Genotoxic Evaluation of Fe3O4 Nanoparticles in Different Three Barley (Hordeum vulgare L.) Genotypes to Explore the Stress-Resistant Molecules

Sustainable agricultural practices are still essential due to soil degradation and crop losses. Recently, the relationship between plants and nanoparticles (NPs) attracted scientists’ attention, especially for applications in agricultural production as nanonutrition. Therefore, the present research was carried out to investigate the effect of Fe3O4 NPs at low concentrations (0, 1, 10, and 20 mg/L) on three genotypes of barley (Hordeum vulgare L.) seedlings grown in hydroponic conditions. Significant increases in seedling growth, enhanced chlorophyll quality and quantity, and two miRNA expression levels were observed. Additionally, increased genotoxicity was observed in seedlings grown with NPs. Generally, Fe3O4 NPs at low concentrations could be successfully used as nanonutrition for increasing barley photosynthetic efficiency with consequently enhanced yield. These results are important for a better understanding of the potential impact of Fe3O4 NPs at low concentrations in agricultural crops and the potential of these NPs as nanonutrition for barley growth and yield enhancement. Future studies are needed to investigate the effect of these NPs on the expression of resistance-related genes and chlorophyll synthesis-related gene expression in treated barley seedlings.


Introduction
According to a Food and Agriculture Organisation (FAO) report, many people still experience food shortages. Moreover, the requirements for food will gradually increase in the world, at least until 2030 [1]. Permanent pesticide, herbicide, and fungicide use, and climate change induce soil degradation and crop losses [2,3]. Therefore, sustainable agricultural practices are essential for humankind [3]. Recently, the relationship between plants and nanoparticles (NPs) has attracted scientists' attention, especially to its application in agricultural production as nanonutrition. It has been shown that a small number of different NPs are present in agricultural soil which have been shown to accumulate in plants [1].
Research has explored that NPs, due to their small size (<100 nm), can pass plant biological barriers, thus allowing efficient delivery of nutrients/micronutrients [4]. Therefore, NPs, as a versatile resource of plant nanobiotechnology, can improve the management of crop disease and crop loss, thereby increasing plant resistance to various environmental biotic and abiotic stressors. Moreover, this method reduces the use of chemical fertilisers and soil contamination and degradation, promoting sustainable agricultural production [3][4][5][6][7][8].

Preparation of Fe 3 O 4 Nanoparticles and Their Characteristics
Fe 3 O 4 NPs with an average size of 25 nm were provided by G. Libert's Center of Innovative Microscopy, Daugavpils University. Fe 3 O 4 nanoparticles were obtained by the co-precipitation (Massart) method [36] using ferric chloride (II) and (III) in a ratio of at least 1:2 and aqueous ammonium hydroxide solution. For this purpose, 0.167 g of FeCl 3 ·6H 2 O and 0.0429 g of FeCl 2 ·4H 2 O were dissolved in 50 mL of distilled water. 0.27 mL of 25% NH 4 OH was added dropwise to the solution under constant stirring. The obtained nanostructures were stabilized with an aqueous citric acid solution (40 mg/mL, 2 mL). The resulting precipitate was separated with a permanent magnet and rinsed three times with distilled water to remove residual reagents. Schematically, the production of Fe 3 O 4 nanostructures can be described by the following equation:  The crystalline structure of the samples was defined by an X-ray diffractometer (Smart Lab, Rigaku, Japan) and corresponds to magnetite (Fe 3 O 4 ). The size of individual nanoparticles was determined using PDXL software according to the Williamson-Hall method and its value was 25 nm.

Barley Seedling Cultivation
Latvian origin spring barley genotypes 'Sencis' (mla 13), 'Abava' (lack of resistance genes), and commonly used high yielding spring malting England variety 'Quench' (mlo 11) were grown for the research. The seeds were provided by the Institute of Agricultural Resources and Economics, Stende Research Center (Priekuli, Latvia). The seeds were rinsed with deionized water and transferred for germination to a hydroponic tray for 8 days at 22 • C. The seedlings were transferred to tubes with tap water (control) supplemented with different Fe 3 O 4 NPs solutions (experimental). An equivalent number of plants (n = 30) were used in each treatment (control and three experimental groups) and each stage of the experiment (for morphological parameters, chlorophyll, genotoxicity, and miRNA analysis). Barley seedlings were grown in NP solution conditions for 8 days.
Nanoparticles were diluted in water to 1, 10, and 20 mg/L and sonicated for 30 min to split the formed nanoparticle agglomerates into individual NPs before use in the experiment.

Detection of Fe 3 O 4 Nanoparticles in Barley Using Confocal Laser-Scanning Fluorescence Microscopy (CLSM)
Fluorescent Fe 3 O 4 NPs (25 nm) were purchased from Sigma-Aldrich (Germany). The experiment with fluorescent Fe 3 O 4 NPs was prepared as described in Section 2.1. CLSFM was performed with a Nikon Eclipse Ti-E microscope (Nikon, Tokyo, Japan). Fluorescence was excited at λ = 457.9 nm, and emission was detected at 464-499 nm. Confocal images were obtained with NIS-elements Advanced Research 3.2 64-bit software (Nikon). Parameters for magnification at 200× and 600× were the same for all images and were as follows: HV = 154, offset = −34, laser power = 63.4. Detection of NPs was performed on day 1 and 6.

Effect of Fe 3 O 4 Nanoparticles on Barley Morphological Parameters
The shoot length and root length of barley seedlings were measured, and the number of roots was counted after 8 days of growth in water (control) with the application of different concentrations of Fe 3 O 4 NPs (1, 10, and 20 mg/L). The mean length values were calculated using three biological replicates.

Effect of Fe 3 O 4 Nanoparticles on Chlorophyll Content and the Steady State Absorption and Fluorescence Spectra of PSII and PSI in Treated Barley Seedlings
Chlorophyll extraction from control and treated barley seedlings' leaves was performed to determine chlorophyll content and to estimate the fluorescence spectra of PSII and PSI. Leaves (300 mg fresh weight) from each group were cut into pieces, ground with a mortar and pestle, and mixed with 5 mL 96% ethanol. Thereafter, the mixture was incubated in the dark for 30 min, followed by centrifugation for 10 min at 3000 rpm. The collected supernatant was kept in the dark until sample analysis. Three replicates for each treatment were performed.
Samples were analysed with a UV-Visible two-beam spectrophotometer SHIMADZU UV-2550PC (Shimadzu Corporation, Kioto, Japan). The absorbance of each treatment sample was recorded at 645 and 663 nm wavelengths for chlorophyll a and chlorophyll b, respectively. The measurement error was less than 2%. Chlorophyll content was estimated by Arnon's Equations (1) and (2) [37,38]: where V-volume of extract in mL; W-fresh weight of leaves in mg; A663-solution absorbance at 663 nm; A645-solution absorbance at 645 nm. Steady-state fluorescence measurements of the samples were performed in ethanol solutions, detecting the photoluminescence signal in a 90 • configuration with a spectrofluorometer from Edinburgh Instruments Ltd. (Livingston, UK), model FLSP920 in the range 600-800 nm (excitation wavelength at 440 nm). The spectroscopic measurements were performed at an ambient temperature, and the solutions were placed in a quartz cuvette with a 1 cm path length and four polished windows. Fluorescence spectra were detected at 675 nm for PSII and at 730 nm for PSI.

Effect of Fe 3 O 4 Nanoparticles on Barley Genome DNA Using the Comet Assay
A comet assay was conducted according to Trevigen's CometAssay ® (Trevigen, Gaithersburg, MD, USA) protocol for an alkaline comet assay with slight modifications. Fresh tissues of roots and leaves were minced with scissors into very small pieces and collected in Eppendorf tubes containing ice-cold PBS. The cell suspensions were combined with molten low melting agarose (LMAgarose) according to the manufacturer's protocol. After electrophoresis, slides were immersed twice in dH 2 O for 5 min each, then in 70% ethanol for 5 min, and dried at 37 • C. Nucleoids were stained with SYBR ® Green and analyzed with a fluorescence microscope (Nikon Eclipse 80i, Tokyo, Japan) using filters at 465-495 nm (excitation). The comet "tails" were detected and analysed using the LUCIA Comet Assay TM (LUCIA, Praha, Czech Republic) software, scoring 300 comets for each treatment group under a 40× magnification.

Effect of Fe 3 O 4 Nanoparticles on Barley miRNA Expression Using qRT-PCR
The total RNA was isolated and purified from fresh, treated barley seedling leaves and roots using a Universal RNA/miRNA Purification Kit (EURx, Gdańsk, Poland) according to the manufacturer's protocol. The RNA concentration and quality were determined using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at OD 260/280 and OD 260/230 absorbance ratios. The first-strand cDNA was synthesized from 1 µg of total RNA using a miRCURY LNA RT Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
Barley HvsnoR14 was used as a reference gene for data normalization. The relative expression of miRNAs in different samples compared to that of the controls was calculated using the 2−∆∆Ct method [39], for which the Ct value was the average of three biological replicates with three technical replicates.

Statistical Analyses
The results were expressed as the mean of the measurements and presented as the mean ± standard deviation (SD). A two-way analysis of variance (ANOVA) was conducted to determine the significance of barley genotype, doses of Fe 3 O 4 nanoparticles, and their interaction on barley seedling's reaction in regard to morphological parameters, chlorophyll content, and genotoxicity based on a comet assay.
The hypothesis presumes no impact of genotype, dose of Fe 3 O 4 nanoparticles, or interactions on the estimated parameters. The significant differences were assessed at a p-value of 0.05 and 0.01. When ANOVA gave a significant result, Tukey's HSD test was performed at the 0.05 level to compare the mean values in cases where the hypothesis was rejected [40]. The obtained results were subject to statistical analysis using the Statistica program, version 13.3.

Fe 3 O 4 Nanoparticle Translocation in Barley Seedlings
Engineered nanoparticles can penetrate plant root cells by different mechanisms, such as through aquaporins, membrane transport proteins, endocytosis, or creating new pores [41,42]. Our experiment with purchased fluorescent Fe 3 O 4 NPs that were 25 nm in diameter (this was the smallest size available for purchase due to the pandemic) showed penetration into barley roots and translocation to leaves ( Figure 2). Moreover, there was a difference in fluorescence intensity between leaves exposed to NPs for 1 ( Figure 2C,D) and 6 days ( Figure 2E-H); longer treatment with NPs showed greater fluorescence in leaves in comparison to 1-day treatment. Fluorescence in control roots and leaves was not detected.  The cell wall and plasma membrane are selective barriers that control the entry of NPs into plant cells. Therefore, the penetration of NPs into plant cells depends on the composition and size of the NP, plant species, diameter of cell wall pores, and endocytic vesicles [42][43][44][45]. Moreover, there is a possibility that different plant species have different cell pore sizes [43,45]. For example, in cotton seedlings, NPs up to 20 nm may enter leaf cells; however, for maize seedlings, NPs entry is limited to 11 nm [45]. Ma et al. (2010) [46] suggested that 20 nm AgNPs from intercellular spaces can be transported into plant cells through plasmodesmata. Tombuloglu et al. (2019) [47] confirmed the uptake and translocation of 13 nm Fe 3 O 4 NPs in barley seedlings. Palocci et al. (2017) [42] identified NP size limits to enter the cell wall and plasma membrane in grapevine cell cultures. Researchers have shown that poly(lactic-co-glycolic) acid NPs with diameters up to 50 and 540 nm are able to cross the cell wall and plasma membrane, respectively. Further analysis confirmed that these NPs are taken up by endocytic vesicles [42]. Investigations performed by Bandmann and Homann (2012) [48] revealed that the diameter of tobacco protoplast vesicles was between 80 and 220 nm. It seems that different species have different sizes of endocytic vesicles [42]. Interestingly, researchers have also presumed that NPs interact with the cell wall and induce the formation of new and large pores that allow penetration into plant cells [46]. Wang et al. (2012) [49] suggested that the pore sizes of plant walls are only 3-8 nm.
Our experiment with purchased fluorescent Fe 3 O 4 NPs that were 25 nm in diameter (this was the smallest size available for purchase due to the COVID-19 pandemic) showed penetration into barley roots and translocation to leaves ( Figure 2). Moreover, there was a difference in fluorescence intensity between leaves exposed to NPs for 1 and 6 days; longer treatment with NPs showed greater fluorescence in leaves in comparison with 1-day treatment. Fluorescence in control roots and leaves was not detected. According to some scientists, it is possible that 25 nm NPs are able to penetrate plant cells [42,48]. Experiments with 52 nm Fe NPs demonstrated that these NPs had not been transported into red pepper leaves by the vascular tissues. In contrast, these NPs penetrated the extracellular space of red pepper plants and reached the endodermis, which indicates these NPs can move through the apoplastic pathway in the roots [50]. Furthermore, agglomerated Fe 2 O 3 and Fe 3 O 4 NPs can migrate to the endodermis and accumulate in the vacuole in corn via the apoplastic pathway [51,52]. In red pepper plants, Fe NPs can be modified into iron ions and transported through the vascular tissues to leaves [50]. In our case, we could not demonstrate the penetration of Fe 3 O 4 NPs (25 nm) into plant cells, but it seems that these NPs were transported to barley leaves through the apoplastic pathway.

Effect of Fe 3 O 4 Nanoparticles on Barley Seedling Growth
To study the effect of  Our previous study on two different barley genotypes showed a significant increase in seedling growth when they were treated with 17 mg/L Fe 3 .O 4 NPs [53]. Konate et al. (2017) [54] observed the slight enhancement of root and shoot length in wheat seedlings exposed to small (7 nm) Fe 3 O 4 NPs compared to the control. Wang et al. (2019) [55] found that initially, 400 mg/L Fe 3 O 4 NPs had an inhibitory effect on muskmelon growth, but after the third week of treatment, growth promotion was observed. According to Rahmatizadeh et [47] revealed that Fe 3 O 4 NPs at concentrations up to 250 mg/L enhanced the growth of barley seedlings after 3 weeks of treatment. Leaf length was increased by 27%, and root length increased 125% compared to control seedlings. Yellow medick seedlings exposed to the same NPs at 4 mg/L for 5 weeks showed significantly increased root length and number of leaves [12]. Even low Fe 3 O 4 NPs concentrations (1, 2, and 4 mg/L) significantly increased shoot and root length in garden rockets exposed to NPs for 5 weeks [58]. Yuan et al. Plant growth reduction could be related to Fe NP aggregation on the surface of roots, which interrupts water uptake [59]. Phytotoxicity can occur if NPs penetrate cell walls and plasma membranes in plant roots and then enter vascular tissues [49]. Fe 3 O 4 is affected by oxidation in the presence of water and oxygen, which results in the accumulation of OH radicals and cell wall loosening followed by cell elongation. There can be limited NP transfer to leaves of seedlings [47]. Our results clearly indicated that Fe 3 O 4 NPs had a significant impact on plant height compared with the control.

Content of Chlorophyll in Barley Seedlings after Treatment with Fe 3 O 4 Nanoparticles
Chlorophyll (Chl) is an important photosynthetic pigment in plants that participates in photosynthesis and thus plant growth [61]. Chl content is a key parameter for many plant biological studies [62]. To identify the effects of the Fe 3 O 4 NPs on the barley seedling Chl level, Chl quality (fluorescence, ×10 6 ) and quantity (content of chlorophyll, mg/g) were determined. All tested concentrations significantly affected Chl fluorescence ( Figure 4A,B), especially in the case of 'Sencis'. Fe 3 O 4 NPs increased the Chl a and b fluorescence levels to more than 0.04 to 0.3 × 10 6 compared to control samples. 'Quench' seedlings had increased levels (0.61 to 0.63 × 10 6 ) depending on the nanoparticle concentration. The 20 mg/L NP dose was optimal for increasing Chl a and b fluorescence to 0.84 and 0.42 × 10 6 , respectively, in treated 'Abava' plants in comparison to the control (0.77 and 0.32 × 10 6 , respectively). The content of Chl a and b in comparison to the control was higher after application of 10 and 20 mg/L Fe 3 O 4 NPs ( Figure 4C,D). Total Chl concentration increased in all treated cultivars ( Figure 4E). The highest increasing of total Chl concentration was observed in 'Quench' seedlings, above 12.22 to 32.1 mg/g in comparison to control. However, the lowest increasing was observed in 'Abava' seedlings, above 2.49 to 9.91 mg/g compared to control samples. Moreover, it was observed that Chl content was inversely related to the Fe 3 O 4 NPs concentrations, there was an observed decrease in total Chl concentration (in three different barley cultivars) with increasing NP concentrations from 1 to 20 mg/L. Previously, significant mean Chl fluorescence increases were detected in yellow medick seedlings treated with Fe 3 O 4 NPs at 1, 2, and 4 mg/L [12]. Chloroplasts are very sensitive to iron oxide NPs [61]. Fe NPs may enhance the photosynthetic efficiency of crop plants [50,63]. Moreover, Fe ions released from iron oxide NPs may be utilised as a nutrition source in plants [61]. According to Yuan et al. (2018) [50], Fe NPs at lower concentrations significantly increased chloroplast number per mesophyll cell. Moreover, experiments showed that chloroplast ultrastructure could be changed by different concentrations of Fe [50]. The same researchers suggested that increased Chl content may promote photosynthesis activities in plant cells, which was also demonstrated in our study; Chl quality increased in barley seedlings after treatment with Fe 3 O 4 NPs. Pariona et al. (2017) [52] indicated that Chl content in oak plants treated with Fe 3 O 4 NPs (up to 160 nm) for 12 weeks had been significantly increased (up to 29.8%). This suggests that Fe 3 O 4 NPs provide iron, which is involved in increasing Chl concentration [52]. Ghafariyan et al. (2013) [60] suggested that the biosynthesis of Chl a and b is influenced differently by iron oxide NPs. The presence of Fe 3 O 4 NPs (80-100 nm) at a very low concentration (10 −3 mg/L) enhanced Chl a and b content, biomass, grain yield, and activity of antioxidant enzymes in barley plants [64]. Li et al. (2021) [65] found that foliar spraying with appropriate concentrations of  [66], where Chl content was lower in sunflower seedlings grown hydroponically and exposed to Fe 2 O 3 NPs for 5 days compared to the control. However, stress was not detected in treated sunflower plants. The authors explained this by the possible reduction of root hydraulic conductivity and thereby nutrient uptake [66]. Our results of total Chl content can also be explained with the above-mentioned reason. Differences in application position, growth environment, and plant species may explain these differences in the impact of Fe 3 O 4 NPs on Chl content [65].
The assessment of Chl content and fluorescence parameters are needed to estimate the photosynthetic rates of CO 2 assimilation under stress and determine plants' response or tolerance to environmental stress [61,65,67,68]. Moreover, Yang et al. (2017) [69] and Li et al. (2021) [65] demonstrated significant linear correlations between Chl fluorescence/content and photosynthesis, indicating that increased Chl in plants subsequently increased plant photosynthetic efficiency [13,65]. Our results suggest that Fe 3 O 4 NPs at low concentrations could be successfully used as nanonutrition for increasing barley photosynthetic efficiency and possibly enhancing yield.

Effect of Fe 3 O 4 Nanoparticles on Genotoxicity in Barley Seedlings
The comet assay is widely used to study the genotoxicity of various NPs in plants. Comet assay results are most often expressed as % tail DNA [70,71]. To the best of our knowledge, there are a limited number of studies performed on genotoxicity evaluation of Fe 3 O 4 NPs in plants. Compared to the control, Fe 3 O 4 NP treatments (1, 10, and 20 mg/L) significantly increased DNA damage ( Figure 5A,B) in roots and shoots, particularly after treatment with a higher concentration of nanoparticles. In general, higher genotoxicity was observed in roots of both treated and control seedlings. Genotoxicity level increased with increasing of Fe 3 O 4 NP concentrations. 'Quench' seedlings were the least sensitive to changes in DNA compared to the other two cultivars, there was observed about 7.8% higher genotoxicity at 20 mg/L compared with control samples. Genotoxicity level increased above 11.6% and 11.9% in 'Abava' seedlings' shoots and roots, respectively, and above 11% in both 'Sencis' seedlings' shoots and roots.
The same study indicated that Fe 3 O 4 NP concentrations up to 70 mg/L did not show significant genotoxicity in barley seedlings treated with NPs for 2 weeks [53]. A study on garden rocket seedlings grown hydroponically with the addition of 1-4 mg/L of Fe 3 O 4 NPs for 5 weeks presented an insignificant genome template stability decrease [58]. Furthermore, interesting genotoxicity results were obtained from yellow medick seedlings exposed to the same NPs hydroponically. Generally, genomic template stability significantly decreased in seedlings treated with NPs; however, there was an observed decrease in genotoxicity with increasing NP concentrations from 1 to 4 mg/L [12]. Moreover, our previous study on flax tissue cultures showed very low genotoxicity in callus cultures exposed to 0.5-1.5 mg/L Fe 3 O 4 NPs [72]. Saquib et al. (2016) [70] indicated the genotoxicity of Fe 2 O 3 NPs (22 nm) in radish. Dose-dependent DNA damage was observed with NP concentrations from 0.25 to 2 mg/mL. Fe 3 O 4 NPs in culture media take part in various oxidation-reduction reactions, which lead to Fe 3+ and Fe 2+ ion formation with the subsequent formation of different ROS (reactive oxygen species) [73,74]. ROS formation caused by the Fe NP oxidation process can also be destructive to plants [75]. However, oxidative stress from Fe deficiency is much stronger than that from Fe 3 O 4 NPs [61]. During the NP sonication process, Fe ions are also released, however, Trujillo-Reues et al. (2014) [59] found that the concentration of Fe ions released from Fe 3 O 4 NPs (at concentrations up to 20 mg/L) during sonication for 30 min is negligible, suggesting that the sonication process in our study (1-20 mg/L of NPs) did not affect the release of iron ions. It seems that the genotoxicity observed in this study could be related to Fe 3+ and Fe 2+ ion formation in the hydroponic medium before they were absorbed by barley seedlings.

Effect of Fe 3 O 4 Nanoparticles on Barley miRNA Expression
The response of miRNA to metals depends on plant and metal species [76]. All concentrations of Fe 3 O 4 NPs significantly increased miR156a and miR159a expression in the three barley varieties ( Figure 6A,B). With an increase in NP concentration, the miRNA expression level also increased and ranged from 1.038-(Abava, 1 mg/L, miR156a) to 1.46-fold (Abava, 20 mg/L, miR159a).
In a Latvian barley variety treated with Fe 3 O 4 NPs at 35 and 70 mg/L, a significant increase in miR156a expression was detected (1.96-and 3.75-fold, respectively) [53]. Additionally, there was a significant increase in miR159c expression in yellow medick after a 5-week exposure to small Fe 3 O 4 NPs (1, 2, and 4 mg/L) [12]. The obtained results of another experiment with garden rocket also showed a slight increase in miR159c expression level [58]. The expression level of miR156 in pineapple plants exposed to MgO NPs at 1-4 g/mL was increased. However, miR159 expression was downregulated in the same plants [77]. Tabatabaee et al. (2021) [36] reported that Cu NPs at concentrations above 100 mg/L significantly increased the expression of miR159 in pepper seedlings. According to a critical review by Yang and Chen (2013) [76], miR156 and miR159 were differently expressed in various plant responses to different heavy metal stressors. Thus, miR159 is downregulated in response to aluminium (Al), arsenic (As), mercury (Hg), cadmium (Cd), and manganese (Mn) in different plants. In turn, miR156 is upregulated by Al, As, and Mn and downregulated under Cd and Hg stress [76]. The same situation is relevant in plant responses to metal NPs. In switchgrass seedlings treated with 0.1-1% TiO 2 NPs, the expression of miR156 and miR159 increased [78]. Yu et al. (2019) [79] indicated that miR156 and miR159 in wild barley seedlings might be related to Cd tolerance. According to Kantar et al. (2010) [28], hvu-miR156a expression in barley leaves was upregulated during dehydration stress. In Arabidopsis, miR156 targets are squamosa promoter binding protein-like (SPL) genes, which control plant development and physiology [80]. Moreover, Cui et al. (2014) [81] demonstrated that this miRNA in Arabidopsis takes part in mechanisms related to abiotic stress tolerance. Thus, miR156 overexpression increased drought stress tolerance. In turn, Križnik et al. (2020) [26] suggested that miR156 in Nicotianas can participate in plant resistance to viral infection. The targets of miR159 are MYB genes [82], which are transcription factor genes that control different processes, including responses to biotic and abiotic stress, as well as plant tolerance to metal stress [83,84]. Yao et al. (2021) [27] found that target genes of hvu-miR156a and hvu-miR159b in Tibetan hulless barley (H. vulgare variety nudum Hook. F.) are NAD(P)H-ubiquinone oxidoreductase B (NDB) and phosphatidylserine decarboxylase (PSD). miR156 and miR159 are fungi responsive miRNAs. Both miRNAs respond to wheat infection with powdery mildew [85]. Researchers found that infection with barley leaf stripe fungi affected the expression of miR156 and miR159 families. This means that these miRNAs can be used to improve resistance to barley leaf stripe [27]. Zhao et al. (2012) [82] reported that target genes of miR156 encoded plant disease resistance proteins, while targets of miR159 encoded peroxidase and cytokinin oxidase proteins. Several studies on miR156 and miR159 in plants showed that target genes of these miRNAs are involved in responses to various environmental stressors, such as fungal infection, cold, dehydration, drought, UV light, and mechanical stress [27,28,80,82,85,86]. miR156 and miR159 may also contribute to the interaction between barley and Fe 3 O 4 NPs. Significant changes were observed between the three different barley genotypes. Therefore, a future study is necessary to explore the impact of Fe 3 O 4 NPs on barley seedlings infected with Blumeria graminis to compare results obtained in this study with the same results from infected seedlings and to determine miR156, miR159, mlo, and mla gene expression in different genotypes of infected barley seedlings under Fe 3 O 4 NPs stress.

Conclusions
Based on the data obtained in this study, 25 nm Fe 3 O 4 NPs entered barley (H. vulgare L.) tissues. Fe 3 O 4 NPs at concentrations of 1, 10, and 20 mg/L directly increased seedlings' root length in three cultivars and reduced shoot lengths of 'Quench'. The number of roots also was reduced after treatments. Total chlorophyll concentration was increased in all cultivars mostly in an inverse manner. Furthermore, the expression level of miRNA156a and miRNA159a in all three H. vulgare cultivars was increased. Moreover, these NPs increased the level of genotoxicity in the tested seedlings. It is important to note that each genotype could have an opposite reaction on the Fe 3 O 4 NPs. These results are important to better understand the potential impact of Fe 3 O 4 NPs at low concentrations in agricultural crops and the potential of these NPs as nanonutrition for enhancing barley growth and yield. The obtained results will be used in the future study of the effect of NPs on barley resistance-related and chlorophyll synthesis-related gene expression.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.