular weight of the isolated proteins was estimated using standard molecular weight markers (standard protein markers, 11–180 kDa; Sigma, St. Louis, MO, USA). The protein bands were stained with Coomassie Brilliant Blue G-250 (Sigma, St. Louis, MO, USA).

#### *2.11. Statistical Analysis*

The experiment was statistically analyzed as a split-plot design according to [37]. The significant differences were statistically evaluated by Duncan's test and one-way analysis of variance (ANOVA) using SPSS, version 18.0 (Statistical Package for Social Science, Copyright 2010, Chicago, IL, USA) to discriminate significance (defined as *p* ≤ 0.05). The least significant differences (LSD) at 5% were calculated for means comparisons.

#### **3. Results**

#### *3.1. Growth Parameters*

Gamma radiation at 25 Gy significantly decreased flag leaf area, shoot dry weight, and root dry weight while showing no significant difference in plant height, root length, no. of leaves, shoot fresh weight, and root fresh weight. Moreover, except for root fresh weight, gamma radiation at 50 Gy significantly decreased (*p* < 0.05) the shoot and root lengths, no. of leaves, flag leaf area, shoot fresh and dry weights, and root dry weight of wheat plants compared with the control group (Table 1).

**Table 1.** Effect of stigmasterol treatments on the growth parameters of wheat plants grown from irradiated grains. The different letters (a–f) show statistical significance at *p* < 0.05. Differences are statistically significant at *p* < 0.05; vertical bars indicate ±SD.


Foliar application of stigmasterol at 100 ppm showed a significant effect on flag leaf area, root length, and root dry weight but showed no changes in plant height, no. of leaves, shoot fresh and dry weights, and root fresh weight of the non-irradiated. Stigmasterol at 200 ppm increased significantly plant height, root length, no. of leaves, shoot dry weight, and root dry weight while showing no changes in flag leaf, shoot fresh weight, and root fresh weight compared to the control plants.

The treated plants with 25 Gy + stigmasterol at 100 ppm caused significant increases in shoot and root dry weights, decreases in plant height and shoot fresh weight, and no significant effects on root length, flag leaf area, and root fresh weight compared to the corresponding control. Regarding the irradiated plants with 25 Gy, stigmasterol at 200 ppm significantly (*p* < 0.05) improved the shoot length, root length, number of leaves/plant, flag leaf area, shoot fresh and dry weights, and root dry weight over the control values.

The irradiated plants with 50 Gy and stigmasterol at 100 ppm, except for shoot fresh weight, showed significant increases in the following growth parameters: shoot length, root length, number of leaves/plant, flag leaf area, shoot dry weight, and root fresh and dry weights (28.26%, 21.33%, 21.41%, 36.33%, 52.63%, 59.84%, and 55.56%, respectively) of wheat plants over the corresponding control. Regarding the irradiated plants with 50 Gy, stigmasterol at 200 ppm significantly (*p* < 0.05) improved the shoot length, root length, number of leaves/plant, flag leaf area, fresh and dry weight of shoot, and root by 43.5%, 33.3%, 42.8%, 47.4%, 39.1%, 129.0%, 95.0%, and 66.7%, respectively, over the control values.

The effect of stigmasterol wheat plant growth depends on the applied concentration. However, stigmasterol at 100 ppm showed no significant effects on some of the

physiological attributes compared to control plants. Foliar application of stigmasterol at 200 ppm could improve the growth parameters in non-irradiated and 25 Gy-irradiated plants. Moreover, 200 ppm could counteract the effects of 50 Gy gamma radiation on wheat plant growth.

#### *3.2. Photosynthetic Pigments*

Irradiated grains had a significant (*p* < 0.05) influence on the photosynthetic pigments, Chl *a*, Chl *b*, and carotenoids of wheat plants (Figure 1). Radiation at 25 Gy showed no significant effect on Chl *a*, while it decreased significantly Chl *b* and carotenoids compared to the control plants. Radiation at 50 Gy produced progressive damage in the photosynthetic pigments compared to the control plants. On the other hand, the application of stigmasterol at 100 and 200 ppm showed significant (*p* < 0.05) increases in Chl *a* and Chl *b*, and no significant effect in carotenoids in non-irradiated plants compared to the control plants. Moreover, the application of stigmasterol at 100 and 200 ppm significantly (*p* < 0.05) increased Chl *a*-, Chl *b*-, and carotenoid-irradiated plants compared with corresponding control values. Stigmasterol at 200 ppm was the most effective treatment for increasing the photosynthetic pigments.

**Figure 1.** Effect of stigmasterol treatments on photosynthetic pigment; (**A**) Chl *a*, (**B**) Chl *b*, and (**C**) carotenoids contents of wheat plants grown from grain irradiated with gamma rays. The different letters (a–e) show statistical significance at *p* < 0.05; vertical bars indicate ± SD.

#### *3.3. Endogenous Phytohormones*

Data in Figure 2 revealed that gamma radiation (25 and 50 Gy) significantly decreased in GA and IAA compared to the control values. Moreover, 25 Gy showed no significant effect on ABA content, while 50 Gy caused a significant increase compared to the control value. In terms of stigmasterol, 200 ppm seemed to be more effective than the control, while 100 ppm treatments were not analyzed. The results showed that stigmasterol at 200 ppm markedly increased the growth promoter (GA3 and IAA) while decreasing the growth inhibitor (ABA) compared with control (non-irradiated plants).

**Figure 2.** Effect of stigmasterol treatments on endogenous phytohormones; (**A**) IAA, (**B**) GA, and (**C**) ABA of wheat plants grown from grain irradiated with gamma rays. The different letters (a–d) show statistical significance at *p* < 0.05; vertical bars indicate ±SD.
