The Effect of Thermally Heated Carbon Nanoparticles of Oil Fly Ash on Tomato (Solanum lycopersicum L.) Under Salt Stress

Salinity is an abiotic factor that severely limits agricultural yield around the world. Tomatoes are important crops among others due to their high nutritional value; however, when the crop is exposed to abiotic stresses such as salinity, tomato production could be negatively affected. The goal of this study was to measure the morphological and physiological responses of tomato seedlings grown under salt stress when carbon nanoparticle of oil fly ash (COFA) under heat treatment in the concentration (5 mg L−1) was applied to the leaves. In this study, three carbon nanoparticles (COFA, COFAH-J, COFAH-R) were applied to tomato seedlings under three different salt (NaCl) treatments: 0 mM, 20 mM, and 40 mM. For each treatment, three biological replicates were conducted, with each replicate containing at least three plants. Our findings demonstrated that salt-stressed tomato plants had considerably lower length of shoot and root, biomass, and photosynthetic pigments over control plants. Furthermore, salinity greatly enhanced the proline concentration, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities. However, the application of thermally treated carbon nanoparticles resulted in increases in the quantities of photosynthetic pigments and plant growth conditions. However, the tomato seedlings treated with COFA, COFAH-J, and COFAH-R increased SOD activity by 65%, 53%, and 45%; CAT activity by 67%, 63%, and 65%; and APX activity by 51%, 52%, and 41%, respectively, when seedlings were exposed to 40 mM. Overall, our data suggest that heated carbon nanoparticles of oil fly ash may improve tomato plants’ salt tolerance by enhancing their antioxidant defense systems. The beneficial impacts of thermally treated carbon nanoparticles in tomato plants offer up new avenues for their potential innovations in novel agricultural methods, particularly while plants are grown to saline conditions.


Introduction
Tomato (Solanum lycopersicum L.) is one of the most economically significant horticulture crops globally, with 181 million tons produced in 2019 (Panno et al. 2021). Tomatoes are currently produced all throughout the world, with its origins in the Andes region of South America. Because of the economic importance of tomatoes, securing predicted yields is a difficult task. Several environmental factors, such as salinity, drought, heavy metals, and high temperature stress, may have an impact on tomato productivity (Agius et al. 2022).
Salinity affects more than 6% of the world's land, accounting for more than 800 million hectares. High salt concentrations have a deleterious impact on 7% of the total land surface and 5% of cultivated land (Shahid et al. 2020). Salinity is an abiotic stress factor that disrupts plant cellular homeostasis by causing osmotic and ionic stress, causing plant production to decline . The plant adjusts its osmotic pressure, lowers cell expansion, causes stomatal closure, and reduces leaf area, all of which limit gas exchange rate (Rahman et al. 2016;Jahan et al. 2021a). Ionic stress is caused when plants are subjected to salt stress, which causes the plants to store massive amounts of (Na + ) (Rahman et al. 2016). Plants' protein synthesis is hampered by these high levels of salt (NaCl) (Rahman et al. 2016;Parvin et al. 2019). To prevent oxidative damage, plants have evolved many defense mechanisms as well as signaling acts to regulate both the generation and elimination of reactive oxygen species (ROS) (Hasan et al. 2020(Hasan et al. , 2021aJahan et al. 2021b). The antioxidant defense method effectively scavenges surplus ROS through the coordinated action of antioxidant enzymes (Soares et al. 2019;Hasan et al. 2021b).
Nanotechnology has been investigated as an innovative methodology; especially, nanoparticles (NPs), which are materials at least a dimension less than 100 nm, have been widely used ). Because of their small size and high surface-to-volume ratio, NPs exhibit high levels of both bioactivity and biosafety . A variety of applications in several economic sectors, including agriculture, medicines, and food technology, have made substantial use of NPs (Baz et al. 2020). Carbon nanotubes (CNTs), fullerene, SiO 2 , TiO 2 , CeO 2 , Al 2 O 3 , Ag, ZnO, Fe 3 O 4 /Fe 2 O 3 , and CuO (Prasad et al. 2017) have been the most extensively investigated nanoparticles in past years (Abdel Latef et al. 2017). The usage of above NPs can lead to a rise in enzyme activity, allowing the plant to tolerate salinity. Applying SiNPs to tomatoes increased the photosynthesis process by increasing salinity tolerance (Haghighi and Pessarakli 2013). SiO 2 NPs also mitigated salt stress in Glycine max L. by enhancing the production of enzymatic and non-enzymatic substances (Farhangi-Abriz and Torabian 2018). Oil fly ash waste is found to be a potential source of carbon nanostructures for the synthesis of carbon nanostructures (Salah et al. 2016;Al Malack et al. 2013;Li et al. 2021;Wu et al. 2022). Numerous studies have tried to obtain nanostructured carbon through using high-energy ball milling technology as an ecofriendly method of producing carbon nanostructures Alluqmani et al. 2021a, b;Alluqmani et al. 2021a, b). To date, however, there has been less investigation into the heat treatment and implementation of synthetic nanostructured carbon derived from oil fly ash for agricultural applications.
So far, only little works have been reported that demonstrate carbon nanoparticles' ability to defend against salt stress (Martínez-Ballesta et al. 2016). After taking into consideration the past findings related to the application of nanoparticles, our hypothesis is that the exogenous application of carbon nanoparticles will induce plant antioxidant systems while also increasing plant tolerance to salt stress. Thus, the current work was conducted with the goal of recording the physiological responses of tomato plants subjected to salt stress after being treated with carbon nanoparticles derived from oil fly ash.

Synthesis and Characterization of Carbon Nanoparticles
Using high-energy ball milling, carbon nanoparticles were synthesized from two raw samples of oil fly ash collected from power plants in the Saudi Arabian cities of Jeddah and Rabigh (Fig. 1). The dry ball milling process was carried out in a horizontal oscillatory mill (PM 400; Retsch, Hann, Germany) with a frequency of 25 Hz, for 45 h, with some grams of oil fly ash samples being ground in the machine under a nitrogen flow. The oil fly ash was stored in a 250-mL agate-based jar with a tight-fitting lid. The milling balls being used have been made of pure agate and weighed approximately 11 g each and measured 15 mm in diameter.

Fig. 1
The steps involved in carbon nanoparticles (carbon nanoparticles of oil fly ash, COFA; carbon nanoparticles of oil fly ash, carbon nanoparticles of oil fly ash sample from Jeddah, COFAH-J; carbon nanoparticles of oil fly ash sample from Rabigh, COFAH-R) for spraying on common tomato plants. Samples of carbon nanoparticles were prepared from raw oil fly ash and thermally treated under nitrogen gas for the colloidal solution, the milled powder was suspended in double-distilled water and sonicated for 30 min prior to use as thermal treatment under nitrogen gas. The sample treatments were sprayed as a foliar spray to tomato seedlings The milled powders of the Jeddah and Rabigh samples were then thermally heated for 700 °C in a nitrogen atmosphere for a total of 15 min. The milled samples were subjected to nitrogen gas analysis and were identified as carbon nanoparticles of oil fly ash (COFA). The sample from Jeddah that was treated with heat after ball milling was designated as COFAH-J, and the sample from Rabigh was designated as COFAH-R. The final step was to examine the morphological characteristics and chemical properties of the synthesized samples using field-emission scanning electron microscopy (FESEM) and energy-dispersive X-ray spectroscopy (EDS) analyses performed on the samples using a JEOL-7600F FESEM (Figs. 2a-f and 3a-c). Using these photos, we were able to determine that the shape of oil fly ash was reduced.
The outputs of X-ray photoelectron spectroscopy (XPS) were evaluated using a PHI 5000 VersaProbe instrument (Chigasaki, Japan). The prepared samples were subjected to a monochromatic Al K radiation (hv = 1486.6 eV) to determine their composition. A 0.25 eV/step was used to carry out the survey scans in this study. Following the heat treatment under nitrogen flow, a significant increase in the C1s peak was observed, which corresponded to a decrease in the concentration of oxygen-containing functional groups on the surfaces of the carbon nanostructures.

Experimental Setup
The foliar application studies were carried out at the green house and laboratories of Saudi Arabia's Umm Al-Qura University and Imam Abdulrahman Bin Faisal University. Four percent of sodium hypochlorite was employed to sterilize the tomato (Solanum lycopersicum L. cv. Hasawi) seeds. Tomato seedlings (3 per pot, 15 cm × 18 cm diameter) were germinated and grown in plastic pots with an equal amount of soil (2.5 kg). The physical properties of used soil were analyzed, and it includes sand (45%), silt (29%), clay (26%), organic carbon (0.9%), electrical conductivity, EC (1.6 mmhocm −1 ), pH (7.5), etc. Growing tomato seedlings were watered twice weekly prior to the application of experimental treatments. During the studies, three salt treatments were used: 0 mM as a control, 20 mM, and 40 mM. On 35-day-old seedlings, the plants were treated three times per week to various salt treatments (0 mM as a control, 20 mM, and 40 mM). Three times each treatment was carried out. The tomato seedlings were grown in a 12-h light period with a relative humidity (RH) of 65% and a temperature range of 24 °C ± 2.

Sample Treatment
Prepared milled powder was suspended in double distilled water and homogenized by sonication for 30 min before use. The treatments used in the present experiments were control, COFA, COFAH-J, and COFAH-R. The sample treatments (5 mg L −1 ) were sprayed twice as a foliar spray to seedlings at 10 and 20 days. The tomato seedlings were sprayed individually until the leaves become entirely moist and stopped before the dripping point. To prevent any touch with the soil, the pot surfaces were covered by plastic film during the foliar spray treatments.

Growth Conditions
The root and shoot lengths were determined before the harvest time using a measurement scale expressed in centimeters (cm). Fresh and dry mass were collected and meticulously measured without any damage after harvest period.

Chlorophyll Measurements
The contents of chlorophyll were extracted using Arnon (1949) method. A 0.25 g mature leaf was collected and crushed with 80% acetone (5 mL). A centrifuge machine was used to centrifuge the leaf extract at 3000 × g for 15 min at 40 °C. The quantities of chlorophyll pigments were determined by measuring the absorbance of the supernatant at 663 and 645 nm, respectively.

Proline Determination
The proline content of tomato seedlings was determined using a technique described earlier by Bates et al. (1973).
The tomato mature leaf sample (0.5 g) was collected, and it was homogenized with 3% sulfosalicylic acid (10 mL). The filtered sample was collected and mixed with 2 mL of ninhydrin solution and glacial acetic acid (2 mL). The mixture was chilled in an ice bath after being heated at 95 °C for almost an hour. The mixture was separated using 10 mL of toluene as a chromophore and cycled continuously via an air stream for 1-2 min to separate the aqueous phase from the toluenecontaining chromophore. Subsequently, the absorbance of the separated colored phase was measured at 520 nm using a spectrophotometer after it had dried for 2-3 min at room temperature.

Antioxidant Enzyme Activity Assay
Antioxidant enzymes were extracted using a technique developed by Mukherjee and Choudhuri (1983). 0.5 g of newly picked mature leaves were extracted in 10 mL of phosphate buffer (pH 7). The homogenate was then centrifuged at 15,000 × g for 10 min at 4° C. To test the activity of antioxidant enzymes, the supernatant was kept at 20° C.

Measurements of Superoxide Dismutase (SOD) Activity
SOD activity was determined using the El-Shabrawi et al.

Measurements of Catalase (CAT) Activity
CAT activity was measured based on the methods of Aebi (1984). In a 40 mL volume, the enzyme extract was mixed with 0.016 mL H 2 O 2 (30%) and a 10 mM phosphate buffer solution (pH 7). The absorbance at 240 nm was evaluated using a spectrophotometer (LKB-Biochrom 4050).

Measurements of Ascorbate Peroxidase (APX) Activity
The Nakano and Asada (1981) approach was used to determine the APX activity. 0.1 M potassium phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM EDTA, 1.0 mM H 2 O 2 , and 20 µl enzyme extract (2.22 mL) were added to the reaction mixture. The activity of APX was evaluated using the 2.8 mM −1 cm −1 enzyme coefficient.

Statistical Analysis
The analysis of variance (ANOVA) was carried out on the basis of the MINITAB 17 software (ANOVA), with the findings presented as treatment mean ± SE (n = 3). One-way analysis of variance was used to analyze all the data. The bar represents the standard error (SE) of three replicates, and the different letters denote the treatment differences that are statistically significant at the p ≤ 0.05 level.

Results
When tomato seedlings were exposed to varied salt concentrations (20 and 40 mM), shoot length, root length, fresh, and dry weight were dramatically reduced (p ≤ 0.001) compared to control (Fig. 4a-d). In contrast, when tomato seedlings were subjected to salt stress, the application of carbon nanoparticles (CNPs) resulted in considerable improvements in these growth parameters.

Discussion
Salt stress is a serious environmental problem that has an impact on crops all over the world. Our study demonstrates that salt stress inhibited tomato plant growth.  (Bhati et al. 2018). Carbon nanotubes (20 mg L −1 ) enhanced the dry mass of Zea mays after 1 week of exposure to optimal conditions (Tiwari et al. 2014).
After being treated with CNPs (10-150 mg L −1 ), wheat (Triticum aestivum) plants were measured, and the results revealed that the 50 mg L −1 treatment had the greatest effect, with shoot and root lengths up to 3 times longer than the controls (Saxena et al. 2014). The impact of our treatments on tomato plant growth differed dramatically with salt application rates in this study. The CNPs significantly boosted shoot and root length, as well as fresh and dry weight. The most effective treatments for tomato seedling growth were COFAH-R. Aquaporins are involved in the much more rapid uptake and transfer of nutrients and water, which promotes agricultural growth for NPs (Servin et al. 2015). Nutrient ions in the rhizosphere that have been loaded with NPs are transferred to the xylem through the epidermis, cortex, and stele under salt stress (20 and 40 mM). The bar represents the standard deviation (SD) of three replicates, and the different letters denote the treatment differences that are statistically significant (Fincheira et al. 2020). NPs also have a large surface area and absorption capacity, allowing them to act as nutritional ion repositories with control over the rate of release Achari and Kowshik 2018). Apart from plant growth, the effect of CNPs on crops is mostly manifested by changes in chlorophyll content, as chlorophyll concentration is a critical predictor of plant growth (Ghoto et al. 2020). Our study found that treated tomato seedlings had a greater chlorophyll content at nearly all doses, implying that the prepared samples stimulate chlorophyll production. Wang et al. (2018) did a similar investigation and proved that carbon dots enhanced the photosynthesis in mung bean sprouts. He demonstrated that treated crops had 14.8% higher photosynthetic pigments than untreated crops. In another investigation, Baz et al. (2020) found that after applying CNPs (250 mgL −1 ) to salt-stressed lettuce plants, the amount of chlorophyll content increased. This is likely because CNPs boost photosystem activity (Rubisco activity and chlorophyll content) by speeding electron transport (Wang et al. 2018).
The accumulation of numerous organic osmolytes of low molecular weight, like proline, by plants in response to adverse environmental conditions has been documented (Munns and Tester 2008). Such molecules are referred to as compatible solutes since they do not interfere with any of the normal metabolic processes occurring in plant cells. They also serve a critical role in reducing the harmful effects of high Na + levels on enzymes, proteins, and membranes when exposed to high salinity conditions. These may also have a role as oxygen radical scavengers, which could help to minimize the harmful effects (20 and 40 mM). The bar represents the standard deviation (SD) of three replicates, and the different letters denote the treatment differences that are statistically significant of reactive oxygen species (ROS) when exposed to high salinity (Munns and Tester 2008). Our findings clearly demonstrated that the buildup of proline reduces significantly when carbon nanoparticles are used. SOD, CAT, and APX are the most important antioxidant enzymes, which protect biological cells by reducing hydrogen peroxide (H 2 O 2 ) and (ROS) (Mahawar and Shekhawat 2016). While detoxifying ROS, the SOD enzyme was responsible for the first reaction, which deactivates superoxide radicals into H 2 O 2 and O 2 (Ali et al. 2006). In our study, the activity of all antioxidants, including SOD, CAT, and APX, increased when tomato seedlings were exposed to the prepared samples, demonstrating that tomato seedlings have a strong response to thermally heated carbon nanoparticles of oil fly ash. Antioxidant activity followed a similar pattern in Vigna radiata treated with CNPs (Shekhawat et al. 2021).

Conclusion
Following the findings of this study, it can be concluded that salt stress inhibits the growth of tomato seedlings, whereas the exogenous application of COFA, COFAH-J, and COFAH-R results in an improvement of the morphological parameters. The seedlings treated with COFA, COFAH-J, and COFAH-R decreased proline accumulation and increased chlorophyll accumulation under salt stress. The results of these studies indicate that foliar spraying of COFAH-R, that boost the plant's antioxidant systems, is a promising approach for regulating and/or alleviating salt stress-induced damage in tomatoes. As a result, we recommend that COFAH-R could be used to reduce the disruption of antioxidant mechanisms in tomato plants caused by salinity. The positive growth and physiological response of tomato plants to COFAH-R suggest novel applications for Data Availability All data and materials included in this work are available.
Code Availability Not applicable.

Declarations
Ethics Approval Not applicable.

Conflict of Interest The authors declare no competing interests.
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