# Enhanced Germination and Growth of Alfalfa with Seed Presoaking and Hydroponic Culture in Fe2O3 Magnetic Nanoparticles

Authors: Yunjiao Yang; Runtian Li; Ling Wang; Hongmei Bi; Zhimei Peng; Junru Li; Changhong Guo; Yingdong Bi; Yongcai Lai; Donglin Guo
Journal: Journal of Nanomaterials
 (2023)

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## Abstract

Nanofertilizers can be used as potential contenders to provide macro- and micronutrients to plants. Specifically, iron-oxide-based engineered nanoparticles are promising candidates for next-generation iron-deficiency fertilizers. Aiming to investigate the effect of Fe2O3 magnetic nanoparticles on plant germination and growth, alfalfa seeds were presoaked by a series of concentrations (0, 50, 100, 150, and 200 mg/L) of Fe2O3 NPs in this study. The germination rate, root length, chlorophyll content, and activity of the antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)) were detected. Further, hydroponic alfalfa young seedlings were treated with 50 mg/L Fe2O3 NPs, with 50 mg/L Fe-EDTA as the control. The growth index, chlorophyll content, metal (Fe, Cu, Zn, and Mn) content, and ferric-chelate reductase activity were detected. The results indicated that the presoaking of Fe2O3 NPs promoted alfalfa seed germination in all treatments. Short-time of Fe2O3 NPs presoaking has not affected chlorophyll and iron content while increasing antioxidase (SOD, POD, and CAT) activities at 50 and 200 mg/L concentrations. Fe2O3 NPs of 50 mg/L have the most beneficiary impact on alfalfa seed germination by presoaking. Fe2O3 NPs of 50 mg/L enhanced the seedlings’ growth and increased chlorophyll content, Fe content, and Mn content of hydroponic alfalfa compared with the control. Moreover, ferric chelate reductase activity was significantly lower in alfalfa roots exposure to Fe2O3 NPs than in the control. The results showed that 50 mg/L Fe2O3 NPs could be used as an appropriate and effective treatment concentration for seed pretreatment and fertilizer application. The growth-promoting effect of Fe2O3 NPs on alfalfa is related to its nanostructured properties. More in-depth research is still needed to understand the growth-promoting mechanism of Fe2O3 NPs and the impact of Fe2O3 NPs on the field environment.


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## Body

## 1. Introduction

Nanotechnology has emerged as the most innovative and revolutionary scientific field in decades that deals with extremely small particles in the range of nanoscale (1–100 nm), termed nanoparticles (NPs) [1]. NPs exhibit unique physical, chemical, and biological characteristics that differ from equivalent bulk materials and atomic or molecular structures due to enhanced solubility and a greater proportion of surface atoms relative to the interior of a structure [2, 3]. Metal and metal oxide nanoparticles (metallic NPs) are widely applied in various fields of science and technology. Recently, agriculture has been facing challenges such as stagnation in crop yields, low nutrient use efficiency, multinutrient deficiencies, and water shortage [4]. Owing to their unique properties, NPs are gaining considerable attention in agriculture. Metallic NPs can be used as nanofertilizers, nanopesticides, nanoherbicides, and nanobactericides to improve agrochemical efficiency, enhance plant growth, compensate for plant nutrition deficiencies, and defend against diseases. Extensive research on the metallic NPs’ effect on plants has been carried out in the last few decades. Most metallic NPs have positive and negative effects on crops at physiological, morphological, biochemical, and molecular levels [5]. The positive morphological effects of metallic NPs include enhancement of germination rate, length of root and shoot, vegetative biomass, physiological parameters, and alleviating the damage from abiotic and biotic stress [6, 7]. Therefore, the appropriate application of metallic NPs improves the growth, yield, and quality of important crops and development, and utilizing metallic NPs as fertilizers is an effective method of agriculture [8, 9]. However, this progress has been accompanied by unknown risks of phytotoxicity. The major mechanisms involved in the negative effects of metallic NPs include a multiplicative combination of reduced substrate supply, altered mineral nutrition and photosynthesis, and direct water stress [10]. Meanwhile, NPs generate free radicals that cause oxidative stress and induce genotoxicity in crops. Excessive release of NPs may cause potential harm to ecosystem components [11], as NPs could get transferred to the food chain and pose a threat to human and environmental health, and safety risks associated with introducing pristine metallic NPs into the environment remain to be addressed [1].Iron (Fe) is an essential trace metal element for plants’ growth and development. It plays a vital role in physiological and biochemical reactions, such as cell metabolism, photosynthesis, and respiration [12]. For example, chlorophyll synthesis in chloroplasts requires Fe and certain enzymes need Fe as a cofactor [13, 14]. Although iron is rich in soil, it is difficult to be absorbed by plants due to its insolubility. Plants often suffer from iron deficiency, which causes chlorosis and biomass reduction [15]. Therefore, an appropriate iron supplement is necessary for crop production, especially for crops cultivated in alkaline soils. Inexpensive and sustainable methods are urgently needed to utilize NPs as a fertilizer for solving iron deficiency-induced chlorosis in plants [16–18]. However, in the case of the Fe fertilizers available nowadays, these objectives are far from being reached.Nanoparticles are powerful adsorbents due to their unique structure and electronic properties [19]. Studies have demonstrated that iron and iron oxide NPs (INPs) have high application potential in the field of fertilizers for seed coatings or foliar applications. Ferric oxide nanoparticles (Fe2O3 NPs) are a common type of iron oxide nanoparticles with a strong surface-to-volume ratio, superparamagnetism, and inherent biocompatibility-like characteristics [20, 21]. Previous studies have shown that low concentrations of Fe2O3 NPs could promote the growth and germination of Lactuca sativa [22], soybean [23], and peanut [16], increase SPAD value, soluble sugar, and soluble protein content in legumes [24–29] and watermelon [21]. When in high concentrations, Fe2O3 NPs induced oxidative stress in plants such as watermelon [21], citrus [30], rice [31], peanut [16], and soybean [23]. Trujillo-Reyes et al. [32] showed that iron INPs did not affect the catalase (CAT) and APX activities in lettuce. These results showed that the toxicity of Fe2O3 NPs was closely related to their properties, size, and plant species [33]. The Fe contents in Fe2O3 NPs treatments were higher in citrus plants than in the control group [30]; the foliar application of INPs had a significant effect on leaf Fe content, stem Mg content, plasma membrane stability, and chlorophyll content of Vigna unguiculata [27]. The content of organic matter, proteins, lipids, and energy recorded higher levels in fruits of squash plants sprayed with Fe nano-oxide [34]. Martinez-Fernandez et al. [35] investigated that the concentrations of Ca, K, Mg, and S in the shoot were reduced in Fe2O3 NPs treatment relative to the control plants, which resulted in lower contents of chlorophyll pigments. The treatment with Fe2O3 reduced the Mo and Zn concentrations in tomato shoots [36]. Boutchuen et al. [37] reported a modified seed presoak strategy using a drop of Fe-enriching hematite nanoparticle dispersion to enhance plant growth and production in four legume species. Yuan et al. [38] found that most Fe NPs were aggregated into the cell wall of pepper and transported through root apoptotic pathways, which might prevent iron transfer when in high Fe NPs concentration. It was also revealed that Fe NPs regulated the expression of Fe transporter genes in plants [39]. Fe3O4 NPs and nanoscale zero-valent iron were also observed to have a beneficiary impact on plants and improve germination at lower concentrations [16, 40–42]. Although Fe2O3 NPs have been studied in the above crops, their application scope and mechanism should be further expanded. Despite the benefits of Fe2O3 NPs, the potential risks still need to be fully elucidated [43, 44].Alfalfa (Medicago sativa) is the largest cultivated area of high-quality perennial legume grass and is used for food and medicine [45, 46]. Alfalfa can be widely planted in alkaline soil and increased with appropriate iron supplements, achieving good economic value and ecological benefits. The effects of ZnO NPs, CuO NPs, Au NPs, and K NPs on alfalfa seedlings were investigated in previous studies [47–49]. In Iannone et al.’s [50] work, citric-acid-coated Fe3O4 NPs were readily absorbed and scarcely translocated from the roots to the shoots in alfalfa. The citric-acid-coated Fe3O4 NPs’ effects on alfalfa plant growth, with no symptoms of oxidation but CAT increased. Kim et al. [51] noted that 500 mg/L nanozerovalent iron could increase the root elongation of exposed alfalfa because this nanomaterial promotes cell wall loosening. Whereas there is a lack of research on Fe2O3 NPs in alfalfa. In this study, the effects of Fe2O3 NPs on alfalfa growth are discussed to provide a theoretical basis and reference for the application of Fe2O3 NPs for the fertilization of crops.


## 2. Materials and Methods

### 2.1. Fe2O3 NPs Solution Treatment

Hydrophilic Fe2O3 NPs were purchased from Zhongye New Material Science and Technology Ltd. The diameters of Fe2O3 NPs were 20.2 ± 2.1 nm with the carboxyl (COOH) groups on the surface. The crystal structure shows cubic-phase Fe2O3. The magnetic saturation value was 62.8 emu g−1. Fe2O3 NPs were weighed, and a series of Fe2O3 NPs treatment solution with concentrations of 50, 100, 150, and 200 mg/L was prepared. The treatment solution was dispersed using an ultrasonicator (100 W, 40 kHz) for 30 min before use.


### 2.2. Plant Material and Culture Conditions

Alfalfa seeds were provided by the Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Sciences. The seeds were sterilized with 10% NaClO, washed with distilled water, and then soaked with varying concentrations of Fe2O3 NPs treatment solutions overnight along with the control (distilled water) on a petri dish at room temperature. After being removed from the soaking and cultured in distilled water for 5 days, the germination rate was observed and recorded. Alfalfa seeds were germinated in distilled H2O for 5 days for hydroponic culture, and then the young seedlings were transferred into 1/2 Hoagland solution for 7 days. Then, seedlings were treated with 50 mg/L Fe2O3 NPs in a de-iron Hoagland solution at room temperature for 7 days, with a Hoagland solution containing 50 mg/L Fe-EDTA as the control.


### 2.3. Phenotype and Growth Index Observation

The germination rate and main root length of Fe2O3 NPs presoaked alfalfa seeds were recorded and calculated as follows: germination rate = total germinated seeds/total seeds × 100%. The main root length, lateral root number, and fresh weight of the hydroponic cultured alfalfa seedlings were measured and statistically analyzed.


### 2.4. Chlorophyll Content Measurement

To measure the chlorophyll content, 0.1 mg of alfalfa leaf sample was taken and added into a 15 mL centrifuge tube, and then 10 mL 95% ethanol was added. The test tube was placed in the dark for 14 hr. The absorbance values at 645 and 663 nm were determined by a Metash ultraviolet–visible spectrophotometer model UV-5100 (Metash Instruments Co., Shanghai, China).


### 2.5. Antioxidant Enzyme Detection

The antioxidant enzyme activity was determined by spectrophotometry of a Metash ultraviolet–visible spectrophotometer model UV-5100 (Metash Instruments Co.). According to Li et al. [52], the superoxide dismutase (SOD) activity was determined at 560 nm. The peroxidase (POD) activity was determined by the absorbance at 470 nm due to guaiacol oxidation, according to Shi et al. [53]. The CAT activity was determined by monitoring the degradation of H2O2 at an absorbance of 240 nm, according to Aebi et al. [54].


### 2.6. NBT, DAB, and Evans Blue Staining

The alfalfa leaves were immersed in NBT dye solution for 2–3 hr, in Evans blue dye solution for 8 hr, and in DAB dye solution for 8 hr in the dark, respectively, and then transferred to the eluent and boiled to decolorize. The staining leaves were observed and photographed.


### 2.7. Metal-Content Measurement

The alfalfa leaf samples were dried and ground into powder, and 0.05 g sample was put into the digestion tube, 4 mL 65% nitric acid was added, and was digested at 120°C for 90 min. When the sample temperature dropped to room temperature, 1 mL of 30% hydrogen peroxide was added into the digestive tube and heated at 120°C for 90 min. After dropping to room temperature, 750μL 30% hydrogen peroxide was added and kept until transparent. Finally, ddH2O was used to constant volume to 50 mL. The metal content was determined by an inductively coupled plasma emission spectrometer (PE ICP-OES8000).


### 2.8. Ferric-Chelate Reductase Activity Detection

The FCR activity was determined by the kit method. The 0.1 g material was added with 1 mL distilled water, homogenized in an ice bath, centrifuged at 10,000 rpm 4°C for 10 min, and the supernatant was taken. Absorbance was determined at 562 nm by a Metash ultraviolet–visible spectrophotometer model UV-5100 (Metash Instruments Co.). The FCR activity was expressed as nmol/min/g.


### 2.9. Statistical Analysis of Data

All results were conducted with three replicates. The experimental data were statistically evaluated by one-way analysis of variance (ANOVA), and the mean values for each treatment were compared using Duncan’s test atP<0.05 level.


## 2.1. Fe2O3 NPs Solution Treatment

Hydrophilic Fe2O3 NPs were purchased from Zhongye New Material Science and Technology Ltd. The diameters of Fe2O3 NPs were 20.2 ± 2.1 nm with the carboxyl (COOH) groups on the surface. The crystal structure shows cubic-phase Fe2O3. The magnetic saturation value was 62.8 emu g−1. Fe2O3 NPs were weighed, and a series of Fe2O3 NPs treatment solution with concentrations of 50, 100, 150, and 200 mg/L was prepared. The treatment solution was dispersed using an ultrasonicator (100 W, 40 kHz) for 30 min before use.


## 2.2. Plant Material and Culture Conditions

Alfalfa seeds were provided by the Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Sciences. The seeds were sterilized with 10% NaClO, washed with distilled water, and then soaked with varying concentrations of Fe2O3 NPs treatment solutions overnight along with the control (distilled water) on a petri dish at room temperature. After being removed from the soaking and cultured in distilled water for 5 days, the germination rate was observed and recorded. Alfalfa seeds were germinated in distilled H2O for 5 days for hydroponic culture, and then the young seedlings were transferred into 1/2 Hoagland solution for 7 days. Then, seedlings were treated with 50 mg/L Fe2O3 NPs in a de-iron Hoagland solution at room temperature for 7 days, with a Hoagland solution containing 50 mg/L Fe-EDTA as the control.


## 2.3. Phenotype and Growth Index Observation

The germination rate and main root length of Fe2O3 NPs presoaked alfalfa seeds were recorded and calculated as follows: germination rate = total germinated seeds/total seeds × 100%. The main root length, lateral root number, and fresh weight of the hydroponic cultured alfalfa seedlings were measured and statistically analyzed.


## 2.4. Chlorophyll Content Measurement

To measure the chlorophyll content, 0.1 mg of alfalfa leaf sample was taken and added into a 15 mL centrifuge tube, and then 10 mL 95% ethanol was added. The test tube was placed in the dark for 14 hr. The absorbance values at 645 and 663 nm were determined by a Metash ultraviolet–visible spectrophotometer model UV-5100 (Metash Instruments Co., Shanghai, China).


## 2.5. Antioxidant Enzyme Detection

The antioxidant enzyme activity was determined by spectrophotometry of a Metash ultraviolet–visible spectrophotometer model UV-5100 (Metash Instruments Co.). According to Li et al. [52], the superoxide dismutase (SOD) activity was determined at 560 nm. The peroxidase (POD) activity was determined by the absorbance at 470 nm due to guaiacol oxidation, according to Shi et al. [53]. The CAT activity was determined by monitoring the degradation of H2O2 at an absorbance of 240 nm, according to Aebi et al. [54].


## 2.6. NBT, DAB, and Evans Blue Staining

The alfalfa leaves were immersed in NBT dye solution for 2–3 hr, in Evans blue dye solution for 8 hr, and in DAB dye solution for 8 hr in the dark, respectively, and then transferred to the eluent and boiled to decolorize. The staining leaves were observed and photographed.


## 2.7. Metal-Content Measurement

The alfalfa leaf samples were dried and ground into powder, and 0.05 g sample was put into the digestion tube, 4 mL 65% nitric acid was added, and was digested at 120°C for 90 min. When the sample temperature dropped to room temperature, 1 mL of 30% hydrogen peroxide was added into the digestive tube and heated at 120°C for 90 min. After dropping to room temperature, 750μL 30% hydrogen peroxide was added and kept until transparent. Finally, ddH2O was used to constant volume to 50 mL. The metal content was determined by an inductively coupled plasma emission spectrometer (PE ICP-OES8000).


## 2.8. Ferric-Chelate Reductase Activity Detection

The FCR activity was determined by the kit method. The 0.1 g material was added with 1 mL distilled water, homogenized in an ice bath, centrifuged at 10,000 rpm 4°C for 10 min, and the supernatant was taken. Absorbance was determined at 562 nm by a Metash ultraviolet–visible spectrophotometer model UV-5100 (Metash Instruments Co.). The FCR activity was expressed as nmol/min/g.


## 2.9. Statistical Analysis of Data

All results were conducted with three replicates. The experimental data were statistically evaluated by one-way analysis of variance (ANOVA), and the mean values for each treatment were compared using Duncan’s test atP<0.05 level.


## 3. Results

### 3.1. Germination and Root Length of Alfalfa Seeds Presoaked by Fe2O3 NPs

As shown in Table1, the alfalfa seed germination rates were higher in all Fe2O3 NPs treatments than that in the control.Table 1
The germination rate and root length of Fe2O3NPs presoaked alfalfa seeds.
Treatment concentrationGermination rate (%)Root length (cm)0 (CK)73.445.43 ± 0.34c50 mg/L82.226.50 ± 0.37ab100 mg/L80.006.87 ± 0.32a150 mg/L75.556.01 ± 0.42bc200 mg/L77.785.62 ± 0.08cDifferent letters indicate significance of the treatments atp<0.05.The alfalfa seed germination rate was the highest in 50 mg/L Fe2O3 NPs treatment, indicating that 50 mg/L Fe2O3 NPs had the most remarkable germinate promotion effect. The main root length of alfalfa in all Fe2O3 NPs treatments was higher than that of the control. The root length of alfalfa was significantly higher than that of the control at 50 and 100 mg/L and reached the maximum at 100 mg/L. The results indicated that 50 and 100 mg/L Fe2O3 NPs had a strong promoting effect on root elongation.


### 3.2. Chlorophyll Content of Fe2O3 NPs Presoaked Alfalfa

As shown in Figure1, the total chlorophyll content, chlorophyll a, and chlorophyll b of alfalfa treated with 150 mg/L Fe2O3 NPs were significantly higher than in other treatments. The results showed that 150 mg/L Fe2O3 NPs had a strong promoting effect on chlorophyll synthesis. The chlorophyll a/b values of Fe2O3 NPs from 0 to 200 mg/L were 2.25, 2.02, 2.52, 2.33, and 2.51, respectively. The chlorophyll a/b value of 50 mg/L Fe2O3 NPs was the lowest, which was conducive to the absorption of light energy under weak light.Figure 1
The chlorophyll content of Fe2O3 NPs presoaked alfalfa. Error bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.


### 3.3. Iron Content of Fe2O3 NPs Presoaked Alfalfa

As shown in Figure2, the iron content was higher in Fe2O3 NPs treatments than that in the control with no significant difference. The iron content reached the highest value in 100 mg/L Fe2O3 NPs treatment.Figure 2
Fe content of Fe2O3 NPs presoaked alfalfa. All dates show the means ± SE of three individual plants. No significant difference was observeed in treatments.


### 3.4. Antioxidant Enzyme Activity of Fe2O3 NPs Presoaked Alfalfa

The activity of antioxidant enzymes directly reflects the degree of stress on plant tissues or cells, as shown in Figure3.Figure 3
Antioxidant enzyme activites (SOD, POD, and CAT) of Fe2O3 NPs presoaked alfalfa. Error bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.The SOD activity of alfalfa seedlings treated with 50 mg/L Fe2O3 NPs was significantly higher than that of the control. Then the SOD activity decreased with the increase of Fe2O3 NPs concentration, reaching a minimum value of 150 mg/L. The POD activity of alfalfa leaves was significantly lower at 100 mg/L Fe2O3 NPs treatment than that of the control, while significantly higher at 200 mg/L Fe2O3 NPs treatment than that of the control. The POD activity at 50 and 150 mg/L Fe2O3 NPs treatment showed no significant difference from the control. The CAT activity of 50–200 mg/L Fe2O3 NPs was significantly higher than the control, with the highest value at 200 mg/L and relatively high at 50 mg/L. These antioxidant enzymes’ activity showed an “upward–downward–upward” trend, which indicated that the antioxidant system of alfalfa was disturbed by different concentrations of Fe2O3 NPs treatments.


### 3.5. NBT, DAB, Evans Blue Staining of Fe2O3 NPs Presoaked Alfalfa

In Figure4, alfalfa leaves stained by NBT, the blue color was lighter in 50 and 100 mg/L Fe2O3 NPs treatments while deeper in 150–200 mg/L Fe2O3 NPs treatments, compared with the control. The results indicated that the low concentration of Fe2O3 NPs-treated alfalfa contained less O2− and the high-concentration-treated alfalfa contained more O2−.Figure 4
Histochemical staining (NBT, DAB, and Evans blue) of Fe2O3 NPs presoaked alfalfa.In alfalfa leaves stained by DAB, the yellowish-brown was deeper in 50–150 mg/L Fe2O3 NPs treatment than that of the control, indicating that H2O2 in leaves was less than the control, indicating more H2O2 and severer oxidative damage. There was no appreciable yellow color in Evans blue-stained alfalfa leaves, which means that the leaf cells remained active in all treatment groups.


### 3.6. Growth Index of Fe2O3 NPs Hydroponic Cultured Alfalfa

The alfalfa young seedlings’ growth status under treatment of 50 mg/L Fe2O3 NPs and the control (50 mg/L Fe-EDTA) is shown in Table 2 and Figure 5.Figure 5
Fe2O3 NPs hydroponic cultured alfalfa seedlings.Table 2
Root length and fresh weight of Fe2O3 NPs hydroponic cultured alfalfa seedlings.
TreatmentRoot length (cm)Lateral root number (cm)Fresh weight (mg)CK11.09 ± 1.45b6.22 ± 1.30b49 ± 0.7b50 mg/L Fe2O3 NPs15.37 ± 3.49a8.89 ± 3.21a57 ± 1.5aError bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.The root length, lateral root number, and fresh weight of alfalfa treated with Fe2O3 NPs were higher than that of the control, with a significant difference (p<0.05). The results showed that 50 mg/L Fe2O3 NPs could promote the growth of alfalfa.


### 3.7. Chlorophyll Content of Fe2O3 NPs Hydroponic Cultured Alfalfa

As shown in Figure6, the total chlorophyll contents of alfalfa treated with 50 mg/L Fe2O3 NPs were significantly higher than that of the control. The chlorophyll a/b values of the control and Fe2O3 NPs were 2.14 and 1.63, respectively. The results showed that 50 mg/L Fe2O3 NPs could promote the synthesis of alfalfa chlorophyll and might improve the photosynthetic capacity of plants.Figure 6
Chlorophyll a, chlorophyll b, and total chlorophyll content of Fe2O3 NPs hydroponic cultured alfalfa seedlings. Error bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.


### 3.8. Metal Content of Fe2O3 NPs Hydroponic Cultured Alfalfa

As shown in Figure7, the Fe and Mn contents of alfalfa in Fe2O3 NPs treatments were significantly higher than that of the control (p<0.05). Cu and Zn contents of alfalfa in Fe2O3 NPs treatments showed no significant difference from the control. The results showed that alfalfa under Fe2O3 NPs treatment absorbed and accumulated more Fe and Mn, whereas it did not absorb and accumulate more Zn and Cu.Figure 7
Metal content, Fe, Cu, Mn, and Zn content, of Fe2O3 NPs hydroponic cultured alfalfa seedlings. Error bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.


### 3.9. Ferric-Chelate Reductase Activity of Fe2O3 NPs Hydroponic Cultured Alfalfa

As shown in Figure8, the FCR activity of alfalfa treated with 50 mg/L Fe2O3 NPs was significantly lower than that of the control (p<0.05). The results showed that Fe2O3 NPs treatment reduced the FCR activity in alfalfa, indicating that absorbtion and accumulation of Fe in alfalfa under Fe2O3 NPs treatment may not rely on the reduction of FCR in plant roots.Figure 8
Ferric-chelate reductase activity of Fe2O3 NPs hydroponic cultured alfalfa roots. Error bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.


## 3.1. Germination and Root Length of Alfalfa Seeds Presoaked by Fe2O3 NPs

As shown in Table1, the alfalfa seed germination rates were higher in all Fe2O3 NPs treatments than that in the control.Table 1
The germination rate and root length of Fe2O3NPs presoaked alfalfa seeds.
Treatment concentrationGermination rate (%)Root length (cm)0 (CK)73.445.43 ± 0.34c50 mg/L82.226.50 ± 0.37ab100 mg/L80.006.87 ± 0.32a150 mg/L75.556.01 ± 0.42bc200 mg/L77.785.62 ± 0.08cDifferent letters indicate significance of the treatments atp<0.05.The alfalfa seed germination rate was the highest in 50 mg/L Fe2O3 NPs treatment, indicating that 50 mg/L Fe2O3 NPs had the most remarkable germinate promotion effect. The main root length of alfalfa in all Fe2O3 NPs treatments was higher than that of the control. The root length of alfalfa was significantly higher than that of the control at 50 and 100 mg/L and reached the maximum at 100 mg/L. The results indicated that 50 and 100 mg/L Fe2O3 NPs had a strong promoting effect on root elongation.


## 3.2. Chlorophyll Content of Fe2O3 NPs Presoaked Alfalfa

As shown in Figure1, the total chlorophyll content, chlorophyll a, and chlorophyll b of alfalfa treated with 150 mg/L Fe2O3 NPs were significantly higher than in other treatments. The results showed that 150 mg/L Fe2O3 NPs had a strong promoting effect on chlorophyll synthesis. The chlorophyll a/b values of Fe2O3 NPs from 0 to 200 mg/L were 2.25, 2.02, 2.52, 2.33, and 2.51, respectively. The chlorophyll a/b value of 50 mg/L Fe2O3 NPs was the lowest, which was conducive to the absorption of light energy under weak light.Figure 1
The chlorophyll content of Fe2O3 NPs presoaked alfalfa. Error bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.


## 3.3. Iron Content of Fe2O3 NPs Presoaked Alfalfa

As shown in Figure2, the iron content was higher in Fe2O3 NPs treatments than that in the control with no significant difference. The iron content reached the highest value in 100 mg/L Fe2O3 NPs treatment.Figure 2
Fe content of Fe2O3 NPs presoaked alfalfa. All dates show the means ± SE of three individual plants. No significant difference was observeed in treatments.


## 3.4. Antioxidant Enzyme Activity of Fe2O3 NPs Presoaked Alfalfa

The activity of antioxidant enzymes directly reflects the degree of stress on plant tissues or cells, as shown in Figure3.Figure 3
Antioxidant enzyme activites (SOD, POD, and CAT) of Fe2O3 NPs presoaked alfalfa. Error bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.The SOD activity of alfalfa seedlings treated with 50 mg/L Fe2O3 NPs was significantly higher than that of the control. Then the SOD activity decreased with the increase of Fe2O3 NPs concentration, reaching a minimum value of 150 mg/L. The POD activity of alfalfa leaves was significantly lower at 100 mg/L Fe2O3 NPs treatment than that of the control, while significantly higher at 200 mg/L Fe2O3 NPs treatment than that of the control. The POD activity at 50 and 150 mg/L Fe2O3 NPs treatment showed no significant difference from the control. The CAT activity of 50–200 mg/L Fe2O3 NPs was significantly higher than the control, with the highest value at 200 mg/L and relatively high at 50 mg/L. These antioxidant enzymes’ activity showed an “upward–downward–upward” trend, which indicated that the antioxidant system of alfalfa was disturbed by different concentrations of Fe2O3 NPs treatments.


## 3.5. NBT, DAB, Evans Blue Staining of Fe2O3 NPs Presoaked Alfalfa

In Figure4, alfalfa leaves stained by NBT, the blue color was lighter in 50 and 100 mg/L Fe2O3 NPs treatments while deeper in 150–200 mg/L Fe2O3 NPs treatments, compared with the control. The results indicated that the low concentration of Fe2O3 NPs-treated alfalfa contained less O2− and the high-concentration-treated alfalfa contained more O2−.Figure 4
Histochemical staining (NBT, DAB, and Evans blue) of Fe2O3 NPs presoaked alfalfa.In alfalfa leaves stained by DAB, the yellowish-brown was deeper in 50–150 mg/L Fe2O3 NPs treatment than that of the control, indicating that H2O2 in leaves was less than the control, indicating more H2O2 and severer oxidative damage. There was no appreciable yellow color in Evans blue-stained alfalfa leaves, which means that the leaf cells remained active in all treatment groups.


## 3.6. Growth Index of Fe2O3 NPs Hydroponic Cultured Alfalfa

The alfalfa young seedlings’ growth status under treatment of 50 mg/L Fe2O3 NPs and the control (50 mg/L Fe-EDTA) is shown in Table 2 and Figure 5.Figure 5
Fe2O3 NPs hydroponic cultured alfalfa seedlings.Table 2
Root length and fresh weight of Fe2O3 NPs hydroponic cultured alfalfa seedlings.
TreatmentRoot length (cm)Lateral root number (cm)Fresh weight (mg)CK11.09 ± 1.45b6.22 ± 1.30b49 ± 0.7b50 mg/L Fe2O3 NPs15.37 ± 3.49a8.89 ± 3.21a57 ± 1.5aError bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.The root length, lateral root number, and fresh weight of alfalfa treated with Fe2O3 NPs were higher than that of the control, with a significant difference (p<0.05). The results showed that 50 mg/L Fe2O3 NPs could promote the growth of alfalfa.


## 3.7. Chlorophyll Content of Fe2O3 NPs Hydroponic Cultured Alfalfa

As shown in Figure6, the total chlorophyll contents of alfalfa treated with 50 mg/L Fe2O3 NPs were significantly higher than that of the control. The chlorophyll a/b values of the control and Fe2O3 NPs were 2.14 and 1.63, respectively. The results showed that 50 mg/L Fe2O3 NPs could promote the synthesis of alfalfa chlorophyll and might improve the photosynthetic capacity of plants.Figure 6
Chlorophyll a, chlorophyll b, and total chlorophyll content of Fe2O3 NPs hydroponic cultured alfalfa seedlings. Error bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.


## 3.8. Metal Content of Fe2O3 NPs Hydroponic Cultured Alfalfa

As shown in Figure7, the Fe and Mn contents of alfalfa in Fe2O3 NPs treatments were significantly higher than that of the control (p<0.05). Cu and Zn contents of alfalfa in Fe2O3 NPs treatments showed no significant difference from the control. The results showed that alfalfa under Fe2O3 NPs treatment absorbed and accumulated more Fe and Mn, whereas it did not absorb and accumulate more Zn and Cu.Figure 7
Metal content, Fe, Cu, Mn, and Zn content, of Fe2O3 NPs hydroponic cultured alfalfa seedlings. Error bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.


## 3.9. Ferric-Chelate Reductase Activity of Fe2O3 NPs Hydroponic Cultured Alfalfa

As shown in Figure8, the FCR activity of alfalfa treated with 50 mg/L Fe2O3 NPs was significantly lower than that of the control (p<0.05). The results showed that Fe2O3 NPs treatment reduced the FCR activity in alfalfa, indicating that absorbtion and accumulation of Fe in alfalfa under Fe2O3 NPs treatment may not rely on the reduction of FCR in plant roots.Figure 8
Ferric-chelate reductase activity of Fe2O3 NPs hydroponic cultured alfalfa roots. Error bars show means of standard error (±SE) of three replicates (n = 3) followed by different alphabetic letters showing statistical significance at p<0.05.


## 4. Discussion

Nanoparticulate forms of plant nutrients show the advantages of being easily absorbed by plants and slowly released into the environment compared with traditional fertilizers. However, the potential ecological risks still need further research [16, 55–58]. Most studies show that nanoparticles are toxic to plants at higher concentrations. Phytotoxicity is a significant consideration in understanding the potential environmental impact of nanoparticles [59–61]. Germination rate and root length are important indications of the growth promotion of NPs and are suggested to utilize to indict the phytotoxicity by the Environmental Protection Agency. In this study, to evaluate the safety and applicable concentration of Fe2O3 NPs on alfalfa, alfalfa seeds were presoaked by gradient concentrations of Fe2O3 NPs.Seed germination is the first critical step in plant growth and development. It is probable that nanoparticles penetrate the seed coat and exert a beneficial effect on the seed germination process. Previous studies concluded that presoaking Fe2O3 NPs significantly promoted the germination and root elongation of legumes. Abusalem et al. [62] found that 40 nm green synthesized Fe2O3 NPs significantly improved the vigor index of tomatoes by wetted seeds. Karunakaran et al. [63] showed that the root length enhanced on treatment with 56 nm Fe2O3 nanoparticles. The 20 mg/L Fe2O3 NPs promoted the germination of watermelon and maize seeds, and 20 and 50 mg/L Fe2O3 NPs could accelerate root elongation [64]. Although nanoparticles have several advantages in germinate promotion, negative effects have been reported. A higher concentration of iron oxide nanoparticles showed root growth reduction. Ghafariyan et al. [26] found that various concentrations of superparamagnetic iron oxide NPs do not significantly affect the germination index. In the present investigation, the germination rate reached the highest value in 50 mg/L Fe2O3 NPs presoaking and decreased in 100–200 mg/L Fe2O3 NPs presoaking, with still higher than that of the control. The root length was significantly higher than the control at 50–150 mg/L and reached peak value in 100 mg/L Fe2O3 NPs presoaking (Table 1). The results proved that the promoting effect of presoaking Fe2O3 NPs on alfalfa seeds was dose-dependent. Our results support that Fe2O3 NPs improve alfalfa germination and early seedling growth in this concentration range. It could partly be explained as a consequence of NPs being easy to deliver in cells or tissue, assisting with water uptake. Moreover, Fe is a micronutrient that improves plant growth and changes metabolic activity.The chlorophyll content is frequently used as a key indicator for reflecting iron status in plants [65]. No significant difference was observed in Fe2O3 NPs presoaked alfalfa except for 150 mg/L (Figure 1). Similarly, Hu et al. [64] found that the chlorophyll content of maize exposure to 20–100 mg/L Fe2O3 NPs was higher than the control in the first week but had no positive effects in the next 2 weeks [64]. As well known, Fe is involved in chlorophyll biosynthesis, and chlorophyll is positively correlated with leaf’s Fe status. In this study, we found that the iron contents were not significantly modified in Fe2O3 NPs presoaking treatment alfalfa plants (Figure 2), which exhibited the same trend as the chlorophyll content. Similarly, Boutchuen et al. [37] found that 22 and 1,100 mg/L of Fe-enriching hematite nanoparticle drops do not affect the iron content of green gram, black bean, red bean, and chickpea. The present results suggest that the short-time duration of Fe2O3 NPs presoaking had little effect on the chlorophyll and iron content of alfalfa seedlings.One of the most common toxic mechanisms of metal NPs is reactive oxygen species (ROS) production [66]. To protect themselves, plants use antioxidant defense systems to scavenge excess ROS. Antioxidant enzymes such as SOD, POD, and CAT are essential components of intracellular antioxidant mechanisms. In previous studies, a high concentration of Fe2O3 nanoparticles (100 and 1,000 mg/L) has enhanced Flax’s POD and CAT activities [63]. The increase of antioxidant enzyme activities in Fe2O3 NPs-treated plants significantly concerning the controls, showing a response leading to prevent oxidative damage. In other research, Fe2O3 NPs treatment did not show any inhibition effect on CAT activity in the corn leaves, indicating oxidative stress did not happen in leaf tissues. POD activity decreased in corn roots treated with 20 mg/L Fe2O3 NPs and increased at the 100 mg/L of Fe2O3 NPs concentration. We found a tendency of “increase– decrease–reincrease” in the activities of protective enzymes under Fe2O3 NPs presoaking treatments (Figure 3). Meanwhile, the enzyme activity results and leaf staining results corroborate each other to some extent (Figure 4). A possible explanation for the higher level of antioxidant enzyme activities at 50 mg/L is that Fe acts as the co-factor for enzymes. For example, SOD is metalloenzymes, found in three different molecular forms containing Cu, Zn, Mn, or Fe as prosthetic metals. Additionally, CAT and POD are heme-containing enzymes with Fe in their structures, so their activities strongly depend on Fe status, suggesting that a proper amount of Fe2O3 NPs addition supplement can improve the activities of antioxidant enzymes. In this study, the observed enhancement in SOD, CAT, and POD activities of alfalfa seedlings in 200 mg/L Fe2O3 NPs treatment (Figure 3) suggests an adverse effect of the high concentration of Fe2O3 NPs on plant growth. It might be due to the excess iron ions participating in the Fenton reaction, which generates oxidizing species and causes oxidative damage. Fe2O3 NPs could activate an antioxidant defense in alfalfa by inducing oxidative stress, and the plant could regulate antioxidase activity when excess ROS is induced.From the results of the above experiments, the data on germination and growth suggest that Fe2O3 NPs presoaking has a stimulatory effect on alfalfa at low concentrations while being toxic or inhibitory at high concentrations. Because Fe has a positive effect on plants as microelements and plant growth regulators, it is difficult to determine the results due to the characteristics of nanoparticles or Fe. For further understanding of the method of Fe2O3 NPs on plant growth, we selected a nonphytotoxicity dose of 50 mg/L for follow-up assessment, comparing the effects of Fe2O3 NPs and EDTA-Fe on hydroponic alfalfa seedlings.The hydroponic experiment has been carried out in plants to detect the effect of Fe2O3 nanoparticles on the plants. Rostamizadeh et al. [67] found that Fe2O3 nanoparticles at concentrations ranging from 10 to 100 mg/L showed statistically significant stimulation in both root and shoot biomass of barley. Abusalem et al. [62] found that green synthesized Fe2O3 nanoparticles significantly improved the seed vigor index, shoot length, and fresh and dry weight of the tomato seedlings. Ghafariyan [26] found that superparamagnetic iron oxide NPs show a significant positive influence on root elongation. Jeyasubramanian et al. [68] suggested that the inclusion of nano-Fe2O3 results in an enhanced growth rate of spinach in a dose and time-dependent manner. These studies highlight the enhanced growth rate of plants due to the uptake of Fe2O3 nanoparticles. Conversely, Hu et al. [69] showed no obvious sign of Citrus maxima growth inhibition under Fe2O3 NPs exposure at experimental concentrations at the seedling stages. The present study demonstrated that alfalfa growth was promoted when cultivated in 50 mg/L Fe2O3 NPs hydroponic conditions. Better growth parameters, including root length, lateral root number, and fresh weight, were observed in Fe2O3 NPs hydroponic treatment of alfalfa, compared with that of EDTA-Fe treatment (Table 2, Figure 5). Our results confirm that the nanometric form of Fe2O3 can enhance plant growth in agreement with the previous work.Chlorophyll is one of the physiological indexes that reflect the high yield performance of plants. Li et al. [70] found that 20–100 mg/L Fe2O3 NPs could increase the chlorophyll contents of watermelon and maize. Hu et al. [69] found that 50 mg/L Fe2O3 NPs significantly enhanced the chlorophyll content of Citrus maxima. Ghafariyan et al. [26] found that SPIONs entered and translocated in the soybean, increasing the chlorophyll content in subapical leaves of soybean levels. Medina-Pérez et al. [71] revealed that three NPs of Fe3O4, FeOOH·xH2O, and Fe2O3 have no significant effect on the chlorophyll content of common beans [71]. Consistent with the previous report, the chlorophyll content was significantly higher (p<0.05) in Fe2O3 NPs hydroponic treatment alfalfa than that of EDTA-Fe treatment, but not in the content of chlorophyll a and chlorophyll b, individually. Our results confirmed that the nanometric form Fe2O3 at 50 mg/L concentration could enhance chlorophyll synthesis (Figure 6). Based on the positive correlation between chlorophyll and iron contents, we speculated that the iron content would be increased in Fe2O3 NPs hydroponic treated alfalfa. The observed higher iron content is in accordance with our speculation (Figure 7). In agreement with previous studies that iron oxide nanoparticles increased iron content in spinach plants in a dose-dependent manner [68], and the iron present in the whole tomato plant increased with increasing concentration of NPs [33]. Although Li et al. [70] suggested that Fe2O3 NPs could enter plant roots and accumulate, the translocation of NPs from roots to shoots was rarely observed by transmission electron microscopy. Boutchuen et al. [37] found that the hematite NPs were transported through the shoot and localized in the leaves of the legumes. We speculate that the Fe2O3 NPs in appropriate concentrations can be translocated from roots to shoots and improve the photosynthesis by promoting the chlorophyll biosynthesis of the plant.Proper nutrition is the basic need of every living organism. The metals iron (Fe), copper (Cu), manganese (Mn), and zinc (Zn) are micronutrients essential for the growth and development of virtually all plant species. The essential trace metals can shape root systems, interfere with the uptake and signalling mechanisms, target hormone-related processes, and thus induce changes in other trace metal nutrients in plants [72]. Fe competes with other transition metals such as Cu, Zn, and Mn in its uptake, transport, and chemical reaction within plant cells [73]. Plenty of previous studies have focused on the accumulation and translocation of exposed metal NPs. However, rare studies of metal nanoparticles’ effect on other metals are performed in plants. Therefore, we examined the content of three other trace metals, Cu, Mn, and Zn. Interestingly, Mn content in Fe2O3 NPs hydroponic treated alfalfa was significantly higher than that in the control, while Cu and Zn content showed no difference (Figure 7). The synergetic enrichment between Mn and Fe can stimulate photosynthetic efficiency. The broad substrate specificity of most Mn transporters (like IRT1, NRAMPs, YSLs, ZIP, and CDFs/MTP) substantially influences Mn homeostasis. These transporters have also been advocated to have a substrate specificity for Fe2+ [74, 75]. Mn2+ stimulated the expression of some Fe-deficiency response genes such as FRO2, IRT1, and NRAMP3. These findings suggest complex crosstalks exist for Fe and Mn, and pathways are interconnected to regulate metal homeostasis and maintain a stable metabolism. A synergistic transporting of Mn by Fe2O3 NPs deserves intensive investigation.The ferric chelate reductase (FCR), the enzyme that reduced ferric iron (Fe (III)) into ferrous iron (Fe(II)), is known to play important roles in Fe absorption and homeostasis in plants that utilize a strategy I mechanism [76, 77]. Reducing ferric to ferrous iron on the root surface is an obligatory process for iron acquisition from the soil in strategy I plants. Expression of FCR genes was induced in roots under iron deficiency [78]. Moreover, FCR activity was significantly reduced by the influence of Fe2O3 NPs in our analysis (Figure 8). The findings differ from the report that 50 mg/L Fe2O3 NPs and Fe3+ significantly increased expression levels of the FRO2 gene and correspondingly had a higher ferric reductase activity [70]. We conclude that there might be no need for more reduction activity of FCR due to the nano form of Fe2O3 NPs being more easily absorbed and transported than Fe-EDTA.From our results, we consider that Fe2O3 NPs can be absorbed through the alfalfa root tip cells independent of FCR activity. The plausible mechanism of Fe2O3 NPs transport in the plant could be as follows. The Fe2O3 NPs might damage the plant cell wall and plasma membrane to facilitate entry and translocation. Subsequently, the NPs were internalized into cell walls, embedded by new cells, and later entered into the vascular system. The Fe2O3 NPs might move from the cell wall through endocytosis and through symplastic transport. Fe2O3 NPs might be located in the xylem and phloem vessels and in the cell walls of xylem vessels. They might be transported through both the apoplastic pathway to the endodermis and the symplastic pathway to the vascular system. In sum, the Fe2O3 NPs are taken by plant roots, travel through the stem, and are accumulated in leaves.


## 5. Conclusion

In summary, by presoaking at lower concentrations, Fe2O3 NPs have a beneficiary impact on alfalfa seed germination. The 50 mg/L Fe2O3 NPs presoaking has a better growth promotion effect on alfalfa. Short-time presoaking of Fe2O3 NPs seed has not significantly affected chlorophyll and iron content while increasing antioxidase (SOD, POD, and CAT) activities at 50 and 200 mg/L. Hydroponic alfalfa seedlings exposed to 50 mg/L Fe2O3 NPs have longer root length, more lateral and higher fresh weight, and significantly higher chlorophyll and related iron content than in Fe-EDTA treatment. The content of Mn is significantly higher than Fe-EDTA treatment, while Cu and Zn contents show no difference. Moreover, ferric chelate reductase activity was significantly reduced in alfalfa root exposure to Fe2O3 NPs. From the above studies, 50 mg/L Fe2O3 NPs could be used as an appropriate and effective treatment concentration for seed pretreatment and application as fertilizers. In-depth research is still needed to understand how Fe2O3 NPs are uptaken by roots and transported from the roots to the shoots, and it is necessary to study further the impact of Fe2O3 NPs on the field environment.



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*Source: 9783977-2023-02-13.xml*