الفهرس 
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Abstract
The present work aims to study the oxidative stress of cerium dioxide (CeO₂-NPs) nanoparticles on the growth, physiology, and molecular changes of Brassica napus plants and the effectiveness of using melatonin by soaking seeds before planting in alleviating the negative effect of nanoceria on Brassica napus plants at different stages of growth. To achieve this goal, the study included three parts:
1- Green synthesis of cerium oxide nanoparticles using Aloe vera extract and studying the physical properties and biological activity of the nanoparticles specially cytotoxicity and antimicrobial activity.
2- Study the effectiveness of different concentration from cerium oxide nanoparticles and melatonin on growth of B. napus seed till 12 days to select the optimum concentration of both.
3- Foliar application on B. napus leaves with different concentration of cerium oxide nanoparticles (250,500, and 1000 ppm), which presoaked before planting with 25 µM of melatonin for 12 days on morphological, oxidative stress physiological and finally molecular changes on B. napus leaves compared with untreated and treated with different concentrations of nanoceria alone.
The morphological, biochemical and molecular parameters on B. napus leaves at 45 days and at 180 harvest-days, then statistical analysis had been done to evaluate the effectiveness of melatonin hormone to alleviate the possible negative impact of different concentration of nanoceria on canola plant.
The result can be summarized in the following:
1- Cerium oxide nanoparticles obtained by modified green method at a specific range of temperature the size distribution by DLS, CeO₂-NPs in suspension was approximately 19.44±0.5 nm. And its zeta potential was measured to be − 18.8 mV at pH 7.5, and the particles are spherical, and the average diameter of CeO₂-NPs was approximately 3.42±1.05 by TEM technique.
2- The XRD spectrum of CeO₂-NPs showed peaks at 2θ = 28.5°, 33.0 °, 47.4°, 56.2°, 59° and 69.3° and they correspond to (111), (200), (220), (311), (222) and (400) lattice planes, respectively. The XRD pattern was the same after two years, so it confirmed the nanoparticle composition.
3- The FTIR pattern of CeO₂-NPs results indicated the existence of O–H stretching vibration of hydroxyl groups, and Aloe vera FTIR showed phenolic −OH group and carboxyl (CO) group, and also Ce (NO₃) ₃.6H₂O showed -NH₂ so that indicated Ce was stabilized but also oxidized by Aloe vera extract.
4- Bioactivities of CeO₂-NPs showed anti-microbial activity, and with cytotoxicity and also antitumor, so antimicrobial activity showed pure compound CeO₂-NPs showed antibacterial activity on the tested Gram-positive strains S. aureus (NRRLB-767) and B. Subtilis (ATCC-6633), but had no effect against E. Coli (KY 780346.1) and P. aeruginosa (KY54964.1) whereas, showed significantly affect as antifungal activity against A. niger (NRRLA-326) and A. solani (MECC 756) but C. albicans (ATCC6633) showed no effect on.
5- Cytotoxicity of nanoceria size was 1-3 nm nanoceria showed towards cultured human lung fibroblast cells MRC-5 detected using MTT assay under these experimental conditions with CC50 = 52.38 ± 3.95 µg/ml, and nanoceria was toxic towards cultured human lung cancer cells A549 detected using MTT assay under these experimental conditions with IC50 = 27.13 ± 1.29 µg/ml.
6- The seed germination for B. napus seed presoaked with different concentrations of melatonin recorded the best concentration at (25 µm) with 100% of germination, whereas B. napus presoaked with different concentrations of nanoceria and the best concentration of nanoceria was at 500 ppm with 95% of germination.
7- Brassica napus at the age of 45 old days seed pretreated with melatonin then sprayed with of different concentrations of CeO₂-NPs or only sprayed with different concentrations of CeO₂-NPs showed a significant increase in growth parameter. The best concentration is 500 ppm CeO₂-NPs and Mel+500 ppm CeO₂-NPs that dramatically alter (p ≤ 0.05) the fresh, dry weights, shoot, root length, and leaves number at 500 ppm CeO₂-NPs as compared with control. The best concentration of plant recovery was Mel+500 ppm CeO₂-NPs significantly increased fresh, dry weight and length shoot and root and leaf number and Pods by 123.1%, 166.6 %, 119.4 %, 220.5 %, 118.9 %, and 232.1 % respectively compared to the 500 ppm CeO₂- NPs treatment.
8- The results showed that B. napus leaves pre-treated with 25 µM melatonin before planting had a positive effect to reduce the oxidative stress of foliar spraying with different concentrations of nanoceria on membrane stability index and reduce the electrolyte leakage comparing with plants sprayed with CeO₂-NPs alone.
9- The ultra-structural changes in control and treatment using 500 ppm CeO₂-NPs, Mel+500 ppm of CeO₂-NPs B. napus leaves have been shown. At 500 ppm CeO₂-NPs led to alteration of the ultrastructure structure of B. napus leaves led to condensed thylakoid structures, enlarged mitochondria, and big space inside cells could be seen, and at Mel+500 ppm CeO₂-NPs showed a regular nuclear structure with nuclear membrane could be seen in control and treated leaves.
10- The chlorophyll content a, b, also carotenoids, total pigment, so the chlorophyll significantly increased in B. napus leaves treated with different concentrations of CeO₂-NPs or pre-treated with melatonin, especially at 500 ppm CeO₂-NPs and Mel+500 ppm CeO₂-NPs.
11- Carbohydrates content in B. napus leaves to significantly increase soluble, insoluble sugar and total carbohydrate at 500 ppm CeO₂-NPs with 1.63 fold, 1.46 fold and 1.52 fold, respectively, Mel+500 ppm CeO₂-NPs showed the highest values at insoluble sugar and soluble at by 291.2 %, and 202.6%, respectively compared to untreated control.
12- Ce accumulation was increased with increase dose concentration while upon Mel+500 ppm CeO₂-NPs obviously decreased Ce accumulation, while, the nitrogen element was increased significantly at 25 µM melatonin treatment and at Mel+500 ppm CeO₂-NPs. However, the phosphorus content significantly decreased in all treatments except at 25µM melatonin treatment and Mel+250 ppm CeO₂-NPs. Potassium and magnesium element increased at 25 µM melatonin treatment and Mel+250 ppm CeO₂-NPs, but calcium element significantly increased at 25 µM melatonin treatment and 250ppm CeO₂-NPs.
13- The content of nitrogen fractions (TSP, FAA), so the changes in total soluble protein in B. napus leaves sprayed with different concentrations of nanoceria showed an increase at 250 and 500 ppm CeO₂-NPs compared to control. The highest value of TSP was found in Mel+1000ppm CeO₂-NPs treatment and increased by 392.7%, as compared with control. However, the content of free amino acid increased highly at 250 ppm and Mel+1000 ppm CeO₂-NPs treatment but decreased at 500 and 1000 ppm CeO₂-NPs.
14- The analysis by SDS-PAGE electrophoresis pattern of B. napus leaves protein pattern represented by 9 protein bands of molecular weight 9,13,15,18,22,33,35,63,116 KDa whereas under different treatment showed changes as recorded and distributions of bands molecular weight at 48.0,54.0, 72.0,84.0,180.0,245.0 KDa a new band appeared. At 245.0 KDa disappeared only in control, bands at 54 and 180 KDa disappeared in all other treatments except in leaves of B. napus treated with 25 µm Mel and 250, 500 and 1000 ppm CeO₂-NPs. protein bands of molecular weights 84, 72 and 48 KDa exist in all treatments and were not found in control and melatonin alone.
15- NBT stain, which gave dark spots detected by Fluorescence Microscopy. Furthermore, the elevated levels of CeO₂-NPs, more dark spots of blue formazan could be seen in leaves, superoxide anion radical showed that all treatment was significantly decreased Mel+250 ppm CeO₂-NPs, Mel+500 ppm CeO₂-NPs and Mel+1000 ppm CeO₂-NPs decreased by 50%, 16.6%, and 50%, compared to the same concentrations of nanoceria without melatonin.
16- The hydrogen peroxide level at 250, 500, and 1000ppm of nanoceria increased by 230.5%, 363.8%, and 582.3% compared with control, respectively. Also Mel+ 250, and 1000 ppm of nanoceria significantly increased H₂O₂ level by 111 %, and 359.8% compared to control While, Mel+500 ppm of nanoceria was significantly reduced H₂O₂ level by 93.9% compared to control.
17- Foliar application of 250,500, and 1000 ppm of nanoceria has dramatically reduced the content of nitric oxide by 17.1%,23.19%, and 7.48%, respectively, compared to control, however, foliar application of Mel+1000 ppm CeO₂-Nps recorded the most significant increase of nitric oxide content.
18- MDA level increased in comparison to control response to nanoceria, which plays a role as an antioxidant at different concentrations of CeO₂-NPs 1000ppm by 303.5%. This led to a decrease in the MDA contents by 69.2%, 57.7 % and 38.8% in Mel+ 250 ppm CeO₂-NPs, Mel+ 500 ppm CeO₂-NPs and Mel+1000ppm CeO₂-NPs treatments compared to CeO₂-NPs treatments without melatonin. LOX enzyme was significantly decreased by 47.0 % at 1000 ppm CeO₂-NPs compared to control. Application of melatonin as seed soaking increased LOX activity in leaves of Brassica napus sprayed with 250ppm, 500ppm and 1000ppm CeO₂-NPs by 103.0%, 109.0 % and 116.2 %, respectively, compared to the control.
19- The anti-oxidant activity showed a highly significant increase of B. napus plant sprayed with different concentrations of nanoceria or per-soaked with melatonin as compared to control. At 500 ppm+Mel of CeO₂-NPs was ranked as the first with 130.4% than all other treatments.
20- The activity of antioxidant enzymes defence in B. napus leaves. POX and CAT activity showed a significant increase at (Mel+500 ppm CeO₂-NPs) by 201.1%. and 144.7% compared to control and SOD, APX, and GR showed enhanced activity at Mel+500ppm of nanoceria by 357.3%, 319.8% and 493.5% compared to control. Moreover, MDHAR and DHAR recorded a significant enhancement at Mel+500 ppm CeO₂-NPs by 600.4% and 205.3% compared to control B. napus leaves.
21- The maximum content of α tocopherol and ascorbic acid were found in Mel+500 ppm CeO₂-NPs treatment and increased by 432.4 % and 461.5% compared to the control treatment. Total phenol was significantly reduced by 60.9%,66.6%, and 75.8 % in leaves of Brassica napus plants sprayed with 250,500 and1000 ppm CeO₂-NPs, respectively. Also, treatment with melatonin, when applied alone or with CeO₂-NPs, decreased phenol content compared to control treatment. The lowest phenol content was recorded in melatonin treatment only. Flavonoids were increased at 500 ppm CeO₂-NPs, and Mel+250 ppm CeO₂-NPs was the most significant increase of antioxidant compounds, and anthocyanin showed a significant increase in Mel+500 ppm CeO₂-NPs.
22- Hormones related to growth that were evaluated including IAA, Mel, GA3, BR, kinetin and ABA. Application of 25 µM melatonin alone significantly increased IAA level by 121.4 % over the control and also increased this level in treatments of Mel+500 ppm of CeO₂-NPs and Mel+1000 ppm of CeO₂-NPs by 121.3% and 169.2%, respectively compared with treatments without melatonin. Application with melatonin significantly increased GA₃ levels in 250, 500 and 1000ppm CeO₂-NPs by 128.5 %, 180.0 and 448.8 %, respectively, compared with treatments without melatonin. On the other hand, the content of phytohormones such as BR and Kinetin were highly increased at 500 ppm of CeO₂-NPs,25 µM melatonin Mel+500 ppm of CeO₂-NPs than other treatments. ABA treatments with cerium oxide nanoparticles with or without melatonin significantly increased ABA levels in Brassica napus leaves, and the highest result was found in 1000 ppm CeO₂-NPs and increased by 338.3 % as compared with control treatment. Regarding the content of melatonin in Brassica napus leaves, it was noted that there is a slight difference in its level between treatments, except in Mel+250 ppm CeO₂-NPs treatment, where it decreased.
23- The un-saturated fatty acid (PUFA) percentage from B. napus seeds was larger at Mel+500 ppm CeO₂-NPs treatment compared to control by 127.98%, but at 500 ppm of CeO₂-NPs was significantly decreased than control by 70.4%. In comparison, the percentage of saturated fatty acid (SFA) was reduced at Mel+500 ppm of nanoceria by 4.3% than control but highly recorded increase at 500 ppm of nanoceria by 200.6%.
24- The transcription level of antioxidant gene expression (SOD) enhanced at 500 ppm of CeO₂-NPs but down–regulated at 1000 ppm of CeO₂-NPs whereas the transcription level of SOD at Mel+500 ppm CeO₂-NPs was higher (3.7 fold) compared to control and then down-regulation at Mel+1000 ppm CeO₂-NPs compare to control. The stress-related gene expression (MTs) was down-regulated at 500 and 1000 ppm of CeO₂-NPs compared to control. However, at Mel+500ppm, CeO₂-NPs, and Mel+ 1000 ppm, CeO₂-NPs were significantly increased.