*3.6. Lipid Peroxidation*

Gamma radiation at 25 Gy showed a non-significant effect on lipid peroxidation MDA), while 50 Gy significantly increased (*p* < 0.05) MDA compared with the control value. Moreover, foliar application of stigmasterol lowered (*p* < 0.05) the MDA content in treated plants below the control. Stigmasterol at 200 ppm decreased MDA by 25%, 25.2%, and 31.25%, respectively, in the control plants and the 25 Gy- and 50 Gy-irradiated plants.

#### *3.7. Protein Profile*

Irradiated grain and application of stigmasterol induced a synthesis or disappearance of different protein bands compared to control plants (Table 2 and Figure 5). There were 23 polypeptide bands ranging from 11 to 185 kDa in the wheat proteins profile. Control plants had six protein bands with molecular weights of 11, 23, 30, 35, 43, and 48 KDa. The results revealed that 25 Gy induced the new polypeptides with 26 and 51 kDa. In addition, gamma radiation at 50 Gy induced polypeptides with 51 and 96 kDa. Moreover, foliar application of 200 ppm stigmasterol caused the appearance of new polypeptide bands with molecular weights of 185, 150, 141, 137, 89, 74, and 37 kDa.

**Table 2.** Changes in protein profile in leaves of wheat plants originated from irradiated grains and treated with stigmasterol. L1: Control, L2: 25 Gy, L3: 50 Gy, L4: 100 ppm stigmasterol, L5: 25 Gy + 100 ppm, L6: 25 Gy + 200 ppm, L7: 200 ppm stigmasterol, L8: 50 Gy + 100 ppm, L9: 50 Gy + 200 ppm.


**Figure 5.** Changes in protein profile in leaves of wheat plants originated from irradiated grains and treated with stigmasterol. L1: Control, L2: 25 Gy, L3: 50 Gy, L4: 100 ppm stigmasterol, L5: 25 Gy + 100 ppm, L6: 25 Gy + 200 ppm, L7: 200 ppm stigmasterol, L8: 50 Gy + 100 ppm, L9: 50 Gy + 200 ppm.

The combination of 25 Gy and 200 ppm stigmasterol induced the formation of new polymorphic polypeptides with molecular weights of 157, 137, 104, 89, and 79 kDa and unique bands at 137, 105, 89, and 30 KDa. Furthermore, the interaction of 50 Gy and 200 ppm stigmasterol resulted in the appearance of the protein bands at 137, 134, 89, 74, and 38 KDa. The treatment of 50 Gy, stigmasterol, and the interaction (25 Gy and 100 ppm stigmasterol) led to the disappearance of 30 kDa compared to the control plants.

#### **4. Discussion**

The results indicated no significant difference in plant height, root length, no. of leaves, shoot fresh weight, or root fresh weight between 0 Gy and 25 Gy treatments, while the vegetative growth of wheat plants decreased with radiation dose (50 Gy). The changes in the plant growth exposed to gamma radiation are due to the conversation of metabolic energy and metabolites to ameliorate the oxidative stress effects imposed by ionizing radiation [8]. The growth inhibition of wheat plants may be due to gamma radiation inhibition and the photosynthetic pigments, IAA, GA3, and the antioxidant capacity while inducing ABA and MDA, as mentioned in this study. The negative response of wheat plants is dependent on the nature and extent of the disturbance of cellular metabolism and finally cell damage. For the stigmasterol applied treatments after gamma pretreatment, some of the growth parameters did not differ between 0 ppm and 100 ppm of stigmasterol treatments (Table 1). On the other hand, the application of stigmasterol at 200 ppm improved the growth parameters of both irradiated and un-irradiated wheat plants (Table 1). Stigmasterol plays a vital role in regulating plant growth and development [21,23]. Furthermore, they added that these improvements may be due to increasing the efficiency of water uptake and utilization, enhancing cell division, and/or cell enlargement.

Leaf chlorophylls are indicators of chloroplast structure and are considered the central part of photosynthetic systems. Data in Figure 1 indicated that Chl *a*, Chl *b*, and carotenoids decreased significantly with 50 Gy gamma radiation doses. These results agree with the previous study [38] on barley plants. The reduction in photosynthetic pigments may be related to the increasing chlorophyll photo-oxidation and damage to the photosynthetic apparatus. Interestingly, the application of stigmasterol can alleviate the damaging effects of gamma radiation on photosynthetic pigment content. The increase in photosynthetic pigment in response to stigmasterol could be attributed to increased photosynthetic apparatus and antioxidant enzyme activities [5].

Interestingly, gamma radiation modulates and alters the endogenous hormones of wheat plants represented by the inhibition of GA and IAA, associated with the accumulation of ABA. These results were confirmed by the findings recorded by [39] on barley plants using gamma radiation (50 Gy). Moreover, foliar spraying of the irradiated plants with 200 ppm stigmasterol showed a marked increase in growth promoter levels (GA3, IAA), while the growth inhibitor (ABA) was decreased as compared with control plants. Similarly, the authors in [40] found that 150 or 200 ppm of stigmasterol resulted in the highest GA3 and IAA and the lowest ABA in both cultivars when compared to untreated plants. In addition, the authors in [41,42] stated that stigmasterol increases phytohormones, which can act as messengers and regulators of plant growth and development. The changes in the endogenous hormones were correlated with the changes in the activity of antioxidant enzymes, MDA, and proline, suggesting that the application of stigmasterol plays a central role in counteracting the injurious effects of gamma radiation.

In addition, changes in total soluble sugars in wheat plants may indicate that gamma radiation has induced oxidative stress [43]. The accumulation of TSS by stigmasterol may be due to scavenging ROS to maintain them at the optimum level, hence protecting cell metabolism from free radicals. This explanation was supported by the fact that stigmasterol may act as a photosynthesis activator in wheat plants (Figure 1).

Phenolics are important constituents with scavenging ability due to their hydroxyl groups, which hence may contribute directly to their antioxidant properties. Total phenol content was increased in 50 Gy-irradiated wheat plants. The alterations in the effect of gamma radiation on phenols may occur because irradiation can break the chemical bonds of bioactive compounds, releasing soluble phenolics with low molecular weight

and increasing the antioxidant potential of these compounds [44]. On the contrary, stigmasterol increased total phenol content compared to untreated plants. The significant positive correlation between stigmasterol and total phenol content may be induced by protective mechanisms against cell damage resulting from oxidative stress. These findings are consistent with those of [45], who demonstrated that stigmasterol promoted the antioxidant defense mechanism to counteract the negative effects of gamma radiation on faba bean plants.

Regarding proline, gamma radiation leads to a marked decrease in proline content. These results may be due to the radiosensitivity of proline or its oxidation using free radicals generated by gamma irritation. Moreover, stigmasterol stimulated proline content in irradiated and un-irradiated wheat plants. Similar results were obtained by [42] on flax plants. The results may be due to the role of stigmasterol, which stimulates proline content with high antioxidant properties for use in repair mechanisms against radiation effects on plant cell metabolism.

The application of gamma radiation (25 and 50 Gy) decreased the activity of POX enzymes while increasing the CAT activity. Ionizing radiation can trigger the production of ROS by interacting with atoms or molecules in the cell, which is called water radiolysis [38]. The results suggest that the increased activity of CAT induced by gamma irradiation can scavenge excess ROS, especially with the inhibition of peroxidase activity, consequently leading to the enhancement of the antioxidant capacity to overcome oxidative stress induced by water radiolysis. Moreover, treatment with stigmasterol increased POX and CAT activities (Figure 4). It appears that the increase in antioxidant enzymes may be due to the mediated role of stigmasterol in detoxification mechanisms against radio-oxidative stress.

Lipid peroxidation was markedly increased in the 50 Gy-irradiated wheat plants while decreasing with exogenous stigmasterol treatment. The increase in lipid peroxidation due to gamma radiation treatments was confirmed by [46] on the Black gram (*Vigna mungo* L.), who explained that ROS can react with nearly all cell constituents, which triggers free radical chain reactions that eventually cause membrane lipid peroxidation. They added that the membranes lose their stability, and their permeability is enhanced, leading to damage to the cell structure and disturbances of normal physiological functions as a result of free radical reactions. Moreover, treated wheat plants with stigmasterol improved stress tolerance by decreasing membrane lipid peroxidation in comparison to the corresponding control. This indicates that stigmasterol has a protective role in counteracting the damage induced by gamma radiation, resulting in the induction of antioxidant enzymes (POX and CAT) and non-enzymatic compounds (phenols and proline) associated with lower lipid peroxidation of wheat plants.

Gamma radiation induced the new polypeptides with molecular weights (Table 2 and Figure 5). These results agree with [47] on fenugreek and [38] on barley plants. They indicated that the formation of new bands (unique) is frequently caused by various DNA structural changes (e.g., breaks, transpositions, and deletions), which cause changes in amino acids and, as a result, the protein generated. Furthermore, proteins may play a role in signal transduction, anti-oxidative defense, and osmolyte production, all of which are crucial to the function and growth of plants [48]. In this regard, the authors in [49] suggested that a band with a molecular weight of 51 kDa could be linked to the Rubisco activase enzyme. Ribulose-1, 5-bisphosphate carboxylase activase is a key enzyme that initiates both photosynthetic and photo-respiratory carbon metabolism. Moreover, according to [45], a protein with a molecular weight of 26 KDa appears to be osmotically expressed in flax and sunflower plants under salinity stress to aid survival in stressed environments.

#### **5. Conclusions**

Gamma radiation at 25 Gy showed no significant difference in some growth parameters, Chl *a*, ABA, soluble phenols, and MDA compared to the control values. Gamma radiation at 50 Gy caused growth inhibition and lowered the photosynthetic pigments, promoting hormone and antioxidant capacity while inducing ABA hormone and lipid

peroxidation. The foliar application of stigmasterol, especially at 200 ppm, on wheat plants originating from gamma-irradiated grains, improved the photosynthetic pigments, induced an accumulation of osmolytes, phenols, the activity of antioxidant enzymes, and new polypeptides, as well as resynthesized the missed bands by radiation. It is also noteworthy that the stimulatory effects persisted in plants pretreated with stigmasterol throughout, promoting hormones while lowering the ABA hormone. Overall, the results proved the effectiveness of stigmasterol at 200 ppm application in alleviating the adverse effects of radiation stress on wheat plants, as indicated by their protective and stimulatory effects on growth attributes and biochemical constituents.

**Author Contributions:** Conceptualization, H.-A.A.H.; methodology, H.-A.A.H., S.K.M.K., F.M.E. and A.M.A.; software, H.-A.A.H., S.O.A. and S.K.M.K.; validation, H.-A.A.H. and A.A.R.; formal analysis, H.-A.A.H., F.M.E. and A.A.R.; investigation, H.-A.A.H. and F.M.E.; resources, H.-A.A.H., S.O.A., S.K.M.K. and A.M.A.; data curation, H.-A.A.H., S.O.A., F.M.E. and A.A.R.; writing—original draft preparation, H.-A.A.H. and A.A.R.; writing—review and editing, H.-A.A.H. and S.O.A.; visualization, H.-A.A.H. and S.O.A.; supervision, H.-A.A.H., S.O.A., S.K.M.K. and A.M.A.; project administration, H.-A.A.H. and S.O.A.; funding acquisition, H.-A.A.H., S.O.A., S.K.M.K., F.M.E. and A.M.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the manuscript.

**Acknowledgments:** The authors are thankful to the Botany and Microbiology Department, Faculty of Science (Girls Branch), Al-Azhar University, and the Biology Department, College of Science, University of Hafr Al-Batin. The authors also would like to thank the Botany Department, Agriculture and Biological Research Institute, National Research Centre.

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

#### **References**

