Herein, we investigated the phytotoxic potential of nano zinc oxide (ZnONPs) on grain yield, focusing on responses that depend on particle size-, morphology-, and concentration of multiple protective antioxidant biomarkers, in soil-grown soybean ( Glycine max cv. Kowsar) during its life cycle. To this end, we synthesized three morphologically unique ZnONPs (spherical/38 nm, floral/59 nm and rod /> 500 nm); all have high purity, triclinic crystal structure and negative surface charge; and compare the toxicity with Zn2+ ions. Each pot received two seeds, which were placed in soil inoculated with N-fixing bacteria ( Rhizobium japonicum) and grown outdoors for 120 days. Our findings demonstrated a significant effect of particle size, morphology and concentration-dependent concentration of ZnONP on grain yield, lipid peroxidation and other antioxidant biomarkers together in soybeans. Spherical 38 nm ZnONPs offer the best protection compared to flower-shaped 59 nm ZnONPs, > 500 nm rod ZnONPs and Zn 2+ ions, particularly up to 160 mg/kg. However, at the highest concentration of 400 mg/kg, the spherical 38 nm ZnONPs produced the highest oxidative stress response (synthetic H 2 O 2 , MDA, SOD, CAT, POX) in soybean compared with two ZnONPs differ in morphology tested. The concentration response curves for the three types of ZnONP and Zn 2+ions were nonlinear (non-monotonic) for all evaluated endpoints. The results also showed the difference between the specific nanotoxicity of ZnONPs nano zinc oxide compared with the Zn 2+ ionic toxicity in soybean. Our NOAEL value higher than 160 mg/kg indicates the potential of ZnONPs to be used as nano-fertilizers for plants in Zn-deficient soils to improve crop yield, food quality, and address crop degradation. nutrition globally.
- Particle size-, morphology- and concentration-dependent effects of nano zinc oxide were tested.
- All Zn compounds (ZnONPs, Zn 2+) promoted grain yield up to 160 mg/kg.
- The spherical 38 nm ZnONPs produced the least oxidative stress, except at 400 mg/kg.
- The concentration-response curves for all Zn compounds are nonlinear.
- ZnONP can be used as a nanofertilizer to enrich Zn-deficient soils.
Nanoscience and nanotechnology implies the study of engineered nanostructures with at least one dimension below 100 nm that have potential applications in many fields, including in agriculture, therapeutics, diagnostics, engineering. , food industry and safety, environmental remediation, and energy infrastructure, among others (National Nanotechnology Initiative, 2005; Du et al., 2019 ; Malea et al., 2005) events, 2019 ). The production of engineered nanoparticles (ENPs) for the above applications is growing exponentially mainly due to the characteristic physicochemical properties of nanoparticles such as high surface area to volume ratio, greater response, high electrical conductivity, high mechanical strength and antibacterial properties ( Hou et al., 2018 ;Pokhrel et al., 2012 ; Rajput et al., 2018 ).
The growing interest in the use of ENPs in food and agricultural safety may also be due to their unique size and surface-related behavior ( Nuruzzaman et al., 2016 ; Dimkpa et al. , 2019 ). ENPs can be used in agriculture as nano-fertilizers, growth regulators or as nano-bactericides to tackle infectious diseases from animals and plants. Furthermore, ENPs can serve as sensors for early detection of pathogens and/or higher reactivity-induced pesticide residue depletion ( Wang et al., 2016b ; Xiong et al., 2017 ). Nano zinc oxide ZnONP with unique physicochemical properties can be used as a novel fertilizer to improve crop yield and food quality ( Dimkpa and Bindraban, 2018 ; Hou et al., 2018 ;White and Gardea-Torresdey , 2018 ).
Plants are the primary producers in almost every ecosystem on which consumers rely ( Ma et al., 2010 ). Thus, plants can also absorb and transport various toxic substances including ENPs from biological solids, soil and irrigation water into edible parts (roots, leaves, flowers, fruits, seeds). Andreotti et al., 2015 ; Ogunkunle et al., 2018 ; Yusefi-Tanha et al., 2020 ) and as a source of exposure to humans and other plant-eating organisms. Finally, toxic substances can be transported through different trophic levels in the food chain ( Zhu et al., 2008 ; Ghasemi Siani et al., 2017). ENPs can interact with the root surface while in soil or hydroponic media, from where ENPs can pass through apoplastic and symphonic pathways in plant cells and from there move up to shoot through the xylem flask ( Deng et al., 2014 ; Lin and Xing , 2008 ; Lin et al., 2009 ; Schwab et al., 2016 ). It is beginning to be understood that the toxicity of ENPs on plant growth, metabolism, defense systems and productivity may depend on the route of exposure, environmental composition, dose, morphology, granulation (size, shape), particle composition and surface chemistry ( Cao et al., 2017 ; Deng et al., 2017 ; Pagano et al., 2017 ; Pokhrel et al., 2012 ;del Real and et al., 2017 ), including the chemical composition of the subcellular sites and plant biological analyzes ( Dietz and Herth, 2011 ; Lin and Xing, 2008 ; Nuruzzaman et al., 2016 ; Kranjc et al. , 2018 ; Xiong et al., 2017 ). Improved knowledge about the potential factors affecting phytotoxicity, phytotoxicity mechanisms, and mitigation measures will aid in future risk assessment of ENPs.
Several studies have reported positive effects of nanoparticles on germination, growth and performance of plants. For example, increased seed germination and seedling growth, improved photosynthetic efficiency, biomass and total protein, sugar, nitrogen and microelements were observed in some crops; for example, Vigna radiata and Cicer arietinum ( Mahajan et al., 2011 ), Spinacia oleracea ( Srivastava et al., 2014 ), Cucumis sativus ( Moghaddasi et al., 2017 ), Solanum lycopersicum ( Faizan), et al. and Triticum aestivum ( Zhang et al., 2018 ).C. sativus cells grown in gel chambers showed increased shoot and root biomass with ZnONPs (1 mg/L), and increased shoot length with ZnONPs compared with large granular ZnO ( Moghaddasi et al., 2017 ). When ZnONP and their derivatives, including ZnCl 2 , are available in the soil in excess, potential toxicity can result in plants ( Liu et al., 2015 ; Mukherjee et al., 2014a ; Wang et al., 2015) et al., 2016a ), including inhibitory effects on seed germination, growth, photosynthesis, physiological and biochemical characteristics, yield characteristics and nutritional quality ( Du et al., 2017 ; Zuverza- Mena et al., 2017 ).
Previous studies have reported potential oxidative stress upon exposure to different ENPs in plants ( Mukherjee et al., 2016 ). Oxidative stress occurs when the balance between reactive oxygen species (ROS) and defense systems in plants is impaired.
ROS, generated as a by-product of light reactions in chloroplasts during photosynthesis, is associated with ENP-induced toxicity ( Ma et al., 2015 ) because excess ROS can lead to damage DNA damage, protein oxidation, lipid peroxidation, membrane damage, electrolyte leakage and ultimately cell death ( Demidchik, 2015 ; Wang et al., 2018). To maintain homeostasis, plants have developed different strategies that allow protection of cellular and subcellular components from the potential toxicity of free radicals through neutralization or elimination. ROS. These mechanisms include both non-enzymatic antioxidants, including low molecular weight compounds (phenolics, ascorbates, a-tocopherols, glutathione, carotenoids, and proline) and enzymes such as superoxide dismutase (SOD). , catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX), among others ( Getnet et al., 2015 ; Ozyigit et al., 2016 ). These antioxidants reduce ENP-related oxidative damage in plants ( Siddiqi and Husen, 2017). MDA is one of the by-products of oxidative stress and ROS is the result of oxidation of unsaturated fatty acids on cell membranes ( Gaschler and Stockwell, 2017 ).
We have recently shown the particle size (25, 50 and 250 nm) – and concentration-dependent effects of CuONP on lipid peroxidation and antioxidant biomarkers including SOD, CAT, POX and APX, in soybeans grown in mycorrhiza-rich soil for some time. for 120 days in an outdoor environment ( Yusefi-Tanha et al., 2020 ). In particular, the impact of 25 nm CuONPs was found to be consistently higher for most of the antioxidant biomarkers tested compared with the two larger size CuONPs treatments (50 nm CuONPs, 250 nm CuONPs) or Cu 2+ ions. We also show that the concentration-response curves for 25 nm CuONPs and Cu 2+ ions are linear, unlike for the larger sized CuONPs (50 nm CuONPs, 250 nm CuONPs), the relationships The relationship was non-linear, for most of the antioxidant biomarkers evaluated. The concentration-response curves for grain yield for all types of Cu compounds were linear (R 2 > 0.65). The soybean seed yield also showed a particle size and concentration-dependent inhibition with CuONP, and the inhibition of the 25 nm CuONP was significantly higher than that of the two larger CuONP or Cu 2+ ions at all levels. all concentrations tested. Our results show a difference between nanospecific toxicity versus Cu 2+ ionic toxicity in soybean ( Yusefi-Tanha et al., 2020 ).
Zinc (Zn) is an essential micronutrient for all plants because it plays an important role in many physiological activities such as in the biosynthesis of chlorophyll, proteins and enzymes, including in metabolism ( Singh et al., 2018 ). For example, Zn is present in cellular copper/zinc-SOD enzymes and chloroplasts have an important role against oxidative stress ( Yruela, 2015 ). Zn metalloproteins are known to participate in DNA replication and transcription, thereby regulating gene expression ( Barker and Pilbeam, 2015 ). In addition, Zn is a structural part of the ribosome and is responsible for its structural integrity, and is involved in amino acid synthesis and nitrogen metabolism ( Barker and Pilbeam, 2015 ; Kobraee and Shamsi, 2015 ). Zn is also found in the structure of zinc finger proteins (Znf – relatively small protein tissues containing many finger-like protrusions that make parallel contacts with their target molecule) CCCH type, genes encoding Their chemistry increases the oil content of soybean seeds through the activation of genes involved in lipid biosynthesis ( Li et al., 2017 ). In addition, Zn stimulates N2 fixation in legumes, such as French beans ( Phaseolus vulgaris L.), by promoting the number of root nodules ( Hemantaranjan and Garg, 2015 ). Zn deficiency in plants is a matter of global concern ( Impa et al. 2013 ), thus highlighting the potential of Zn supplementation in the form of nanoparticles as fertilizer ( Dimkpa et al., 2015 ; Du et al. , 2019). However, it should be noted that at higher concentrations, ZnONPs nanozinc oxide can also be toxic to plants (Dimkpa et al., 2015 ; Liu et al., 2015 , wang et al., 2016a ).
Soybean, one of the most cultivated and economically important food crops, is rich in protein and oil content and is consumed globally (FAO, 2019; Kanchana et al., 2016 ). Soil fertilization can affect the nutritional quality of crop seeds such as soybean ( Zulfiqar et al., 2019 ). In this study, we investigated whether nano zinc oxide (ZnONPs) could act as a novel nanofertilizer that could improve plant health and yield. We tested grain size, morphology and concentration-dependent responses on seed yield and antioxidant defense system in soybean soil ( Glycine maxcv. Kowsar) over its life cycle of 120 day. To achieve this goal, we synthesized three morphologically different ZnONPs (spherical/38 nm, flower-like/59 nm and rod-like /> 500 nm); all have high purity, triclinic crystal structure and negative surface charge.
Materials and methods
Synthesis and characterization of ZnONPs nano zinc oxide
Nano zinc oxide with three different morphologies (sphere/38 nm, flower shape/59 nm and rod /> 500 nm) were prepared by ball milling and sol-gel. Analytical zinc acetate dehydration (Zn (CH3COO)2 .2H2O) and citric acid (C6H8O 7 ) were purchased from Merck and used as received. The details of the synthesis have been previously described by Zandi et al. (2011) . Briefly, zinc acetate and citric acid (in powder form) were mixed in a 1:1 molar ratio and ground for 1 h at room temperature. The milled powder was calcined at 530 °C for 10 h to obtain ZnONPs (S 1). ZnONP samples with larger particle sizes were also prepared by the gel sol method. For this purpose, a 1:1 molar ratio of zinc acetate and citric acid was dissolved in distilled water. The solution was stirred with a magnetic stirrer at 80 °C when a viscous gel was obtained. The gel was dried at 100°C to convert to a powder, which was then divided into two batches; each batch is fired at 800°C (S 2 ), or 1000°C (S 3) in the atmosphere. The phase formation and crystal structure of the samples were characterized using X-ray diffraction (XRD) over the scanning angle range 2θ = 20-80° using a Philips X’Pert PRO X-ray diffractometer using Cu-Kα X-ray source (λ = 1.5406 Å). Field emission scanning electron microscopy (FE-SEM) was used to determine the morphology and diameter of the particles. Dynamic light scattering (DLS) was used to estimate the hydrodynamic diameter (HDD) and zeta potential ζ (of the synthesized ZnONPs.
Our experiments follow a two-dimensional factorial design, including: ZnCl2 (Zn2+ ; positive control) and ZnONP nano zinc oxide with three different morphologies (ZnONP; spherical/38 nm, floral/59 nm and 500 nm rods) and five different concentrations (0, 40, 80, 160 and 400 mg Zn/kg). To avoid any spatial effect, treatments followed a completely randomized design (RCD) with three replicates for each treatment, for a total of 60 experimental units with two plants each. taste ( n = 120 plants). All experiments were performed at Shahrekord University, Iran.
Characteristics of the land
Soil is taken from the surface layer (depth 0-30 cm). To separate wood chips, clumps and stones, the soil was air-dried and sieved (2 mm mesh). The background zinc content in the soil was 0.892 mg/kg. The main physicochemical characteristics of this soil are as follows: classified as silty soil (16% sand, 58% alluvium and 26% clay), with a pH of 7.44, EC of 0.47 dS/ m, 9.24 g/kg organic carbon, 0.88 g/kg total N, and 11.7 and 405 mg/kg P and K, respectively.
Soil improvement with zinc compounds
For soil improvement, each zinc compound (Zn 2+ and ZnONP: sphere / 38 nm, flower shape / 59 nm and rod /> 500 nm) was weighed and suspended in 100 mL of distilled water to achieve the desired concentration ( 0, 40, 80, 160 and 400 mg Zn/kg soil). ZnONP zinc oxide nano-suspensions and Zn 2+ ions were stirred with a magnetic bar for 30 min at 25°C before being added to the soil. Then, different Zn treatments (ZnONP and Zn 2+ ) were added to the soil. ZnCl 2 was added to the soil as an aqueous solution (positive control) to determine the potential effect of Zn 2+ on soybean. Untreated soil served as a negative control. Three replicates of each treatment were prepared. Nano zinc oxide and Zn 2+ ions in water suspension were mixed by hand with 1 kg of soil for 30 min, mixing three times with 1 kg of soil each time for a total of 4 kg of soil per pot (to allow mixing homogenized) and equilibrated in the open air for 24 h before subsequent planting.
Crop management through maturation and seed production
In brief, the control and improved soil samples (4 kg) were placed in PE (polyethylene) bags and in PE pots (20 cm diameter x 20 cm height). Each pot has an inner PE mesh lining (all bags have 50 5 mm holes for drainage) and the bottom is filled with a layer of washed gravel (500 g), which facilitates ventilation and Drainage. This design allows the root system to remain in the bag/pot and facilitates easier removal of the plant from the pot during harvest. Soybean seeds (Kowsar variety) were obtained from the Plant Variety Improvement Institute, Karaj, Iran. They are soaked in distilled water for 24 hours to facilitate germination. Two seeds of uniform size were grown after inoculating soybean symbiotic bacteria ( Rhizobium japonicum) at a depth of 2.5 cm of soil for 24 h after soil amendment. This study was conducted under miniature conditions to better understand the true impact of NPs in the environment. During the growing period, the pots are irrigated according to the capacity of the field. At each irrigation, a subsample of water was measured for Zn concentration by inductively coupled plasma optical emission spectroscopy (ICP-OES). At maturity (120 days after planting), the plants and seeds are harvested. The seeds were air-dried and weight recorded.
Determination of biochemical parameters
The two youngest leaves per pot (one of two leaves per pot) were sampled to determine all biochemical parameters examined ( Yusefi-Tanha et al., 2020 ).
Measure the concentration of malondialdehyde
The degree of lipid peroxidation was determined by measuring the formation of malondialdehyde (MDA) with thiobarbituric acid (TBA) by the method of Heath and Packer (1968) . Briefly, fresh leaf tissue samples (0.1 g) were homogenized in 1.5 ml of 0.1% trichloroacetic acid (TCA). The resulting homogenate was centrifuged at 10,000 × g for 10 min, and 1 mL of the supernatant was added to 2 mL of 20% TCA containing 0.5% TBA. The extract was heated in a water bath at 95 °C for 30 min and then rapidly cooled in an ice bath. Then centrifuge at 10,000 × g for 10 min. The absorbance of the supernatant was read at 532 and 600 nm against the blank. MDA concentrations were expressed in nmol/g FW (using the 155 mM -1 cm -1 extinction factor) (Narwal et al., 2009 ).
Estimated hydrogen peroxide (H2O2 )
Hydrogen peroxide generated in plant leaves was measured according to the method described earlier by Nag et al. (2000) . Fresh leaf tissue (1 g) was pulverized with liquid nitrogen and homogenized in 12 mL of cold acetone. Then, the homogenizer was filtered through Whatman filter paper. The mixture was diluted using 4 mL of 16% titanium reagent and 0.2 mL of 28% ammonium hydroxide. The tissue extract was further centrifuged at 8500 rpm for 5 min at 4 °C. The supernatant was separated and then washed twice with 5 mL acetone. Finally, add 2 mL of sulfuric acid (1M), and the absorbance was measured at 410 nm. The hydrogen peroxide content was calculated using a standard curve prepared in the same way and expressed as nmol/g FW.
Estimated superoxide dismutase (SOD)
Tissue samples (1 g fresh leaf tissue) were frozen in liquid nitrogen and homogenized in 10 mL of 0.1 M potassium phosphate buffer (pH 7.5). The tissue extract was further centrifuged at 20,000 rpm for 30 min at 4°C. The supernatant was collected, separated into several fractions, and stored at -20°C for activity determinations. of antioxidant enzymes. SOD activity was determined by measuring inhibition of photochemical reduction of nitroblue tetrazolium (NBT) ( Narwal et al., 2009 ). 1.95 mL 0.1 M potassium phosphate buffer (pH 7.5), 250 μL 150 mM methionine, 250 μL 1.2 mM Na 2EDTA, 250 μL 24 μM riboflavin, 250 μL 840 μM NBT, and 50 μL Plant extracts were prepared. The reaction was initiated by illumination and the absorbance was read at 560 nm. One unit of SOD activity was defined as the amount of enzyme that caused a 50% inhibition of oxidative reactions per milligram of protein in the extract.
Estimation of catalase activity (CAT)
CAT activity was determined according to Narwal et al. (2009) by measuring the decrease in absorbance at 240 nm after the decomposition of hydrogen peroxide (H2O2). The reaction mixture consisted of 50 μL of supernatant, 1.95 ml of 0.1 M potassium phosphate buffer (pH 7.0) and 100 μL of 264 mM H2O2. Absorption reduction was detected over a period of 100 s with a period of 5 s at room temperature (25 °C). One unit of CAT activity corresponding to 1 mMol H2O2 was consumed per minute per mg of protein using a shutdown factor of 40 mM -1 cm -1 .
Estimation of guaiacol peroxidase (POX) activity
The activity of guaiacol peroxidase (POX) was estimated according to the method previously developed by MacAdam et al. (1992) . Briefly, 50 μL of the plant extract was added to 1.35 mL of 0.1 M potassium phosphate buffer (pH 6.0), 100 μL of 45 mM guaiacole, and 500 μL of 44 mM hydrogen peroxide. We then measure the kinetics of changes in absorbance at 470 nm at 10 s intervals for 300 s at 25°C using a UV-Vis spectrometer. One unit of POX activity corresponding to 1 mMol tetraguaiacol is consumed per minute. per mg of protein using a reduction factor of 26.6 mM -1 cm -1 .
Estimation of ascorbate peroxidase (APX) activity
APX activity was measured by monitoring the rate of ascorbate oxidation with H2O2, according to the method developed by Narwal et al. (2009) . Ascorbic acid reduction, starting from a mixture of 2.4 mL 0.1 M potassium phosphate buffer (pH 7.0), 250 μL 1.2 mM Na2EDTA, 50 μL 35 mM H2O2, 100 μL 15 mM ascorbic acid , and 200 μL of supernatant was measured at 290 nm over a period of 500 s at a time interval of 10 s at room temperature (25 °C). Activity was calculated using a damping factor of 2.8 mM -1 cm -1 . One APX unit is defined as 1 mMol ascorbate oxidized per minute. per mg of protein.
Estimated amount of H2O2 obtained
The H2O2 collection was measured according to the method of Benkebila (2005), which measures the decrease in UV light absorption at 230 nm due to the H2O2 collection activity of the tested substances/extracts. Each sample was prepared by adding 1 ml of plant extract and 600 μL of 2 mM freshly prepared H2O2 in phosphate-buffered saline (PBS). In another tube, a control sample was prepared by adding 1 mL of PBS and 600 μL of 2 mM freshly prepared H2O2 in PBS. Then the mixture is left for 10-15 minutes. at room temperature. Then the absorbance was measured at 230 nm.
Data were analyzed with two-way analysis of variance (ANOVA) to test for interactions between factors using the least significant difference test using SAS (SAS Inc., version 9.4). . Differences were considered significant at p<0.05 and means were separated using Fisher’s protected LSD test. The results are presented as mean ± standard deviation (SD). To determine whether concentration-response curves are linear (monotonic) or non-linear (non-monotonic), we combine a visual inspection of the curves with a simple decision rule: if the coefficient of determination (R squared) value for the linear regression curve is 65 % or higher the concentration-response curves are considered linear, indicating that the response of the plants varies linearly with concentration is applied according to the relationship: y = ax + b; where y represents the dependent variable, x represents the independent variable, and a and b are the parameters of the model. The calculated R-squared values are presented in Table S1 Supplementary Information (SI).
Features of nanoparticles
Analysis of the XRD samples revealed that the samples were in the form of triclinic crystals with the p63mc space group, without any noticeable impurity traces. The XRD patterns of the powder samples are shown in Figure 1 . The morphology and average particle diameter of the nano zinc oxide ZnONPs were measured by field emission scanning electron microscopy (FE-SEM). Representative FE-SEM microscopic images of ZnONPs samples and their particle size distribution (PSD) fitted to a normal distribution function (as shown in equation 1 below; Rostamnejadi et al., 2017 ) is shown in Figure 1 :
Figure 1. FE-SEM image of three morphologically distinct nano zinc oxide: A) S1 (sphere / 38 nm diameter); B) S2 (flower variety / diameter 59nm); and C) S3 (rod diameter /> 500 nm and length > 1μm). Particle size distribution (PSD) of sample S1 (D) and sample S2 (E). F) XRD patterns of powder samples of three morphologically distinct ZnONPs: S1, S2 and S3.
In this relation <d> is the mean particle size and σ is the standard deviation. Our analysis of the FE-SEM micrographs of the S1 samples showed that the particles were almost spherical in shape with an average particle diameter of 38 nm but with dense packing appearing as likely. conglomerate. The S2 samples had a flower-like morphology with an average grain diameter of 59 nm. The S3 samples showed spherical particles with mean diameter greater than 500 nm on top of microrods with a diameter of about 1 μm and a length of 10-50 μm. All zinc oxide (S1-S3) nanosamples are highly negatively charged with zeta potential above 44.0 mV but have variable hydrodynamic diameter (HDD) ( Table 1 ), a result consistent with FE-SEM analysis
Table 1. Dynamic light scattering (DLS) measurement of hydrodynamic diameter (HDD) and zeta potential values for: (S 1 ) spherical ZnONP-38 nm, (S2) flower-shaped ZnONP-59 nm and (S3) ZnONP rod > 500 nm.
Impact on seed yield
Our results show that the influence of zinc compounds ( Zn type ), zinc concentration (C) and the interaction of Zn type × C are statistically significant on the grain yield in soybean ( p <0 .0001) ( Table 2 ). In general, the grain yield of all Zn compounds increased concentration-dependently up to 160 mg/kg, but at the highest concentration tested (400 mg/kg) the grain yield decreased significantly for all Zn compounds ( Figure 2 ). The grain yield was highest for the spherical 38 nm ZnONPs compared with the flower or rod 59 nm ZnONPs > 500 nm. Seed yield was generally lowest for Zn 2+ ion treatment compared with all three ZnONPs treatments ( Figure 2), indicating an inhibitory effect of Zn 2+ ion on seed yield compared with ZnONP tested. . Furthermore, the highest grain yield was 160 mg/kg for all Zn compounds. In general, the concentration response curves for all Zn compounds are nonlinear ( Figure 2 ; see Table SI S1 for R-squared values) and that the responses depend on the particle size. and morphology.
Figure 2. Seed production in soybean soil treated with nano zinc oxide and zinc ions (sphere ZnONP-38 nm, flower ZnONP-59 nm, rod ZnONP > 500 nm and Zn 2+ ions) function number of concentrations (0, 40, 80 160, 400 mg Zn/kg soil). The error bars represent the mean ± SD. Different letters above the bar indicate a significant difference at p <0.05.
Table 2. Analysis of variance ( p- value) for seed production and biochemical parameters of soybean grown in soil treated with different concentrations of zinc compounds.
Impact on production of hydrogen peroxide (H2O2 )
Our ANOVA showed that the effects of Zn, C and Zn × C types on leaf H2O2 production in soybean were significant ( p <0.0001) ( Table 2 ). We observed the effect of particle size-, morphology- and concentration-dependent concentration of ZnONP on H2O2 production in soybean leaves ( Figure 3 ). With increasing concentrations, the H2O2 yield decreased significantly up to 160 mg/kg for all Zn compounds; while at 400 mg/kg, the H2O2 yield increased significantly for all Zn compounds, except that treatment of rod ZnONP >500 nm showed no significant change compared with the control ( Figure 3 ). Overall, these results show that all Zn compounds are able to protect soybean up to 160 mg/kg and the responses are non-linear ( Figure 3 ; see Table SI S1 for values) R squared).
Figure 3. Changes in hydrogen peroxide (H2O 2 ) production in leaves ( A ) and malondialdehyde (MDA) content in leaves ( B ), in soybean soil treated with zinc oxide nanoparticles and zinc ions ( Spherical ZnONP-38 nm, flower-like ZnONP-59 nm, rod-like ZnONP > 500 nm, and Zn 2+ ions) as a function of concentration (0, 40, 80, 160, 400 mg Zn/kg soil). The error bars represent the mean ± SD. Different letters above the bar indicate a significant difference at p <0.05.
Impact on malondialdehyde (MDA) accumulation
The accumulation of the MDA-TBA complex was measured to estimate lipid peroxidation in leaf tissue. Our results show that MDA content is affected by type Zn ( p <0.0001), C ( p<0.0001) and their interaction ( type Zn ×C) ( p <0.0001) ( Table 2). We observed the effect of particle size-, morphology- and concentration-dependent concentration of nano zinc oxide on MDA levels in soybean leaves ( Figure 3 ). With increasing concentrations, the MDA content decreased significantly up to 160 mg/kg for all Zn compounds; while at 400 mg/kg, the MDA content increased significantly for all Zn compounds, except for the >500 nm rod ZnONP treatment which showed no significant change compared with the control (Fig. ). As expected, the MDA results reflect the results of H2O2 production in leaves suggesting a mutualistic relationship between H2O2 production and MDA-TBA accumulation upon lipid peroxidation. In general, lipid peroxidation reactions are nonlinear ( Figure 3 ; see Table SI S1 for R-squared values).
Impact on the activity of superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POX)
The activities of multiple antioxidant enzymes (SOD, CAT and POX) in soybean leaves exposed to different Zn compounds are presented in Figure 4 . The effect of type Zn ( p <0.0001), C ( p <0.0001) and interaction term ( type Zn × C) ( p <0.0001) were statistically significant on the performance of the antioxidant enzymes (SOD, CAT and POX) in soybean leaves. Similar to the H2O2 production and subsequent MDA accumulation outlined above, we observed the effect of particle size-, morphology- and concentration-dependent concentration of nano zinc oxide ZnONP on the activities of ZnONPs. SOD, CAT and POX activity in soybean leaves ( Figure 4). With increasing concentrations, the activities of this antioxidant enzyme were significantly reduced up to 160 mg/kg for all Zn compounds; while at 400 mg/kg, the activities of this enzyme increased significantly for all Zn compounds, with the exception of rod ZnONP treatment > 500 nm which showed no significant difference in the activity of Zn compounds. enzyme compared with the control ( Figure 4 ). Overall, these results suggest that all three activities of antioxidant enzymes are consistent with the trends towards H2O2 synthesis and MDA accumulation, and that the reactions of the enzymes are non-linear. ( Figure 4 ; see SI Table S1 for R- squared value).
Figure 4. Changes in leaf superoxide dismutase (SOD) ( A ), catalase (CAT) ( B ), and guaiacol peroxidase (POX) ( C), activities in the soybean soil treated with nanoparticles zinc oxide and zinc ions (spherical ZnONP-38 nm, flower- like ZnONP-59 nm, rod ZnONP > 500 nm and Zn 2+ ions) as a function of concentration (0, 40, 80, 160, 400 mg Zn/kg) soil). The error bars represent the mean ± SD. Different letters above the bar indicate a significant difference at p <0.05.
Effects on ascorbate peroxidase (APX) and H2O2 . scavenging activities
The effects on APX and H2O2 collection activities in soybean leaves exposed to different Zn compounds are depicted in Figure 5 . ANOVA showed a statistically significant effect of Zn concentration (C) and interaction duration ( type Zn × C) on APX activity ( p <0.0001) in soybean leaves. Meanwhile, the effects of Zn, C and interaction terms (type Zn × C) are statistically significant on the process of H2O2 collection ( p <0.0001) in soybean leaves. Overall, the APX response of soybean when exposed to different types of Zn compounds contrasted with that of the observed scavenging H2O2 .
Figure 5.Changes in peroxidase ascorbate (APX) ( A ) and H2O2 ( B ) absorption activities in soybean soil treated with different types of zinc compounds (spherical ZnONP-38 nm, ZnONP-59 nm). plant form, rod ZnONP > 500 nm, and Zn 2+ ions) as a function of concentration (0, 40, 80, 160, 400 mg Zn/kg soil). The error bars represent the mean ± SD. Different letters above the bar indicate a significant difference at p <0.05.
For APX activity, measuring the rate of ascorbate oxidation in the presence of H2O2, we observed a particle size-dependent effect, morphology and concentration of nano zinc oxide in soybean leaves (Figure 5A ). With increasing concentrations, APX activity was significantly reduced up to 160 mg/kg for all tested Zn compounds; while at 400 mg/kg, APX activity significantly increased for all Zn compounds compared with control ( Figure 5A). Furthermore, the spherical 38 nm zinc oxide nanosheets had the best protection compared to all other Zn compounds tested (flower-shaped 59 nm ZnONP, rod > 500 nm ZnONP and Zn 2+ ions) up to 160 mg/kg; but at the highest concentration (400 mg/kg) the toxicity trend reversed, i.e., Zn 2+ ions were the most protective compared to the spherical 38 nm ZnONPs. Overall, at 400 mg/kg all Zn compounds showed significantly higher APX (protective response) activity than the control ( Figure 5A ). Furthermore, the APX responses for all Zn compounds were nonlinear ( Figure 5A ; see Table SI S1 for R-squared values).
Our analysis of the H2O2 scavenging ability of different ZnONPs revealed a particle size, morphology and concentration-dependent effect of ZnONPs in soybean leaves ( Figure 5B ). With increasing concentrations, the collection of H2O2 was significantly reduced up to 160 mg/kg for all tested Zn compounds; while at 400 mg/kg, the collection activity was significantly increased for all Zn compounds compared with the control ( Figure 5B ). Furthermore, the spherical 38 nm ZnONPs had the lowest efficiency in removing H2O2 from leaves compared to all other Zn compounds tested (floral ZnONP 59 nm, rod ZnONP > 500 nm and Zn 2+) ions) up to 160 mg/kg; but at the highest concentration (400 mg/kg) the scavenging trend reversed for the NPs, i.e. the spherical 38 nm ZnONPs were most effective in scavenging H 2 O 2 in leaves compared with two nanometers. other zinc oxide tested (flower-like 59 nm ZnONPs, rods > 500 nm ZnONP). Meanwhile, at 400 mg/kg, Zn 2+ ion showed the most potential for H 2 O 2 recovery compared to all three ZnONPs. Overall, the leaf H2 O 2 gathering reactions for all Zn compounds were non-linear ( Figure 5B ; see Table SI S1 for R-squared values).
Previous studies have documented different toxicity of nano zinc oxide in soybean and other plants. However, most of these studies have focused only on a single nanoscale of zinc oxide, thus limiting our understanding of how some key physicochemical factors such as size, morphology (shape) and electrical How surface area might play a role in nanotoxicity. For example, exposure to 18 nm ZnONPs resulted in increased chlorophyll content, shoot height and grain yield in winter wheat, while plant biomass remained unaffected (Dimkpa et al., 2018 ). The foliar application of zinc oxide, CuO and B2O3 nanocompounds showed a significant increase in soybean biomass, seed number, grain biomass and absorption of micro and macro nutrients ( Dimkpa et al. events, 2017a). The frequency of pods was increased in soybeans exposed to ZnONPs, while the average size and number of seeds per pod did not differ between treatments ( Priester et al., 2012 ). Wheat biomass and grain yield were increased by 72% and 55%, respectively, when treated with ZnSO 4, while ZnONPs zinc oxide nanotreatment resulted in an increase in biomass and grain yield of 63% and 56%, respectively, compared with control ( Du et al., 2019 ). However, in Vigna unguiculata grown in soil supplemented with soluble Zn 2+ ions or nano zinc oxide showed no significant difference in plant growth between the two Zn compounds ( Wang et al., two thousand and thirteen). Plant uptake and NP transport to aerial plant parts have been previously documented for other types of NP exposure (Mueller and Nowack, 2008; Shang et al., 2019), resulting in decrease in plant biomass (Ma et al., 2015a; Shang et al., 2019). As discussed previously, Zn is required by plants to perform many physiological activities such as protein and enzyme biosynthesis, chlorophyll, and normal functioning of metabolic processes ( Singh et al., 2018 ). In this study, the grain yield was highest for the spherical 38 nm ZnONPs compared with the flower 59 nm ZnONPs or the rod > 500 nm ZnONPs ( Figure 2 ) up to 160 mg Zn/kg in the treatment. Seed yield was generally reduced with Zn 2+ ion treatment compared with all three ZnONPs treatments ( Figure 2), indicating the inhibitory effect of Zn 2+ ions on soybean seed development compared with ZnON2+ ions treatment. ZnONPs were evaluated. Although we did not measure ZnONP uptake in soybean, it is possible that the biochemical processes discussed above (Chandra et al., 2014; Shaw and Hossain, 2013; Da Costa and Sharma, 2016) may have been altered by exposure to solutes Zn 2+ and ZnONP ions, capable of promoting (at 40-160 mg/kg) or inhibiting (at 400 mg/kg) grain yield. Taken together, our results show distinct effects of ZnONPs compared with Zn 2+ ( Figure 2 ) ion treatments. Furthermore, the decreased energy requirement due to decreased antioxidant enzymes (SOD, CAT, POX and APX) ( Kurutas, 2016 ; Yusefi-Tanha et al., 2020 ) was expressed up to 160 mg/kg of the treatments. ZnONP or Zn 2+ ion treatment could explain the increased grain yield and reversibility at 400 mg/kg of ZnONP or Zn 2+ ion treatment.
Plant tolerance to exposure to metal-based nanoparticles is determined by their ability to protect effective antioxidant systems to remove or eliminate ROS (H 2 O 2 , OH – ) in excess in cells and tissues. Previous studies have shown that the toxicity of bulk ZnO is lower than that of zinc oxide nanoparticles ( Amooaghaie et al., 2016 ; Mukherjee et al., 2014a ), and the small ZnONPs were transformed into aggregates are larger in plant cells, complicating the assessment of toxicity ( Lee et al., 2013 ). Toxicity induced by higher concentrations of Zn (400 mg Zn/kg) was manifested as a potential interference with major antioxidant enzyme activities as described in Fig. 2- 5A . H 2 O 2 is toxic to cells, especially at higher levels, so plants must maintain a balance in their cells to minimize its levels to avoid oxidative stress and lipid peroxidation. latent ( Das and Roychoudhury, 2014 ). Previous works showed that ROS was increased by exposure to higher concentrations of ZnONP <100 nm (500 mg Zn/kg) in wheat ( Dimkpa et al., 2012 ) or 90 nm ZnONP (800 mg/kg soil) in maize ( Liu et al., 2015 ). The hydroxyl radical (OH – ), the most reactive of all the ROS, is generated from H2O2 as a result of molecular reduction, and can separate the hydrogen atom from the methylene group (CH2) in the side chain of polyunsaturated fatty acids of membrane lipids, thereby initiating lipid peroxidation (Shaw et al., 2014). Our observation of the grain size, morphology and concentration-dependent effects of ZnONP on MDA accumulation in soybean as a response to H2O2 synthesis and lipid peroxidation in leaves is unique for nanotoxicity studies ( Figure 3 ). Nair and Chung (2014a) observed a significant increase in H2O2 in soybean roots when exposed to different concentrations of CuONP. Reduced lipid peroxidation (MDA levels) observed with exposure to rod ZnONP >500 nm, especially at the highest concentration (400 mg/kg), may indicate a lack of seed transport in the plant due to The larger particle size and rod-like morphology, could provide significant protection for membrane integrity compared with the other two types of ZnONP and Zn 2+ ions. These results are consistent with previous work that showed a threshold of protective effects beyond the manifest toxicity for ZnONPs in onion root cells ( Kumari et al., 2011 ).
Hernandez-Viezcas et al. (2011) reported increased CAT activity in roots, shoots and leaves at different concentrations (500-4000 mg/L) of the 10 nm ZnONPs treatment, but at concentrations higher than 500 mg/L APX activity was reduced . Allium cepa exposure to ~85 nm ZnONPs (200, 400 and 800 mg/L) resulted in decreased CAT activity, but did not affect ROS levels and glutathione peroxidase activity ( Ghosh et al., 2016 ). In the aquatic fern ( Salvinia natans ), exposure to 25 nm ZnONPs (1-50 mg/L) was not shown to affect plant growth, but resulted in increased SOD and CAT activities at high concentrations. The highest levels tested ( Hu et al., 2014), showed an increased oxidative stress response of the fern at 50 mg Zn/L. At 500 and 750 mg Zn/kg, the CAT enzyme activity decreased in stems and leaves have been reported for different Zn compounds in alfalfa ( Bandyopadhyay et al., 2015 ). Our findings showed that MDA and many antioxidant biomarkers (SOD, CAT, and POX) were altered in a similar fashion by Zn compound type, concentration and their interactions ( Table 2 , 3 ; Figure 3). More specifically, the results show the effect of particle size, morphology and concentration-dependent concentration of nano zinc oxide ZnONP on MDA and antioxidant biomarkers, including SOD, CAT and POX, in beans soybeans are grown for 120 days. Overall, up to 160 mg of Zn/kg of treatment, a significant protective effect was observed in soybean by all types of Zn compounds tested.
Table 3.Analysis of variance ( p -value) for antioxidant enzyme activities of soybean grown in soil treated with zinc oxide nanoparticles and zinc ions at different concentrations.
As an important antioxidant in chloroplasts, cells and mitochondria ( Demidchik, 2015), SOD catalyzes superoxide radical (O2 – ) and conversion to H2O2 and O2 , thus playing a key role in stress resistance. oxidation ( Shekhawat, 2013 ; Ahmed et al., 2018 ). The results showed that the increased leaf SOD activity (O2 – + 2H + → H2O2 + O2 ) at 400 mg Zn/kg may have promoted the production of H 2 O 2 as shown in Figure 3A. However, the overall SOD activity was up to 160 mg Zn/kg treatment lower for all tested Zn compounds ( Figure 3A ). Superoxide (O2 – ) levels were found to increase with 400-3,200 mg Zn/kg ZnONP (90 ± 10 nm) and a significant increase in SOD activity was observed at the highest dose in maize ( Wang et al., 2016a ) . In Gossypium hirs, an increase in SOD and POX activity was reported along with a subsequent decrease in lipid peroxidation with ZnONPs treatments ( Venkatachalam et al., 2017 ). These results are consistent with our study. Enzymes such as CAT, POX and APX are known to catalyze the reduction of H 2 O 2 to water and oxygen in cellular chloroplasts, cytosols, mitochondria and/or peroxisomes (Anjum et al., 2016). These and other antioxidants may act independently or together through crosstalk and reducing levels of oxidative damage and toxicity in plants ( Ahmed et al., 2017a and 2017b ). The enzymes CAT, POX, and APX seem to work simultaneously. Here, low APX activity ( Figure 5A ) was followed by higher CAT and POX activities for ZnONP and Zn 2+ ( Figure 4B, C ) ions – a finding that is consistent with their previous results. I recorded for the treatment of CuONPs in soybean ( Yusefi-Tanha et al., 2020). The overall trend of APX activity upon Zn treatment was reversed compared with that of MDA accumulation and SOD, CAT and POX responses in soybean leaves ( Figure 5A ). Meanwhile, the collection of H2O2 by Zn compounds showed a similar trend to the MDA, SOD, CAT and POX reactions ( Figure 5B). Furthermore, we noted a clear threshold of 160 mg Zn/kg, which is above the threshold where soybean responses to all Zn compounds were found to be negative for all. endpoints are measured. Our use of the same high purity crystal structure (three layers) and surface charge (negative) for all three sizes and morphologically different zinc oxide nanosheets allows us to control specifically investigated the size and morphology-dependent effects of ZnONP in soybean. Our results show the particle size, morphology and concentration-dependent effects of nano zinc oxide on grain yield, lipid peroxidation and various antioxidant biomarkers in soybean. planted on land. Further findings revealed the unique toxicity of nano zinc oxide compared with Zn 2+ ion in soybean.
An estimated 50% of arable land used in agriculture may contain reduced amounts of soluble Zn, resulting in reduced crop yields and poor nutritional quality of cereals and derivatives ( Moreira et al., 2018 ). Organic matter is known to affect the availability of Zn in the soil to plants with ZnONPs or bulk Zn treatment ( Moghaddasi et al., 2017 ; Medina-Velo et al., 2017 ; Dimpka et al. , 2020). Recently, Dimkpa et al. (2020) recorded the beneficial effects of 18 nm ZnONPs on different criteria (bud length, plant height, shoot biomass, chlorophyll, seed yield, Zn 2+ bud/seed absorption) , grain Ca 2+ and Mg 2+ uptake) were measured in wheat under simulated drought conditions (at 40% field humidity) and/or with compost (cow manure). At half the concentration, ZnONPs (2.17 mg ZnO/kg) showed similar or greater positive effects than the bulk ZnO treatments (powder >1000 nm). These findings are significant in that the higher reactivity of ZnONPs may prove more sustainable (on a mass basis) than the application of large amounts of Zn to soil ( Dimkpa et al., 2020). ). Our findings also show the prospect of ZnONPs to be used as nano-fertilizers (up to 160 mg Zn/kg) for Zn elemental supplementation to plants ( Singh et al., 2013), reducing oxygen stress. and promote seed yield. Then, through edible plant parts such as leaves and seeds, Zn is bioavailable to humans as high as 70% while the average absorption is about 33% ( Turnlund et al., 1984 ; Cousins, 1985 ; FAO/WHO, 2004 ). As an essential micronutrient, Zn deficiency is associated with hypogonadism, anemia and stunted growth in humans ( Roohani et al., 2013 ); therefore, adding nano to plants during growth with the required amount of Zn (this can depend on soil type) could be a viable concept — a game-changing concept, especially especially, for those who rely on a diet rich in plants such as grains and beans and who consume less meat ( Roohani et al., 2013 ).
Evaluation of the R-squared values for all measured endpoints revealed non-linear concentration response curves (R 2 <0.65) (see SI Table S1). Furthermore, our results showed no observed adverse effect level (NOAEL) of 160 mg/kg for all three different nanozinc oxides, including Zn 2+ ions, for all all endpoints are measured (see TOC clause), except for H2O2 collection ( Figure 5B ). The protective effect provided by ZnONP up to 160 mg/kg in soybean may be due to the role of Zn as an essential micronutrient in many enzymes and proteins, which is key for the growth and development of plants. crops and crop yield ( Moreira et al., 2018 ).
In summary, our findings demonstrated the particle size, morphology and concentration-dependent effects of ZnONP on grain yield, lipid peroxidation, and various antioxidant biomarkers identified measured in soil-grown soybeans. The spherical 38 nm ZnONP zinc oxide nanoparticles had the best protection compared to the flower-shaped 59 nm ZnONPs, the > 500 nm rod ZnONPs, or the Zn 2+ ions, particularly up to 160 mg/kg. However, at the highest concentration of 400 mg/kg, the spherical 38 nm ZnONP nano zinc oxide produced the highest oxidation stress response (synthesis of H 2 O 2 , MDA, SOD, CAT, POX) in soybeans. compared with two morphologically different ZnONPs tested. The concentration-response curves for the three types of ZnONP and Zn 2+ions were nonlinear for all evaluated endpoints. Our results also showed a difference between the specific nanotoxicity of ZnONPs and the ionic toxicity of Zn 2+ in soybean. Furthermore, NOAEL values higher than 160 mg/kg indicate the potential to use ZnONPs as nano-fertilizers for plants in Zn-deficient soils to improve crop yield, food quality and address malnutrition. globally.
Elham Yusefi-Tanha, Sina Fallah, Ali Rostamnejadi, Lok Raj Pokhrel