Foliar Spray of Biosynthesized Zinc Oxide Nanoparticles Alleviate Salinity Stress Effect on Vicia faba Plants

Previous studies recorded positive impact of ZnO NPs on plants stressed with salinity. The current work was performed to study the effect of two different concentrations of biosynthesized ZnO NPs (50 and 100 mg L−1) on faba bean plants under salinity stress. The zinc oxide nanoparticles (ZnO NPs) were synthesized using Mentha extract, and their shape and size were characterized using X-ray diffraction and transmission electron microscope while diffuse reflectance spectra were measured using UV–Vis spectrophotometer. The generated ZnO NPs were spherical with a particle size 9.4 nm and had a rod form with particle size 15.2 in length and 3.5 nm in width. The response of faba been plants to the foliar spray of ZnO NPs concentrations (0, 50, and 100 mg L−1) alone and in combination with salt stress at 150 mM NaCl was studied. Salinity induced reduction in faba bean root and shoot length and dry/fresh weights, while an enhancement was recorded in response to foliar treatment with ZnO NPs at 50 and 100 mg L−1 either in presence or absence of salinity stress. The highest amounts of chlorophyll a, b, carotenoids, and total pigments were recorded in plants received 50 mg L−1 ZnO NPs compared to the alternative control. Secondary metabolites (phenols, flavonoids, and tannins) were accumulated in salinity-stressed plants and further accumulation in response to ZnO NPs treatment was noticed. Amino acids, proline, glycine betaine, and total soluble sugars, as well as enzymatic and non-enzymatic antioxidant contents, increased almost onefold in salinity-stressed plants as compared to control plants while the 50 mg L−1 ZnO NPs treatment resulted in higher accumulation of the previously mentioned substances. In contrast, plants oxidative stress was reduced in response to ZnO NPs treatments. The nitrogen, phosphorus, potassium, calcium, zinc, and iron contents of faba bean plants were recorded under salinity stress and in response to the two applied concentrations of ZnO NPs. Faba bean plants stressed with 150 MN NaCl showed growth decline that may be attributed to osmotic stress and low water availability imposed by salinity. The treatment of stressed plants with 50 mg L−1 ZnO NPs induced an enhancement in plant growth as well as an accumulation of antioxidants, osmolytes, and secondary metabolites that could help plants overcome the negative effects of salinity.


Introduction
Salinity is one of the primary abiotic stressors causing plant yield losses. High salt concentrations endanger one-third of the world's agricultural land area as a result of poor water quality, and this threat may worsen in the future as a result of climate changes. The effects of salinity are associated with an increase in the amount of sodium and chloride ions in plants, which causes growth and morpho-physiological process disorders due to osmotic, ion toxicity stress, and water reduction in plant tissues (Hussien and Abou-Baker 2018; Yang et al. 2020). The majority of plants grown in saline conditions exhibited cell membrane damage, ion buildup, the production of reactive oxygen species (ROS) and oxidative free radicals, and a decrease in pigment synthesis (Masilamani et al. 2020).
The application of nanoparticles (NPs) of appropriate size (1 to 100 nm) has been integrated into plant protection through mineral aggregation in subcellular areas, which has prompted regulation of plant development and protection against a variety of abiotic stresses by ultimately improving the physio-compound features that benefit yield creation (Pooja et al. 2020). Nonetheless, the small size, shape, chemical structure, and surface area to volume ratio of NPs play an important role in their performance; on the contrary, NPs cause oxidative stress depending on the size, surface area, reactivity, and concentration used, as well as the age and species of the treated plants (Burman et al. 2013;Pooja et al. 2020). Zinc, as one of the most important micronutrients, is involved in many vital physiological processes such as hormone biosynthesis (auxin, abscisic acid, gibberellin, and cytokinin), chlorophyll synthesis, chloroplast development, and protecting and maintaining the stability of cell membrane structure, and it is also a co-factor of many enzymes associated with transferases, hydrolases, ligases, and isomerases that regulate ion balance and stomata conductance when different nanoparticles pass through roots by apoplastic pathway and are transported in shoots through the vascular system (Sturikova et al. 2018).
The green approach to nanoparticles synthesis is dependent on the plant source and the organic compounds found in plants (enzymes, amino acids, proteins, saccharides, vitamins, and organic acids) that can function as reducing and/or capping agents during metal nanoparticle production (Zafar et al. 2020). Aside from their antibacterial properties, metal oxide NPs have been shown in experiments to serve as fertilizers, improving plant growth and production. ZnO nanoparticles are non-toxic, cheap, and biocompatible when compared to other metal oxides (Sahdev et al. 2013). ZnO is classified as a "generally recognized as safe" (GRAS) substance by the Food and Drug Administration (Espitia et al. 2012).
Zinc oxide nanoparticles (ZnO NPs) are biocompatible, extremely small, and inexpensive nanoparticles that do not interact with the vast majority of pharmaceutically active compounds. ZnO NPs act as co-factors for enzymes and have the potential to significantly increase pigment formation, enzyme mobilization, bioactive compounds induction, and accumulation. ZnO NPs are one of the most commonly used nanoparticles in agriculture because they are associated with the formation of secondary metabolite pigments, protein, and sugar content nutrient translocation, which scavenges free oxygen radicals produced in stressed plant tissues (Zafar et al. 2016;Ali et al. 2021).
Being broad bean (faba bean) (Vicia faba), an important crop in Northern Africa that is rich in organic materials and minerals (proteins, carbs, thiamin, folic acid, and tocopherols), is thought to be one of the most significant sources of human and animal nutrition and is used in the food industry (Hassanein et al. 2012). Furthermore, faba bean plants add nitrogenous compounds to the soil, increasing soil fertility and productivity (Qados et al. 2015). However, a variety of abiotic stressors, such as salinity and water deficiency, have an impact on bean plant development and metabolism.
Mentha spicata (common mint or spearmint) is a valuable medicinal herb, with its essential oils having antioxidant and antimicrobial properties. Its leaves contain significant amounts of phenolic, flavonoids, terpenoid, and steroids, which are responsible for the reduction and stabilization of NPs (Rad et al. 2019). The purpose of this study was to green synthesize ZnO NPs from Mentha spicata extract and to investigate the shape and crystal size characterization of the formed nanoparticles. The studies also focused on applying ZnO NPs to salinity-stressed broad bean plants in order to study the potential role of ZnO NPs in alleviating the negative effects of salt stress and improving plant performance under stressful conditions.

Plant Materials
Broad beans (Vicia faba L. cv. Nubaria, 1) were purchased from Field Crops Research Institute, Agricultural Research Center, Giza, Egypt, and used in the current studies. This cultivar is characterized by early flowering, a high number of seeds per plant, the weight of 100 seed recorded (95 g), and high seed content of both proteins and carbohydrates (24 and 58%), respectively (Qabil et al. 2018). Common mint (Mentha spicata L.) was used as a reducing agent to produce zinc oxide nanoparticles, and it was obtained from the botanical garden of the Faculty of Education, Ain-shams University, Cairo, Egypt.

Leaves Extract Preparation and Green Synthesis of ZnO NPs
The ZnO NPs were synthesized by the method of Ezealisiji and Xavier (2020). To begin the synthesis, 10 g of clean and air-dried Mentha leaves were crushed to a fine powder in a sterile electric blender. The powder was dissolved in 100 mL of double-distilled water and blended at 60 °C for 2 h. The aqueous extract was allowed to cool, filtered through sterile filter paper, and then kept at room temperature in a dry and clean container. Simultaneously, a solution of zinc nitrate (1.5 mM) in 200 mL of double-distilled water was prepared. Following that, for roughly 6 h, a zinc nitrate solution was applied to the plant leaf separate under careful blending at a temperature of 80 °C in dim. The appearance of the faint yellow color indicates the formation of ZnO NPs. The residual was collected and washed many times with deionized water and ethanol. Finally, the item was dried for 8 h at 60 °C before being calcined for 2 h in a muffle furnace (JSMF-45 T Korea) at 350 °C to generate pure ZnO NPs.

Characterization of Zinc Oxide Nanoparticles
X-ray diffraction (XRD) patterns were produced for the obtained powder using a Brucker Axs D8 Advance, Germany, at a scanning rate of 2o in 2/min. A transmission electron microscope (TEM) (JEM-2100CX) was used to study the particle's form and size (JEOL). The diffuse reflectance (DR) spectra of the photocatalysts were measured using a Lambda 365 UV-Vis spectrophotometer, with barium sulfate serving as a control. A wavelength range of 200-650 nm was recorded in diffuse reflectance mode, and the bandgap value was determined using the Kubelka-Munk plot.

Plant Cultivation and Treatments
Broad bean seeds of equal size and weight were sterilized in an aqueous solution of 0.1% HgCl 2 for 1 min with continuous shaking before being thoroughly washed in distilled water multiple times. Ten seeds were sown (5 cm depth) in each plastic pot (25 cm in diameter) in 3.5 kg homogeneous loamy dried soil (sand 80%; silt 15.5%; clay 4.5%; organic matter 0.45%, pH 7.8; EC 0.4 dSm −1 ). The pots were kept under natural climatic conditions of light and darkness (temperature ranging from 28 °C/16 °C, day/night with a relative humidity of 75%) in the glass house of the Faculty of Education Ain, Shams University, Cairo. All plants were watered to maintain the salt level used in the experiment and allowed to grow for the duration of the experiment. The study included six treatments: the first treatment (T1) consisted of irrigating plants with fresh water as a control, the second treatment (T2) was foliar spraying with ZnO NPs (50 mg L −1 ), the third treatment (T3) was spraying with ZnO NPs (100 mg L −1 ), the fourth treatment (S) was with NaCl solution (150 mM), and the fifth and sixth treatments were for the two interactions: S + T2 and S + T3. Each treatment consisted of five replications that were randomly distributed. Both the fresh water and salinity treated pots were irrigated with an equal amount of tap water or 150 mM NaCl solution. The ZnO NPs were sprayed twice every 2 weeks. After 60 days, plant samples were collected and root and shoot length, fresh and dried weight, and other characteristics were measured.

Determination of Photosynthetic Pigments
Photosynthetic pigments were determined using the Lichtenthaler and Buschmann technique (2001). 0.5 g fresh young broad bean leaves were homogenized in 80% acetone for the measurement of chlorophyll a (Chl a), chlorophyll b (Chl b), carotenoids (Car.), and total pigments. The absorbance was measured using a colorimeter (VEB Carl Zeiss, Germany) at 663.2, 646.8, and 470 nm. The quantity of the pigments was determined as mg g −1 leaf fresh weight (FW).

Determination of Total Phenols Content (TPC)
Total phenolic compounds were determined using tannic acid equivalent (TAE) based on the Folins-Ciocalteu technique, as reported by Dihazi et al. (2003). For extraction, 1 g of fresh leaves was combined with 50 mL of 80% cold methanol (v/v) three times at 90 °C. After the extract had been filtered, methanol was used to dilute it to a known volume. 1 mL of methanolic material was mixed with 1 mL (10%) Folins-Ciocalteu reactant and 2 mL of a 25% Na 2 CO 3 solution, and the extract was thoroughly combined and kept in the dark for 60 min. The absorbance was calculated as mg tannic acid g −1 FW and measured at 750 nm using VEB Carl Zeiss, Germany.

Detection of Total Flavonoids Content (TFC)
Flavonoids were assayed using the method of Bushra et al. (2009). 0.5 g of fresh leaves was combined with 10 mL of 80% aqueous methanol before being filtered. 1 mL of extract, 4 mL distilled water, 0.3 mL of 5% NaNO 2 , and 0.3 mL of 10% AlCl 3 were added after 5 min, and the samples were incubated for 6 min before the addition of 2 mL of NaOH (1 M) and 10 mL of H 2 O. Quercetin serious solutions were used as a standard, and the flavonoids level was detected as mg/g FW at 430 nm using VEB Carl Zeiss, Germany.

Estimation of Tannin Content (TC)
Tannin concentration was measured using the Folin-Denis reagent, as reported by Ejikeme et al. (2014). 100 mL of distilled water was added to 1 g of plant tissue, and the mixture was then lightly boiled for 1 h and filtered. For color development, 10 mL of the extract was mixed with 50 mL of distilled water, 5.0 mL of Folin-Denis's reagent, and 10 mL of saturated Na 2 CO 3 solution, and the mixture was left to react in a water bath at 25 °C for 30 min. The absorbance was measured at 700 nm using VEB Carl Zeiss, Germany, and the concentration was estimated using a tannic acid standard curve as follows: Tannic acid (mg/100 g) = C × extract volume × 100/aliquot volume × weight of sample.

Determination of Total Free Amino Acids (TAA) Content
Total amino acids were extracted in accordance with Lee and Takanashi (1966). The leaves were homogenized in ethanol (80%) and simmered for 10 min. The mixture was then stirred at 2000 g for 10 min. A mixture of 0.05 mL supernatant and 2 mL ninhydrin was boiled. The mixture was left to cool before being diluted with distilled water to 10 mL. The color produced was measured at 570 nm with VEB Carl Zeiss, Germany, using glycine as a standard.

Estimation of Proline (Pro) Content
Proline was measured using the Bates et al. (1973) method, which involved crushing 0.5 g of dried bean leaves in 10 mL of 3% sulfosalicylic acid and centrifuging at 10000 g for 10 min. The supernatant was then combined with 2 mL of newly produced acid ninhydrin. The samples were placed in a 90 °C water bath for 30 min before being incubated in an ice bath to stop the reaction. 5 mL of toluene was vortexed for 15 s and left at room temperature for 20 min. The toluene phase was collected, and the developed color absorbance was measured at 520 nm using a spectrophotometer (VEB Carl Zeiss, Germany) and the amount was calculated as μg g −1 FW.

Determination of Glycine Betaine (GB) Content
Glycine betaine was estimated using Grieve and Grattan's (1983) technique. 0.5 g of powdered dry plant tissue was mixed with 20 mL distilled water for 24 h and filtered. The filtrate was diluted with an equivalent amount of 1 M H 2 SO 4 and cooled on ice for 1 h after which 0.2 mL cold KI-I 2 reagent was added. The reactants were mixed, kept at 4 °C overnight, and centrifuged at 12000 g for 15 min at 4 °C to produce periodide crystals. The precipitate was dissolved in 1,2-dichloroethane, and the absorbance at 365 nm was measured using a spectrophotometer (VEB Carl Zeiss, Germany) with glycine betaine dissolved in 1 M H 2 SO 4 as standard after 2 h.

Determination of Total Soluble Sugars (TSS) Content
Total soluble sugars were extracted and evaluated using the Homme et al. (1992) technique. Soluble sugars were extracted by immersing dry tissue in 10 mL of 80% (v/v) ethanol overnight at 25 °C with shaking, and then centrifuged at 6000 g. For the measurement of soluble carbohydrates, the supernatant was fully dried and dissolved in a specified of distilled water. Total soluble sugars were determined by heating 0.1 mL of the ethanolic extract with 3.0 mL freshly prepared anthrone (150 mg anthrone + 100 mL 72% H 2 SO 4 ) in a boiling water bath for 10 min and then cooled. The absorbance at 485 nm was measured using a spectrophotometer (VEB Carl Zeiss, Germany).

Glutathione (GSH) Content
The total non-protein SH group (reduced glutathione) was measured in the supernatant of a fresh plant sample that was extracted in 5% ice-cold sulfosalicylic acid solution (w/v) and centrifuged at 6000 g for 20 min using Ellman's reagent (5,5′-dithio-(bis-2-nitrobenzoic) acid according to the technique of Silber et al. (1992). Mancinelli (1990) method was used to determine the anthocyanin level. Fresh plant samples were crushed in 10 mL of acidified methanol (HCl: methanol, 1:99 v/v). The homogenate obtained was centrifuged at 18000 g for 30 min at 4 °C. The supernatant was filtered using Whatman's filter paper No.1 and incubated in the dark at 5 °C for 24 h. The quantity of anthocyanin was detected at 550 nm using a spectrophotometer (VEB Carl Zeiss, Germany) and expressed as μmol/g FW. The extinction coefficient of anthocyanin ε = 33,000/mol 2 cm was used in the calculation.

Ascorbic Acid (AsA) Content
Ascorbic acid levels were estimated according to Mukherjee and Choudhuri (1983). 2 g fresh materials were homogenized in 6% trichloroacetic acid (TCA) and the extract was filtered and centrifuged at 1000 g for 20 min. Four milliliters of the extract was combined with 2 mL of 2% dinitrophenylhydrazine after which 1 drop of thiourea was added (10% in 70% ethanol). After boiling for 15 min in a water bath, the solution was cooled to room temperature and 5 mL of 80% (v/v) of H 2 SO 4 was added. The absorbance was calculated as µg/g FW using a spectrophotometer (VEB Carl Zeiss, Germany) set to 625 nm.

α-Tocopherol (α-Toc) Content
α-Tocopherol was measured using the method of Philip et al. (1954). Fresh samples were homogenized with 10 mL of a 2:1.6 v/v combination of petroleum ether and ethanol, and the extract was centrifuged at 10000 g for 20 min. One milliliter of the extract and 0.2 m of 2% 2.2-dipyridyl in ethanol were mixed thoroughly and left in the dark for 5 min. The absorbance at 520 nm of the resulting red color in the aqueous layer was measured using a spectrophotometer (VEB Carl Zeiss, Germany). A standard graph with a known quantity of α-tocopherol was used to determine the α-tocopherol content and it was expressed as µg/g FW.

Catalase Enzyme
Catalase (CAT) enzyme (EC 1.11.1.6) activity was determined using the method of Velikova et al. (2000). 0.5 g of frozen plant material was homogenized with 5 mL of ice-cold 50 mM sodium phosphate buffer (pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). The extract was centrifuged at 12500 g for 20 min at 4 °C. The supernatant was used to measure enzyme assays; 2.6 mL of 50 mM potassium phosphate buffer (pH 7.0), 400 μL of 15 mM H 2 O 2 , and 40 μL of enzyme extract were used in the test. The breakdown of H 2 O 2 was accompanied by a reduction in absorbance at 240 nm as measured by a spectrophotometer (VEB Carl Zeiss, Germany).

Polyphenol Oxidase (PPO) Enzyme
For PPO activity measurement, 2 mL of 0.1 M phosphate buffer (pH 6.0), 1 mL of 0.1 M catechol, and 0.5 mL of enzyme extract were combined and incubated at 25 °C for 5 min before the reaction was stopped with 1 mL of 2.5 N H 2 SO 4 . According to Velikova et al. (2000), the absorbance of the purpurogallinproduced color was measured at 495 nm using a spectrophotometer (VEB Carl Zeiss, Germany). At zero-time, 2.5 N H 2 SO 4 was added to the same test mixture as a blank. The polyphenol oxidase activity was expressed as mg −1 protein (U = change in 0.1 absorbance min −1 mg −1 protein).

Determination of Lipid Peroxidation
Lipid peroxidation was measured by the amount of malondialdehyde (MDA) according to the method of Jiang and Zhang (2001). The concentration of malondialdehyde was detected according to the method of Jiang and Zhang (2001). In 5 mL of 0.1% (w/v) trichloroacetic acid, 1 g of tissue samples was homogenized, and the homogenate was centrifuged at 10000 g for 5 min. 1 mL of the supernatant was mixed with 4 mL of thiobarbituric acid (0.5% in TCA 20%) and 10 mL of butylated hydroxyl toluene (BHT) (4% in ethanol). The mixture was heated to 95 °C for 60 min then immediately cooled in an ice bath and centrifuged at 10000 g for 15 min, and the absorbance of the supernatant was measured against a reagent blank at 532 and 600 nm using a spectrophotometer (VEB Carl Zeiss, Germany). The MDA concentration was calculated as μmol·g −1 FW using an absorption coefficient of 155 mM −1 cm −1 .

Determination of Hydrogen Peroxide (H 2 O 2 ) Content
The level of H 2 O 2 was evaluated according to the method of Jana and Choudhuri (1981). A known weight of plant material was ground for 20 min in phosphate buffer (50 mM, pH 6.5), and the homogenate was centrifuged at 8000 g. After diluting the supernatant to 20 mL with phosphate buffer, 0.5 mL of the supernatant was blended with 2.5 mL of 0.1% titanium sulfate in 20% H 2 SO 4 and centrifuged at 8000 g for 10 min. The absorbance of the supernatant was measured using a spectrophotometer (VEB Carl Zeiss, Germany) at 410 nm, and the H 2 O 2 value was estimated using an extinction coefficient of 0.28 mol −1 cm −1 .

Measurement of Electrolyte Leakage (EL)
To eliminate contamination caused by sampling, 20 leaf discs from each subgroup were excised and thoroughly washed with double-distilled water, as described by Lutt et al. (1996). The samples were transferred to a test tube containing 20 mL of deionized H 2 O at 25 °C, and the electrical conductivity (E 0 ) of the solution was immediately measured with an electrical conductivity meter (DDSJ-308A, Shanghai). After 24 h of incubation at 35 °C, the electrical conductivity (E 1 ) was measured again. The tubes were then submerged in a 120 °C water bath for 20 min. The electrical conductivity (E 2 ) was recalculated after the tubes cooled to 25 °C. The percentage of electrolyte leakage (EL%) of leaf cells was calculated as follows:

Assaying of Minerals Content
Dried shoot samples were crushed to pass through a 20-mesh screen and digested with a combination of sulfuric and perchloric acid utilizing microwave radiation (Yildiz and Diżikisa, 2017) before being measured for nitrogen (N), phosphorous (P), potassium (K), zinc (Zn), and iron (Fe). The total nitrogen in plant tissue was measured using the micro-Kjeldahl technique in a solution of H 2 SO 4 and HClO 4 (AOAC 1995). The proportion of inorganic phosphorous was quantified using a colorimeter (Sacala et al. 2008). 0.5 g of plant tissue was extracted in 8 mL of 6% trichloroacetic acid and centrifuged at 18,000 × g for 15 min. A solution of 5 mol sulfuric acid, 2.5% ammonium molybdate, and 0.25% 1,2,4-aminonaphtholsulphonic acid was added. The absorbance was measured at 660 nm after 15 min of incubation at 37 °C. An atomic absorption spectrophotometer was used to measure potassium, zinc, and iron (Ieggli et al. 2011).

Statistical Analysis
The values of all measurements are presented as average ± standard deviation (SD) and analyzed using the twoway analysis of variance (ANOVA) with least significant difference (LSD) at a 5% I-significance level.

Characterization of Zinc Oxide Nanoparticles
The scanning electron microscope (SEM), XRD, and UV-Vis spectroscope spectrum were used to determine the form and real size of the ZnO NPs.  (100), (002), (101), (102), (110), (103), (200), (112), and (201) plane indicated typical zinc oxide crystals (Fig. 1). The XRD revealed no peak other than ZnO NPs peaks, indicating that the ZnO NPs were pure. The rod shape with an average length of 15.23 nm and width of 3.50 nm and the spherical shape with an average radius of 9.42 nm were formed and depicted in Fig. 2 using SEM imaging. Figure 3 depicts the UV-Vis absorption spectra of ZnO NPs measured between 200 and 600 nm. The peaks were detected between 300 and 359 nm, confirming the earlier result that Mentha leaf extract produced ZnO nanoparticles.

Changes in Growth Parameters
Data showed that the effect of salinity stress-induced declines in all studied growth criteria ( Table 1). The visible adverse impact of saltiness on faba bean was shown in terms of the marked reduction in root and shoot length (35% 28%), fresh weight (77% and 47%), and dry weight (29% and 138%), respectively, compared to non-stressed plants (Table 1). Foliar spraying of ZnO NPs resulted in higher shoot and root length and fresh and dry weight under natural and salinity stress conditions in faba bean plants relative to their untreated controls. The lowest concentration of ZnO NPs (50 mg L −1 ) in combination with salt stress caused about 31% and 22% increment in lengths, 39% and 46% in fresh weights, and 25% and 51% in dry weights of root and shoot respectively in comparison to non-stressed plants.
However, treated plants with 100 mg L −1 level of ZnO NPs showed marked reduction in all the above-mentioned parameters compared with control plants and the relating salinity level.

Changes in Photosynthetic Pigments
As shown in Table 2, salinity treatment induced a substantial effect on the pigment content of bean plants when compared to the control. Chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll content decreased in salt-treated plants by 43, 51, 27, and 43.5%. The mentioned pigments content showed significant increment when bean plants were exposed to the lower concentration , and under salinity stress, the contents increased to 2.41, 1.20, 0.743, and 4.36 mg g −1 as compared with salinity-stressed control group (2.13, 0.89, 0.687, and 3.71 mg g −1 ). At the highest concentrations of ZnO NPs, the degree of inhibition was significantly more pronounced when compared to the comparable control.

Changes in Secondary Metabolites Content
The obtained findings indicated that TPC, TFC, and TC significantly increase as compared to control plants in response to NaCl treatment, the contents increased twofold in TPC and TFC while fivefold increment was detected in TC contents (Table 3). Meanwhile, when compared to control

Changes in Osmolytes Compounds
The observed results (Table 4) revealed that in response to salinity osmolytes contents accumulated in faba bean plants, the increment was 95% for TAA, 100% for TSS, 157% for Pro, and 36% for GB. The foliar spraying of bean plants with both ZnO NPs levels (50 and 100 mg L −1 ) generated a significant increase in mentioned osmolytes level: TAA (25.7 and 30.4 mg g −1 ), Pro (27.3 and 33.2 μmol g −1 ), GB (11.5 and 19.7 μmol g −1 ), and TSS (25.2 and 16.7 mg g −1 ) respectively as compared with salinity-stressed plants which gave values 23.13 and 19.7 mg g −1 for TAA and TSS and 30.6 and 14.7 μmol g −1 for Pro and GB contents.

Changes in Non-enzymatic Antioxidants Content
Salinity had a substantial impact on the antioxidant content of the plant sample. The concentrations of free GSH, ACNs, AsA, and α-toc in salinity-stressed plants are considerably higher than in control plants (

Changes in Enzymatic Antioxidants
Under salinity stress, plants produced antioxidant enzymes, mainly catalase and peroxidase and polyphenol oxidase. The activity of CAT, POX, and PPO (

Determination of MDA, H 2 O 2 Contents, and Membrane Leakage
MDA concentration, which is an aftereffect of lipid peroxidation, was used to evaluate lipid peroxidation. The results showed that NaCl caused a significant increase in the stressed plant's lipid peroxidation and H 2 O 2 concentration (42 and 75%) respectively when compared to control plants ( Table 7). The current study revealed that ZnO NPs application at the level 50 mg L −1 promoted 17 and 23% decrease in the malondialdehyde and H 2 O 2 contents respectively when compared with control plants, while in the presence of salinity stress, the decline was 17 and 17.5% as compared with respected control. On the other hand, a substantial increase was detected in both MDA and H 2 O 2 contents in response to the high level of ZnO NPs (100 mg L −1 ) treatment, the increment reached 4 and 10% under normal and 10 and 20% in salinity stress conditions. The impact of salinity on plasma membrane intactness is shown in Table 7. The degree of electrolyte leakage of bean plants produced under salt stress was substantially increased almost twofolds compared to control plants (19.7%), whereas faba bean received low concentration of ZnO NPs treatments showed the least value (16.2%) among all groups. Treating salinity-stressed plants with 50 mg L −1 ZnO NPs stimulated a significant reduction in plasma membrane leakage measured in developed plants' leaves (30.7%) when compared with salinity-stressed group (41.3%). In contrast, as

Changes in Mineral Contents
A significant decrease in K, N, P, Zn, and Fe nutrient content under saline conditions (12,22,12,8, and 1.5%) when compared to control plants was detected as shown in Table 8. In contrast, percentage Na and Cl content increased in faba bean to give values 0.801 and 0.412% compared with control plants which gave values 0.012 and 0.001%. It is worth mentioning that 50 mg L −1 nano zinc particles resulted to positive increases in N, P, and K content while the impact of 100 mg L −1 of ZnO NPs significantly reduced the uptake and aggregation of these mineral ions in bean plants either in the presence or absence of salinity. Furthermore, Cl and Na contents decreased about 0.261 and 0.211% due to low centralization of zinc oxide nanoparticles under saltiness conditions, whereas about 0.412 and 0.801% and 0.372 and 0.362% increment was recorded in plants treated with 150 mM NaCl alone or in combination with 100 mg L −1 ZnO NPs, respectively. In the presence of salinity stress, zinc content dropped considerably compared to control plants; moreover, 50 mg L −1 and 100 mg L −1 ZnO NPs treatments produced an increase in zinc levels in Vicia faba plants. Iron content was reduced in plants subjected to salt stress compared to control plants, whereas it declined completely in plants subjected to ZnO NPs treatments.

Discussion
Salt stress on plants caused physiological and morphological depression, posing a threat to plant life due to water scarcity, osmotic stress, and nutritional deficiency (Jiang et al. 2014). Zinc is essential for plant growth and development, and it plays an important role in mitigating the negative effects of salts in plants by synthesis of auxin, maintaining the integrality of bio-membranes, accumulation of phospholipids and protein, scavenging free oxygen radicals, nutrient translocation from aged cells to newborn cells, decreasing excess Na + and Cl − uptake, and increasing K + aggregation (Hussein and Baker 2018). Nanoparticles can help to improve metal reactive sites, promoting metal absorption in plants. However, the impact of metal nanoparticles on plants is influenced by plant age and species, as well as nanoparticle concentration and size (Burman et al. 2013). Metal nanoparticle production using microbes and biological systems is gaining popularity as an efficient, simple, and environmentally friendly method that avoids the use of cruel, toxic, and expensive synthetic chemicals (Narayanan and Sakthivel 2010). Green nanoparticle synthesis produced pure, smallsized, and defined forms. Furthermore, it was discovered that the optical and electrical properties of metal nanoparticles vary significantly depending on their form (Ankamwar et al. 2005). Mohammadi-Aloucheh et al. (2018) and Abdelkhalek and Al-Askar (2020) discovered that Mentha plant extracts enhanced the production of ZnO NPs. Salinity-stressed faba bean plants had shorter shoot and root lengths, as well as lower fresh and dry weights, according to the findings (Table 1). Similar results were recorded by Assimakopoulou et al. (2015) in three green bean varieties (Corallo, Romano, and Starazagorski) and in Tanacetum parthenium L. (Mallahi et al. 2018), Brassica napus (Ahmadi et al. 2018), and Phaseolus vulgaris L. (Sofy et al. 2020). The decrease in plant vegetative features caused by soil salinity could be attributed to a decrease in leaf photosynthetic pigment content as well as a change in energy in adaption processes via ion exclusion and osmotic adjustment (Kamran et al. 2020). ZnO NPs aid plant growth and development (Hussain et al. 2016). In the current study, faba bean plants exposed to different concentrations of ZnO NPs (0, 50, and 100 mg L −1 ) showed an improvement in growth criteria in response to foliar treatment with 50 mg L −1 ZnO NPs compared to the corresponding control; however, 100 mg L −1 ZnO NPs induced a reduction in growth criteria of stressed plants. Similar to our findings, Kahlel et al. (2020) discovered that    (Cakmak 2008), as well as protect and maintain the structural stability of cell membranes; similarly, Zn could be used in protein production, membrane function, cell prolongation, and tolerance to ecological stresses (Cakmak, 2000 andKahlel et al. 2020), which could explain the enhancement effect of zinc nanotreatment on salinity-stressed faba bean growth. The toxicity of high concentrations of zinc nanoparticles on nucleic acids and cell division, on the other hand, could be explained by the toxicity of high concentrations of ZnO on nucleic acids and cell division; this claim was supported by previous studies on soybean ( Table 2 shows that salinity reduced the formation of photosynthetic pigments in stressed faba bean plants. Selem (2019) also found that salinity-stressed faba bean plants had lower levels of photosynthetic pigments and carotenoids. Reduction in Chl a, b, and carotenoids concentration was observed in common bean plants exposed to salt stress (Sofy et al. 2020). Significant reductions in relative water content, organelle damage, and ROS formation occur as a result of sodium ion accumulation, resulting in a reduction in photosynthesis rate, chloroplast structure damage, pigment-protein complex instability, and chlorophyllase activity (Hussain et al. 2019). In terms of the effect of salt on photosynthetic pigments, our findings indicated an increase in photosynthetic pigment content in faba bean plants treated with 50 mg L −1 ZnO NPs in the presence or absence of salt stress compared to the corresponding control. A 100 mg L −1 ZnO NPs foliar application resulted in a substantial decrease in the same contents. These findings are consistent with those of Alabdallah and Alzahrani (2020), who reported an increase in Chl a, b, carotenoids, and total pigment content in saltstressed okra plants in response to ZnO NP S (10 mg L −1 ) foliar application. The likely cause of this increase in photosynthetic pigments content in plant leaves is ZnO's stimulating effect on carbonic anhydrase, which may have facilitated the CO 2 transport to carboxylation sites in the chloroplast. Zinc is required for proto-chlorophyllide production, chloroplast growth, and repair of photosystem II in plants (Salama et al. 2019). The increase in carotenoid content in plants subjected to salinity stress could be a plant response to the oxidative damage caused by salt stress (Mohsenzadeh and Moosavian, 2017). However, too much zinc had a negative impact on photosynthesis because zinc ions replaced central Mg 2+ in chlorophyll (Adhikari et al. 2020).
Plants demonstrate physiological characteristics to detoxify and adapt to stressful conditions under salinity stress, which are dependent on signaling and metabolic networks (Naeem et al. 2017). According to this study, faba bean resistance to salt treatments is associated with an increase in the secondary metabolite concentration (Table 3). According to Salem et al. (2014), total phenols, flavonoids, and condensed tannins increased with salt up to 10 g L −1 in Carthamus tinctorius L plants. Abd El-Lateef et al. (2020) reported an increase in phenols in sugar beet plants treated with salt stress. Phenols and flavonoids play an important role in protecting plants from the damaging effects of stress and have an antioxidant effect because they are electrondonating agents, reducing reactive oxygen species (Gupta and Pandey, 2020). According to our findings, faba bean plants treated with ZnO NPs at low or high concentrations increased the content of secondary metabolites significantly. In line with our findings, Mahmoud et al. (2019) found that ZnO NPs significantly increased the concentrations of anthocyanins, phenols, tannins, flavonoids, crude protein, and carbohydrates in red radish root. Hezaveh et al. (2020) recorded an increase in phenolic compounds and flavonoids in rapeseed under salt stress and treatment with 20, 40, and 80 mg L −1 ZnO NPs. The use of nanoparticles as oxidative stress producers resulted in the production of secondary metabolites that function as ROS scavengers (Michalak, 2006). The combined effect of salinity and 100 mg L −1 ZnO NPs on faba bean stimulated plants' ability to manufacture secondary metabolites to combat such extreme conditions investigated in this study. In line with our findings, Mahmoud et al. (2019) found that ZnO NPs significantly increased the concentrations of anthocyanins, phenols, tannins, flavonoids, crude protein, and carbohydrates in red radish root. Zinc is also involved in nucleic acid metabolism and is an essential component of many biomolecules such as proteins and auxins; these findings explain the increase in secondary metabolite production in zinc-treated plants (Tsonev and Lidon 2012). Secondary metabolite accumulation, in this context, is part of the adaptive response to salt stress, acting as ROS scavengers either in conjunction with or independently of antioxidative enzymes. The significant increase in these compounds caused by ZnO NPs application suggests that ZnO NPs play a role in reducing salinity stress.
In this study, total free amino acids, proline, and glycine betaine content increased in faba bean plants exposed to salt, but total soluble sugar content decreased (Table 4). Similar results were recorded in lupine plants stressed by 50 mM NaCl salt (Latef et al. 2017), in tomato plants under 30 and 60 mg L −1 (Umar et al. 2018), and in common bean plants stressed by 100 mM of NaCl (Sofy et al. 2020). Proline, free amino acids, and total soluble sugar contents of plant cells play an important role in the osmotic adjustment under stress, protecting the macromolecule structure and membranes of the cell. The foliar application of ZnO NPs on broad bean plants resulted in an increase in these osmolyte contents. Similar findings were reported by Latef et al. (2017) on saline-stressed lupine, by Alabdallah and Alzahrani (2020) on okra, and by Gaafar et al. (2020) on salinitystressed soybean plants. Our results revealed an increment in calcium uptake in response to Zn NPs treatment. This finding is consistent with the notion that calcium could signal the synthesis and accumulation of osmolytes in salinitystressed plants in order to reduce Na ion toxicity (Weisany et al. 2014), which aids in explaining osmolyte profusion in zinc-treated and -stressed plants. External application of zinc improves the Zn finger proteins, which stimulate the production of soluble sugars and proline in plants (Wu et al. 2015), and production of gibberellic acid and amino acids was recorded by Mishra and Abidi (2010) in response to zinc provision in plants.
In this study, the levels of the antioxidants (GSH, ACNs, AsA, and α-Toc) in faba bean plants increased under salinity stress compared to control plants (Table 5). In consistent with these findings, Latef et al. (2017) found an increase in ascorbic acid and anthocyanin content in salt-stressed lupine and rapeseed, Sofy et al. (2020) reported increased glutathione levels in common bean plants treated with 50 mM NaCl, and Al-elwany et al. (2020) reported an increase in ascorbate, glutathione, capsaicin, and phenolic contents, as well as WUE in association with higher Na + and Cl − contents in salinity-affected Capsicum frutescence. Hassan et al. (2021) discovered that non-enzymatic antioxidants and their associated genes (AsA, α-Toc, and GSH) are activated in barely treated with concentrations of 50, 100, and 150 mM NaCl. In response to salinity stress, antioxidants such as GSH, ACNs, AsA, and α-Toc are associated with increasing plant defense system by scavenging ROS, improving membrane integrity, decreasing ion leakage, and inducing the synthesis of defense proteins against stress (Zhou et al. 2016). Our findings, on the other hand, show that under salt stress, faba bean plants exposed to 50 and 100 mg L −1 ZnO NPs showed an increase in the content of non-enzymatic antioxidant activity caused by both NaCl and ZnO NPs treatments. This is consistent with the findings of Hezaveh et al.
(2020) on salinity-stressed rapeseed plants exposed to 40, 60, and 80 mg L −1 ZnO NPs. Zinc ions aid in enhancing the expression of antioxidant genes in stressed plants (Adhikari et al. 2020). In this regard, Zn may be able to aid in the production of non-enzymatic antioxidants (Soliman et. al., 2015). Zinc promotes photosynthesis and increases leaf water potential, allowing stressed plants to produce antioxidants in response to the stressful conditions (Hassan et al. 2020). The protective role of zinc oxide nanoparticles could be attributed to increased iron and copper uptake by plants, which leads to improved physiological processes such as photosynthesis and antioxidant production (Weisany et al. 2014). Table 6 demonstrates that growing faba bean under saline conditions increased the activity of antioxidant enzymes CAT, POX, and PPO, which is a good indicator of ROS production and plant ability to adapt to ROS. In agreement with our findings, Sofy et al. (2020) discovered that under salt stress, the activity of CAT and POX enzymes in common beans was much higher than in control plants. There was also an increase in CAT and POX enzymes in the shoots and roots of wheat plants exposed to salt treatment (Meena et al. 2020). This suggests that the increase in antioxidant enzyme activity may be due to a plant's adaptive defense mechanism against the damaging effects of NaCl. Many studies have reported that exposure to salt conditions can cause ROS production, resulting in increased activity of antioxidative enzymes as  (Farooq et al. 2020), and PPO enzymes catalyze the hydroxylation of monophenols to o-diphenols, which are then oxidized to the corresponding o-quinones (Mishra and Gautam 2016). However, under salt stress, foliar treatments with ZnO NPs at concentrations of 50 and 100 mg L −1 were effective in increasing antioxidant enzyme activity in faba bean plants. Sofy et al. (2020) demonstrated that scavenging ROS by increased activity of different antioxidant enzyme activities leads to better equilibrium of ROS generation. The current findings are consistent with those of Kouhi et al. (2015), who found that using ZnO NPs increased the activity of antioxidant enzymes in Brassica napus. Furthermore, Alabdallah and Alzahrani (2020) found an increase in antioxidant enzymes in saltirrigated okra plants treated with ZnO NPs. According to Weisany et al. (2014), ZnO NPs treatment induced significant increase in Fe and Zn ions uptake, which act as co-factors for enzymes that catalyze many biochemical reactions. Furthermore, zinc promotes the expression of antioxidant enzyme genes, increasing enzyme activity and decreasing ROS accumulation (Mishra and Abidi 2010).
Anion toxicity was caused by the rapid production of ROS and the accumulation of Na and Cl ions in plant tissue, which disrupted plant growth and physiological responses (El-Ramady et al. 2018). When compared to control plants, salinity-stressed faba bean showed a significant increase in MDA and H 2 O 2 contents in the current study (Table 7). Similar results were reported by Gupta and Pandey (2020) and Sofy et al. (2020). Free radicals attack the plasma membrane, causing damage and degradation of cell membrane components, raising the possibility that salt stress will degrade the integrity of the cellular membrane and molecules (proteins and lipids). These findings also revealed that 50 mg L −1 ZnO NPs reduced oxidative stress in faba bean plants when compared to corresponding control plants, but 100 mg L −1 treatment increased oxidative stress in plants. This is similar with the findings of Burman et al. (2013), who found that ZnO NPs elicited defensive effects on bio-membranes in chickpea seedlings against changes in membrane permeability and oxidative stress. Gaafar et al. (2020) claimed a reduction in MDA and H 2 O 2 content in salinity-stressed soybean plants in response to 25 and 50 mg L −1 ZnO NPs but a substantial increase in response to 100 and 200 mg L −1 ZnO NPs. In this regard, Weisany et al. (2014) reported that Zn treatment on plants induced reduction in membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, resulting in less ROS production. Zinc oxide nanoparticles improve membrane integrity and phospholipid accumulation (Jiang et al. 2014), as well as zinc's positive effect on antioxidant formation and photosynthesis process, which contributes to cell protection from oxidative stress (Hassan et al. 2020). While high concentrations of ZnO NPs are hazardous metals that attach to cell essential components (DNA and proteins) and alter their activities, they frequently induce redox disruption and oxidative stress in metal-exposed plants (Sharma and Dietz 2009).
In the current study, salinity stress resulted in a substantial decrease in N, P, and K mineral absorption in faba bean plants, while Na and Cl buildup was observed (Table 8). Similarly, Sofy et al. (2020) reported a significant decrease in N, P, and K content in common bean plants under salinity stress, with further decrease as the salt concentration increase, while Na buildup was observed in plants as salinity stress concentration increased. Furthermore Na and Cl accumulated in common bean plant parts under salinity conditions, but an inverse relationship between potassium and sodium was established by Gulmezoglu and Izci (2020). Plant roots absorb fewer nutrients due to high osmolality caused by salt stress and water scarcity, while Na and Cl deposited in plant tissues compete with numerous plant nutrients such as Ca and K, the uptake of potassium, calcium, and phosphorus may be suppressed due to high levels of salinity in many plants, whereas reduction in Fe and Zn uptake in salinity-stressed plants may induce a depressive effect of salinity on root (Weisany et al. 2014). On the other hand, the present study revealed an accumulation of N, P, and K nutrients associated with a decrease in Na and Cl content in salinity-stressed faba bean plants treated with 50 mg L −1 ZnO NPs, whereas 100 mg L −1 treatment induced a significant decrease in NPK together with an increase in Na and Cl content. Foliar spraying of micro-elements such as zinc is widely recognized to be highly beneficial to plants, as the small size and ease of penetration allow plants to quickly utilize required minerals when compared to absorption through roots (Elsheery et al. 2020). In this context, it was discovered that ZnO NPs treatment reduced the absorption of excess Na and Cl (Jiang et al. 2014). These findings could be explained zinc's critical role in maintaining plasma membrane structure and controlling the access of Na ions in cells (Weisany et al. 2014). Furthermore, ZnO NPs increased the activity of enzymes, such as phosphatase and phytases, which improve phosphorous mobilization and absorption in plants (Adhikari et al. 2020).

Conclusion
It is inferred from the present research results that salinity stress causes sodium and chloride ions to accumulate in plant tissues, resulting in increased osmotic stress, oxidative stress, enzymatic and non-enzymatic antioxidants content, osmolytes, and secondary metabolites content. Plant growth and photosynthetic pigment content were both reduced during salt stress. By increasing the synthesis of antioxidants, secondary metabolites, and osmolytes, a low concentration of biosynthesized zinc oxide nanoparticles helps to reduce the severe effect of salt stress on faba bean plants. However, either in the presence or absence of salt stress, high concentrations of zinc oxide nanoparticles induced a phytotoxic impact on plants. These findings could be explained in the light of the essential role of zinc in maintaining the plasma membrane structure and controlling the access of Na ions in cell and its role in the activity of enzymes that allow plants to absorb NKP and increase enzymes activity and secondary metabolites production.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Conflict of Interest The authors declare no competing interests.
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