Effect of Fe3O4 and CuO Nanoparticles on Morphology, Genotoxicity, and miRNA Expression on Different Barley (Hordeum vulgare L.) Genotypes

Metal nanoparticles (NPs) have an influence on plant growth and development. They can alter plant shoot and root length, fresh biomass production, and even influence the genome. Nanoparticles are also able to affect expression levels of plant microRNAs. MicroRNAs are able to protect plants from biotic stress, including pathogens which cause powdery mildew. In this study, Hordeum vulgare L. varieties “Marthe” and “KWS Olof” were grown in hydroponics with magnetic iron oxide (Fe3O4) and copper oxide (CuO) NPs added at 17, 35, and 70 mg/L. Plant morphology, genotoxicity, and expression of miR156a were investigated. The Fe3O4 and CuO NPs demonstrated different effects on the barley varieties, namely, Fe3O4 nanoparticles increased plant shoot and root lengths and fresh biomass, while CuO nanoparticles decreased them. CuO NPs presence caused larger changes on barley genome compared to Fe3O4 NPs. Thus, Fe3O4 NPs reduced genome stability to 72% in the “Marthe” variety and to 76.34% in the “KWS Olof” variety, while CuO NPs reduced genome stability to 53.33% in “Marthe” variety and in the “KWS Olof” variety to 68.81%. The miR156a expression levels after Fe3O4 NPs treatment did not change in the “Marthe” variety, but increased in the “KWS Olof” variety, while CuO NPs treatment increased miRNA expression levels in the “Marthe” variety but decrease them in the “KWS Olof” variety. As NPs are able to influence miRNA expression and miRNAs can affect the plant resistance, obtained results suggest that tested NPs may alter plant resistance response to pathogens.


Introduction
Nanotechnology and its diverse products are an integral part of the modern lifestyle. Nanoparticles (NPs) are extensively used in agriculture in many cases including microfertilizers, pathogen detection, and pest control. Nanoparticles are also used in cosmetic products such as deodorants, as well as in household care products. Nanoparticles are also widely used in biomedicine as drug carriers [1,2]. e use of NPs offers higher efficiency compared to larger particles, but NPs have unique functional capabilities, electrical and optical properties, high stability, and high adsorption capacity [3]. Nanoparticles have a major influence on plant morphology and genome. A small amount of NPs can increase crop yields, but too much NP exposure can cause physiological disturbances in plants as well as oxidative stress. In addition, NPs are able to reduce the activity of antioxidative enzymes leading to cytotoxicity and genotoxicity [4,5].
Iron is one of the most important plant nutrients for plant development, but copper is a micronutrient which assists in plant metabolism. Fe 3 O 4 and CuO NPs are used in small doses as fertilizers for soil to enrich the essential metal content, thus augmenting crop growth. Iron oxide and copper oxide NPs are used in the form of fungicides in high doses to protect plants against pathogens [6][7][8]. Plants that had CuO NPs applied at 150-340 μg/mL had superior fungal treatment results compared to those treated with Cu 2 O and Cu/Cu 2 O NPs [9]. Application of 50 mg/L CuO NP suspension to rose leaves reduces the growth of Podosphaera pannosa fungi [10]. Fe 3 O 4 NPs can increase Nicotiana benthamiana plant resistance response against the tobacco mosaic virus, developing plant morphological parameters such as plant dry and fresh weights [11]. Metal-based NPs have a major impact on plant morphology. Fe 3 O 4 NPs are able to increase the length of tomato, wheat, and lettuce roots. CuO NPs' different concentrations can reduce shoot and root length in chickpea plants. Seed germination of cucumber, rice, lettuce, and radish was reduced under CuO NPs stress [5,12,13].
Plants have broad-spectrum defense mechanisms against pathogens [14]. Vertical (specific) and horizontal (nonspecific) resistances to plant diseases play a general role in plant protection across infections. Nonspecific resistance protects plant varieties against multiple pathogens, which is encoded by the recessive mutation allele (mlo) of the MLO gene. e MLO gene is a negative regulator of cell death and a regulator of powdery mildew [15,16]. In barley, powdery mildew fungi are able to penetrate the host cell wall by using MLO proteins, leading to spontaneous stem cell death. However, homozygous mutant recessive (mlo) alleles of the MLO genes confer broad-spectrum disease resistance. Plants often show sensitivity, where the MLO genes generally express MLO's respective proteins, which mediate cell-cell communication in plants. Importantly, there is broadspectrum disease resistance when the MLO gene is not expressed or when it expresses dysfunctional proteins [17][18][19][20][21][22].
MicroRNA (miRNA) is an endogenous small noncoding RNA, which consists of 18-24 nucleotides. miRNA plays a crucial role in the regulatory functions responsible for gene expression in eukaryotic organisms-miRNA complementarily binds to the target messenger RNA (mRNA) and facilitates its degradation, thus suppressing gene expression [23][24][25]. Plant miRNA not only regulates various aspects of plant development but has been implicated in resistance to biotic stresses as well as in regulating immunity to pathogens (viruses, bacteria, and fungi) [26]. e miR156 is miRNA which has a regulatory role in plants and responds to biotic stress, hypoxia, salt stress, and stress induced by NPs. e miR156 family is associated with plant response to the pathogens and is able to control bacterial, fungal, and viral diseases in plants [24,[27][28][29].
Unstable weather conditions, especially the humidity developing in agricultural regions, contribute to the spread of pathogenic diseases in plants, such as powdery mildew, which immensely reduces cereal yields and consequentially affects global markets [30]. To avoid pathogenic diseases, the agricultural industry uses different fungicides or potassium fertilizers to reduce plant exposure to fungal infections. Also used is a mix of cultivated crop varieties, which are partially or completely susceptible to pathogens [31,32].
As an example, barley (Hordeum vulgare L.) is a one-year cereal plant which is widely used not only in agriculture, forage, and malt but also in the food industry worldwide [33][34][35][36]. Barley, like other cereals, is affected by various diseases, most often caused by pathogens. e most persistent control against pathogens is the use of resistant barley varieties (for example, varieties with different MLO genes). Using pathogen-resistant varieties automatically increases yield in their growing regions [30]. e aim of this study was to investigate the effects of different concentrations of Fe 3 O 4 and CuO NPs on seedling morphology, genotoxicity, and mlo-resistance-related miRNA expression on different barley varieties. e "Marthe" variety has a mlo11 gene, and the "KWS Olof" variety has a mlo (unknown) gene. e seeds were rinsed with deionized water and transferred to Petri dishes and kept in plates at 22°C for 1 day. e seeds were transferred to a hydroponic solution with 50% Murashige and Skoog (MS) salt solution and kept in a climate chamber 16 h/8 h day/ night photoperiod at 23°C [37]. One-week-old seedlings were divided into six experimental groups and transferred to an aqueous hydroponic solution with NPs: three experimental groups were plants grown in 3 mL of different concentrations of Fe 3 O 4 NPs (17 mg/L, 35 mg/L, and 70 mg/ L), three experimental groups were plants grown in 3 mL of different concentrations of CuO NPs (17 mg/L, 35 mg/L, and 70 mg/L), and control group plants were grown in water. e plants were watered daily with tap water and fertilized with fresh 50% MS salt solution every day. Two-week-old fresh barley seedlings were taken for plant morphology analysis, genotoxicity detection, and miRNA level determination.

Measurement of Seedling Biomass, Shoot, and Root Length.
e morphological parameters of the control and experimental barley (H. vulgare L.) varieties "Marthe" and "KWS Olof" were measured by length of shoot, length of root, and fresh plant biomass using the method followed by [37].

DNA Extraction and Randomly Amplified Polymorphic
Analysis. e genotoxic effects induced by Fe 3 O 4 and CuO NPs were assessed using the randomly polymorphic DNA (RAPD) technique. Genomic DNA was extracted from 140 samples from each variety of fresh barley shoots (20 in each experimental group). Extraction was carried out via Gene-MATRIX Swab-Extract DNA Purification Kit following the purification of total DNA protocol. e quantity and quality of genomic DNA was assessed using a spectrophotometer (NanoDrop One, ermo Scientific, USA).

Detection of Genotoxicity by Estimation of Genomic Template Stability.
e Genome template stability (GTS) value is calculated for each primer using the formula reported by Rocco et al. [38]: where a is the average number of polymorphic bands detected in each sample, and n is the total number of bands in the control samples. e polymorphism in RAPD profiles involves the disappearance of the normal band and the appearance of a new band relative to control. To compare the sensitivity of each parameter, changes in these values are calculated as percentages [38,39]. Genome template stability values were calculated for each experimental group.

Expression Validation of MicroRNA Using
Real-Time qPCR. Two-step quantitative real-time PCR (qRT-PCR) analysis was performed to assess miRNA expression levels in barley varieties "Marthe" and "KWS Olof" grown with different concentrations of Fe 3 O 4 and CuO NPs compared to control plants. miRNAs were extracted from the shoots using a Universal RNA/miRNA Purification Kit (EURx, Poland). RNA was extracted from 140 samples from "Marthe" and "KWS Olof" variety fresh shoots (20 in each experimental group). RNA was quantified and qualified with a spectrophotometer (NanoDrop One, ermo Scientific, USA). Samples with an A260/280 ratio from 1.7 to 2.1 were used for analysis. miRNA target-specific primer hvu-miR156a with locked nucleic acid was designed. e target miRNA hvu-miR156a sequence was 5′-TGACAGAAGAGAGTGAGCACA-3′. HvsnoR14 was used as a reference gene for the normalization of miRNA expression values. Reverse transcription for miRNAs was performed using the ermal Cycler UNO96 (VWR, United Kingdom) and miRCURY LNA RT Kit (Qiagen, Germany) according to the protocol first-strand cDNA synthesis. For miRNA qRT-PCR analysis, miRCURY SYBR Green PCR kit (Qiagen, Germany) was used according the manufacturer's protocol. e Rotor Gene Q Series Software program was used to analyse the miR156a expression level of barley varieties. e results were analysed using the 2 − ΔΔCT method [40].

Statistical
Analysis. e results were expressed as an average for the measurement and were reported with ±SD.
Student's t-test was used to determine statistical differences and significant means of the experimental data examination. In all statistical analyses, the significant differences were determined at a p value of 0.05 or 0.01. All of the experimental values were compared to their relevant control. All of the experiments were repeated three times. Different Fe 3 O 4 NP concentrations insignificantly affected "Marthe" and "KWS Olof" varieties' root length. All CuO NP concentrations significantly (p < 0.05) decreased "Marthe" root length, and all CuO NP concentrations significantly (p < 0.01) decreased "KWS Olof" root length. e "Marthe" control group root length was 7.58 cm, and the group with Fe 3 O 4 NPs at 17 mg/L root length was 7.17 cm, 35 mg/L was 6.33 cm, and 70 mg/L was 9.86 cm. e "Marthe" group with CuO NPs at 17 mg/L was 3.08 cm, 35 mg/L was 5.31 cm, and 70 mg/L was 5.76 cm (Figure 2). Among the "KWS Olof" variety, the control group root length was 10.01 cm, and the group with It is known that the degree of metal bioaccumulation from CuO NPs may be affected by the NP evaporation rate [7]. In general, NPs based on metals such as CuO can dissolve and release metal ions [41]. In the same way, the CuO NPs accumulation in plants depends on the concentration of NPs-bioaccumulation has been shown to increase with increasing CuO NPs concentration in wheat, mung bean, zucchini, and lettuce [42,43]. As a result, experimental plants with CuO at different NP concentrations do not exhibit an exponential decrease of H. vulgare L. root length. Also, Shawn et al. [44] showed that CuO NPs at different concentrations (0.5 mM, 1.0 mM, and 1.5 mM) regularly decrease H. vulgare L. root length and shoot length with increasing NP concentrations compared to control. Similarly, Zakharova et al. [45] presented results where CuO NPs at 0.01 g/L, 0.1 g/L, and 1 g/L reduced Triticum aestivum L. root length. Also, Wrigth et al. [46] observed the same trend in wheat but with different CuO NP concentrations.

Results and Discussion
e AlQuraidi et al. [47] study showed results where CuO NPs concentrations (200, 400, and 800 mg/L) decreased plant fresh biomass and root length in coriander (Coriandrum sativum). Moreover, Margenot et al. [48] showed that 16 nm CuO NP concentrations affected carrot (Daucus carota subsp. sativus cv. Little Finger) and lettuce (Lactuca sativa, cv. Nevada Summer Crisp) root thickness, reducing it. e Wang et al. [49] study results showed that CuO NPs doses (50 and 500 mg/kg) applied to T. aestivum inhibited plant growth, reducing plant biomass and shoot lengths. A study with peanut (Arachis hypogaea L.) showed that CuO NPs 50 mg kg −1 concentration inhibited plant growth, as well as reduced plant biomass and shoot length [50].
In the present study, it was shown that Fe 3 O 4 NPs at 17 mg/L, 35 mg/L, and 70 mg/L increased the shoot length, root length, and fresh seedling biomass of barley varieties. Tombuloglu et al. [51] presented results showing that increasing Fe 3 O 4 NP concentrations (125 mg/L, 250 mg/L, 500 mg/L, and 1000 mg/L) irregularly increased plant shoot length, root length, and fresh H. vulgare L. plant biomass. Konate et al. [12] showed that Fe 3 O 4 NPs at 2000 mg/L increases Triticum aestivum L. root length compared to control by a factor of 1.1, and that wheat shoot length was doubled compared to control. Moreover, the results showed that small concentrations of Fe 3 O 4 NPs (5 mg/L, 10 mg/L, 15 mg/L, and 20 mg/L) also increased wheat plant root length [52]. Relatively low concentrations of NPs were used in this study with barley varieties compared to other studies, where most often the effect of highly concentrated NPs are studied.

Genotoxicity Analysis by RAPD Assay.
e genotoxicity of Fe 3 O 4 and CuO NPs was investigated by observing the band profile after the RAPD assay on 5 replicates per treatment obtained from different barley variety seedlings. All primers created stable RAPD bands.
e genomic changes were noted as appeared (a) and disappeared (b) bands in treated plant DNA compared to control bands (Table 1). Changes in DNA bands (fragment dropouts or new fragment formations) in samples treated with NPs reflect DNA changes in the genome from single base changes (point mutations) to complex chromosome rearrangements considered to be genotoxic [38,53]. (1 mg/L) to 87.5% (4 mg/L) [37]. e same study with Eruca sativa plants with the same Fe 3 O 4 NPs concentrations caused a GTS decrease as NP concentrations increased from 93.9% (1 mg/L) to 87.9% (4 mg/L) [28]. However, Tombuloglu et al. [51] showed that Fe 3 O 4 NPs at 125 mg/L, 250 mg/L, 500 mg/L, and 1000 mg/L concentrations did not show any toxic effect on the experimental H. vulgare L. plants.
As result, the study with Cu NPs at 200 mg/L, 400 mg/L, and 800 mg/L shows that NP made significant changes in C. sativum plant genome using the RAPD technique. Primers OPA-01, OPA-02, and OPA-06 at all Cu NP concentrations showed the disappearance of one band for the genome, but primer OPA-07 has previously shown the formation of a new band at 400 mg/L and 800 mg/L NP concentrations [47]. e other study, which used Cu NPs at 50 mg/L, 100 mg/L, and 200 mg/L concentrations, showed a change to the C. sativus plant genome, where primers OPA-07 and OPA-08 formed new bands in the presence of NP [54]. In contrast, the different concentrations of nano-TiO 2 and NaCl + nano-TiO 2 applied to maize (Zea mays L.) affected GTS. e GTS values ranged from 28.8% to 87.7% [55]. e present study shows that CuO NPs at 17 mg/L, 35 mg/L, and 70 mg/L significantly and insignificantly decreased genome template stability in all experimental H. vulgare L. plants. e effect of CuO NPs on the plant genome and genotoxicity has not been studied previously.

MicroRNA Analysis.
Referring to Gurjar et al. [56], miRNA expression levels are measured by the logarithmic formula Log 2 (treatment/control). Each sample group with contrast between miRNA expression levels. All CuO NP concentrations have a statistically significant result comparing both varieties in all concentrations. CuO NP increased the "Marthe" variety miRNA expression level, but all copper oxide NP concentrations decreased the "KWS Olof" variety miRNA expression level. Different CuO NP concentrations increased only the "Marthe" variety miR156a expression level, increasing their resistance. e "KWS Olof" variety treated with CuO NPs shows only a negative effect on the miRNA expression level and decreased plant resistance (Figure 7). In crop plants such as wheat, powdery mildew infection decreased miR156 expression levels [57]. Su et al. [21] suggested that nov-mir-10 reduced the expression level in sugarcane which can reduce the inhibition of defense response by the MLO protein and improve plant resistance to smut pathogen. Kokina et al. [37] studied Medicago falcata L. plants and showed that Fe 3 O 4 NPs at 1 mg/L, 2 mg/L, and 4 mg/L increased the miR159c expression level at increased NPs concentrations by 0.31-fold, 0.36-fold, and 0.40-fold, respectively. e same study with Eruca sativa with the same Fe 3 O 4 NP concentrations showed decreased miR159c expression levels with increased NPs concentrations by 1.30fold, 1.19-fold, and 1.04-fold, respectively [28]. A study with TiO 2 NPs showed that miR156 in Nicotiana tabacum is upregulated by 0.1% TiO 2 but inhibited by 0.1% nano-TiO 2 [58]. In contrast, 0.1% aluminium oxide NPs upregulated miR156 expression on tobacco plant with an insignificant fold change, but miR159 with 1% Al expression levels is upregulated with a 5.9-fold change [59]. As an example, TiO 2 NPs concentrations of 0.1%, 0.5%, 1%, and 2.5% irregularly affected miR156a expression in Panicum virgatum L. plants, first increasing and then decreasing [60]. e effect of CuO NPs on the miRNA expression level has not been previously studied. e only difference between the treated and control plants was the presence or absence of Fe 3 O 4 NPs or CuO NPs, which supports the theory that the changes in plants were caused by this effect of the NPs. Furthermore, Zhu et al. [61] confirmed that 20 nm Fe 3 O 4 NPs can penetrate into pumpkin cells, translocate, and accumulate in the plant tissues. Moreover, it has been proven that 25 nm Fe 3 O 4 NPs can penetrate flax callus culture cells [62]. Also, the translocation of 40 nm CuO NPs in the rice roots was observed [63].     As copper and iron are often used in agriculture as a nutrient, the presented results could be used in the future to accept new technology for crop plants to increase resistance against fungal pathogens through an increase in miRNA expression levels. Also, nutrition with iron and copper can increase mlo-based resistance, which will increase the resistance of plants to powdery mildew. It is possible that in our study, NP increased the resistance to pathogens in plants with mlo genes because the increasing miR156a level in barley varieties is established. e role of miR156 in barley has not been fully investigated, but the use of NP is likely to increase the resistance of barley to pathogens. It is necessary to complete further studies with barley varieties with mlo genes and without mlo genes and to examine different miRNA as well as miR156a expression and their role in plant resistance to infections.

Conclusion
e results of this study showed that Fe 3 O 4 and CuO NPs had different effects on plant morphology-Fe 3 O 4 NPs compared to CuO NPs irregularly increased plant shoot and root lengths and fresh plant biomass. CuO NPs reduced barley seedling shoot and root lengths. e Fe 3 O 4 and CuO NPs at 17 mg/L, 35 mg/L, and 70 mg/L concentrations mostly insignificantly affected the genome template stability in different varieties of barley. Comparing the effect of Fe 3 O 4 and CuO NP on barley varieties, CuO NPs increased miRNA levels in plants, which is likely to affect plant resistance to pathogens. To the best of our knowledge, this is the first study aiming to determine the genotoxicity of CuO NPs in plants. Also, no previous studies have been performed to detect changes to the barley genome when treated with Fe 3 O 4 NPs. e miRNA expression level of barley varieties using NPs at different concentrations is genome dependent. Future studies are necessary to analyse the effect of miR156 and other miRNA expressions in barley mlo varieties and non-mlo varieties seedlings under NP stress, as well as to assess the potential of using NPs for increasing plant resistance against pathogens.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest.