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. 2018 Oct 2;13(1):66–70. doi: 10.1049/iet-nbt.2018.5212

Investigation of ZnO nanoparticles on proline, anthocyanin contents and photosynthetic pigments and lipid peroxidation in the soybean

Shahla Hashemi 1,2,✉, Zahra Asrar 1, Shahram Pourseyedi 3, Nazi Nadernejad 1
PMCID: PMC8676270  PMID: 30964040

Abstract

The interaction between nanoparticles and plants is inevitable. In this study, the effect of different concentrations of ZnO nanoparticles synthesised using olive extract on the soybean was studied. The soybean seeds were cultured in a Hoagland medium containing agar which was treated different concentrations (0, 200 and 400 ppm) of ZnO nanoparticles. After 21 days, the plants were harvested and the parameters of proline, anthocyanin, malondialdehyde (MDA), hydrogen peroxide (H2 O2), chlorophyll and carotenoid contents and phenylalanine ammonia‐lyase (PAL) and catalase (CAT) activity in soybeans (Glycine max) were measured. The results showed that the levels of chlorophyll a and b and carotenoid at concentrations of 200 and 400 ppm in comparison with control decreased, while carotenoid content at 200 ppm concentration at a concentration of 400 ppm was not significant. The level of anthocyanin and PAL activity increased with increasing concentration of nanoparticles, while proline content decreased. By increasing the concentration of ZnO nanoparticles, the content of MDA and hydrogen peroxide increased compared to control but CAT activity did not change significantly. This research suggests that ZnO nanoparticles synthesised using olive extract in soybean plants may be toxic by reactive oxygen species production.

Inspec keywords: crops, pigments, enzymes, nanoparticles, zinc compounds, toxicology, agricultural safety, nanobiotechnology, hydrogen compounds

Other keywords: carotenoid content, proline content, ZnO nanoparticles, soybean plants, H2 O2 , ZnO, time 21.0 d, reactive oxygen species production, toxicity, catalase activity, phenylalanine ammonia‐lyase activity, chlorophyll content, Hoagland medium, olive extract, lipid peroxidation, photosynthetic pigments, hydrogen peroxide, anthocyanin contents

1 Introduction

Nanotechnology and nanoscience have attracted great attention due to synthesis and application of nanoparticles [1, 2, 3]. Nanomaterials are materials with at least two dimensions between 1 and 100 nm [4]. The sizes of the nanoparticles are located between individual molecules and bulk materials [5]. Unique properties such as a large specific surface area and greater reactivity that result from the small scale of nanomaterials are responsible for their unusual properties such as electronic, catalytic and magnetic activities [6]. The rare properties of nanoparticles permit an extensive range of applications [7]. Nanomaterials can offer many applications in mechanical industries especially in coating, lubricants and adhesive applications [5]. The magnetic nanoparticles such as Fe3 O4 are employed in the biomedical and clinical fields [8, 9]. TiO2 nanoparticles find an application in cosmetics, pigments, sunscreen products, solar cells and photocatalysis [10]. ZnO nanoparticles are one of the most widely used ones in various engineering and biological applications, such as potential pharmacological agents, high‐temperature oxidation protection materials, solid‐oxide fuel cells, catalytic materials and solar cells [11, 12]. The synthesis of nanoparticles was mainly done by physical and chemical methods, and in recent years, biological methods have been added [2, 13]. Although the synthesis of nanoparticles by chemical and physical methods has desirable properties for their application but many uses of nanoparticles have a serious risk to the environment. However, inappropriate handling, accidental and incidental release of nanoparticles could lead to environmental contamination [14]. As such, their environmental behaviour and fate, potential risk and toxicity to ecosystems have attracted attention [15, 16]. An important component of ecosystems is plants. Plants can be used for the assessment of the overall fate, transport and exposure pathways of nanoparticles in the environment [17, 18]. A few research studies have reported the effects of nanoparticles on plants [14, 16, 18]. It has been reported that Fe3 O4 nanoparticles increased the growth and the levels of chlorophyll of Zea mays [19]. Jaberzadeh et al. reported that TiO2 nanoparticles increased wheat plant growth. Increased growth by TiO2 nanoparticles was attributed to the control of the enzymes activity involved in nitrogen metabolism such as glutamine synthase, glutamate dehydrogenase, nitrate reductase and glutamic–pyruvic transaminase by these nanoparticles [20]. Lin and Xing studied the toxicity of five types of nanoparticles (aluminium, alumina, multiwalled carbon nanotube, zinc and zinc oxide) on seed germination and root growth of six plant species. In the root elongation test, all plants were reduced when suspended in nanoparticles. Reduced root growth was due to disruption in the nutrient and water pathways in plants [21]. Gui et al. [22] reported CeO2 nanoparticles had no significant effect on the chlorophyll content of lettuce. Zhang et al. [23] stated that CeO2 nanoparticles decreased relative chlorophyll content but did not affect the average quantum yield of photosystem II in radish. Application of CeO2 nanoparticles significantly reduced the seed germination of corn [24] but significantly increased root elongation of cilantro [25]. Lee et al. indicated the effects of copper nanoparticles on the seedling growth of wheat and mung bean. Their findings explained that mung bean was more sensitive to copper nanoparticles than wheat [26]. However, the nanoparticle effects vary depending on the type of nanoparticles and plant species. On the other hand, because of the high use of ZnO nanoparticles in agricultural products such as, growth regulators [27], fertilisers [28], pesticides and herbicides [29], their effects increased on ecosystems and plants. Several studies have shown that ZnO nanoparticles have an increasing effect on tomato and green peas growth [30, 31]. A study of the hydroponic solution containing ZnO nanoparticles did not have any negative effects on the seed germination and root growth of zucchini seeds [32]. Dimkpa et al. [33] reported that ZnO nanoparticles reduced wheat plant growth with an increased production of reactive oxygen species (ROS).

These investigations have contributed to the perception of effects of ZnO nanoparticles synthesised by chemical and physical methods on plants. Most papers on the synthesis of nanoparticles by plants investigated antibacterial [34, 35, 36], antifungal [37, 38] and anticancer activity [9, 13, 39] of nanoparticles, but their effects on plants were less studied [40]. The purpose of this study was to investigate the effect of nanoparticles of the synthesised nanoparticles of olive (Olea europaea) on proline and pigment contents and phenylalanine ammonia‐lyase (PAL) and catalase (CAT) activities in the soybean (Glycine max).

2 Materials and methods

2.1 Characterisation and preparation of ZnO nanoparticle suspensions

According to the previous study, ZnO nanoparticles were synthesised from olive extract and of zinc nitrate [41]. The synthesis of zinc oxide nanoparticles was determined using a UV–Vis spectrophotometer, X‐ray diffraction (XRD) and transmission electron microscopy (TEM) [41]. Different concentrations (0, 200 and 400 ppm) of ZnO nanoparticles were suspended in deionised water (DI‐water) and dispersed by ultrasonic vibration (100 W, 40 kHz) for 30 min. Before use, to prevent the accumulation of particles, a small magnetic rod was placed within the suspensions for 10 min.

2.2 Treatment of plants with nanoparticles

Soybean seeds were sterilised with 10% (v/v) sodium hypochlorite solution for 5 min and washed thoroughly with distilled water. The seeds were cultured in a Hoagland medium containing agar which was treated at different concentrations (0, 200 and 400 ppm) of ZnO nanoparticles. Plants were harvested after 21 days of exposure to ZnO nanoparticles, and biochemical parameters were analysed.

2.3 Determination of chlorophyll, carotenoid and malondialdehyde (MDA) contents

Chlorophyll and carotenoid contents were determined according to the method of Lichtenthaler. 0.2 g of fresh leaves was homogenised in 80% acetone, and then centrifuged at 4000g for 10 min. The supernatant was used to determine the absorbance 470 nm for carotenoid and at 663.2 and 646.8 nm for chlorophyll content [42]. According to the method of Heath and Packer [43], MDA content was evaluated using thiobarbituric acid as the reactive substance.

2.4 Determination of proline and hydrogen peroxide (H2 O2) contents

Proline content assay was conducted as described [44]. 0.02 g of fresh leaves was weighed to extract proline using 10 ml solution of 3% sulfosalicylic acid, and then centrifuged at 10,000g for 5 min. Two millilitres of the supernatant was mixed with 2 mL of glacial acetic acid and 2 mL of ninhydrin buffer at 100°C for 1 h. After cooling, 4 mL of toluene was added to each sample. Then, the absorbance was read at 520 nm. Content of proline was determined by the calibration curve of pure proline and expressed as µg/g fresh weight. H2 O2 content was determined based on an interaction between H2 O2 and potassium iodide according to Alexieva et al. [45].

2.5 Anthocyanin content and PAL activity

The anthocyanin content analysis was assayed according to the method by Wagner [46]. Briefly, 0.1 g of fresh leaves was homogenised in 10 mL of acidified methanol and placed in the dark for 24 h. After centrifuging, the absorbance of the supernatant was read at 550 nm [46]. PAL activity was assessed according to Beaudoin‐Eagan and Thorpe [47].

2.6 CAT activity

CAT activity was measured by absorption reduction at 240 nm [48]. The CAT activity was assayed according to the method by Dhindsa et al. [48].

2.7 Statistical analysis

All data are reported as means ± standard error. Analysis of variance was used to evaluate the effect of different treatments of ZnO nanoparticles. The Duncan's multiple range test was used to investigate the effect different treatments (P  < 0.05) using SPSS software.

3 Results and discussion

3.1 Characterisation of ZnO nanoparticles

The UV–Vis absorption spectra of ZnO nanoparticles showed that the absorption maxima of nanoparticles were 370 nm (Fig. 1). The hexagonal phase (data not shown) and spherical shape (Fig. 2) of nanoparticles with a mean size of 41 nm were determined by XRD and TEM, respectively [41].

Fig. 1.

Fig. 1

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UV–visible spectroscopy of ZnO nanoparticles [41]

Fig. 2.

Fig. 2

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TEM image of ZnO nanoparticles [41]

3.2 Chlorophyll content

Fig. 3 showed chlorophyll a and b contents in leaves of soybean plants cultured in a Hoagland medium containing agar which was treated at different concentrations (0, 200 and 400 ppm) of ZnO nanoparticles. compared with control and chlorophyll a and b significantly reduced at 200 and 400 ppm of ZnO nanoparticle treatment. The content of chlorophyll a and b at a concentration of 400 ppm nanoparticles decreased in comparison with a concentration of 200 ppm. Our results are consistent with previously reported results with ZnO nanoparticles [31]. Mukherjee et al. [31] found that ZnO nanoparticles reduced the chlorophyll content of green peas. Wang et al. [49] studied the effect of 0–300 ppm ZnO nanoparticles on photosynthesis pigments in Arabidopsis. Compared to control, plants exposure to 300 mg/L ZnO nanoparticles exhibited a 50% reduction in chlorophyll content.

Fig. 3.

Fig. 3

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Effect of treatment of zinc oxide nanoparticles on chlorophyll content in soybeans. Vertical bars indicate ± standard error

3.3 Carotenoid content

The results of the carotenoid content study clearly revealed that ZnO nanoparticles with 200 and 400 ppm concentrations decreased carotenoid content while carotenoid content did not change significantly between 200 and 400 ppm. Similar to our results, Wang et al. [49] reported 3200 mg/kg ZnO nanoparticles decreased carotenoid contents in maize leaves. Content of carotenoids is an indicator for evaluating the physiological state of plants. Carotenoids have a number of polyene chains of conjugated double bonds within the central C40 backbone. Carotenoids play a key role in the trapping radical oxygen species, quenching singlet oxygen and protecting cellular components, such as chlorophylls, proteins, DNA and lipids from oxidative damage [50]. The study suggested that low carotenoid content in nanoparticles treatment failed to protect chlorophyll content from oxidative stress (Fig. 4).

Fig. 4.

Fig. 4

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Effect of treatment of zinc oxide nanoparticles on carotenoid content in soybeans. Vertical bars indicate ± standard error

3.4 Anthocyanin content and PAL activity

The results of the anthocyanin content and PAL activity under the treatment of nanoparticles are shown in Figs. 5 and 6, respectively. The amount of anthocyanin and PAL activity in soybeans increased at 200, 400 ppm concentration of nanoparticles compared with control. The content of anthocyanin and PAL activity at a concentration of 400 ppm was increased in comparison with a concentration of 200 ppm nanoparticles. Similar to the results of this study, Syu et al. [51] reported that nanoparticles increased the anthocyanin production in Arabidopsis. Anthocyanins are pigment acting as efficient scavengers of ROS in plants. Anthocyanin biosynthesis in plants is activated by PAL, a key enzyme of the phenylpropanoid route. In this study, the activity of PAL is increased along with the increase of anthocyanin content.

Fig. 5.

Fig. 5

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Effect of treatment of zinc oxide nanoparticles on anthocyanin content in soybeans. Vertical bars indicate ± standard error

Fig. 6.

Fig. 6

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Effect of treatment of zinc oxide nanoparticles on PAL activity in soybeans. Vertical bars indicate ± standard error

3.5 Determination of proline content

According to Fig. 7, proline content was not significantly different from controls after exposure to 200 ppm nanoparticles, while decreased by 400 ppm. Proline belongs to organic metabolites. Several studies have shown an antioxidant property to proline, activity of ROS scavenging and also act as a singlet oxygen quencher [52]. The author suggests that proline reduction may be due to the ROS scavenging by proline.

Fig. 7.

Fig. 7

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Effect of treatment of zinc oxide nanoparticles on proline content in soybeans. Vertical bars indicate ± standard error

3.6 MDA and H2 O2 contents

The important indicators for measuring stress on plants are the MDA and H2 O2 contents. The results of the MDA and H2 O2 contents in the treatment of nanoparticles are shown in Figs. 8 and 9, respectively. At concentrations of 200 and 400 nanoparticles, the content of MDA and H2 O2 in soybeans increased in comparison with control. Increasing content of MDA and H2 O2 at 400 ppm concentration was significant in comparison with the 200 ppm concentration. According to Mukherjee et al., ZnO nanoparticles increased MDA and H2 O2 contents in green peas. Previous studies reported that zinc induced the production of free radicals in Zea mays [53]. H2 O2 belongs to the ROS group. Hydrogen peroxide is a low‐molecule molecule, but it can easily penetrate the membrane of the cell and produce ROS such as radical hydroxyl, through the reaction of Fenton (H2 O2  + Fe2+ →Fe3+  + OH−  + OH•) [54].

Fig. 8.

Fig. 8

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Effect of treatment of zinc oxide nanoparticles on MDA content in soybeans. Vertical bars indicate ± standard error

Fig. 9.

Fig. 9

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Effect of treatment of zinc oxide nanoparticles on H2 O2 content in soybeans. Vertical bars indicate ± standard error

The imbalance in removal and accumulation of H2 O2 and other ROS molecules leads to oxidative stress with oxidative damage to fats, DNA and proteins. In order to investigate the effect of H2 O2 on membrane damage, lipid peroxidation in soybean plants was analysed. MDA formation in plants exposed to adverse environmental conditions is an indicator of lipid peroxidation in biological systems [43]. Liu et al. [55] stated that MDA formation may be partially due to ROS production in plants.

3.7 CAT activity

In the metabolism of hydrogen peroxide, CAT plays an important role (2H2 O2 →2H2 O + O2) [56]. According to Fig. 10 CAT activity was not significantly different from controls after exposure to 200 and 400 ppm nanoparticles. Therefore, in this study, the CAT enzyme did not have significant activity to eliminate hydrogen peroxide produced at different concentrations of nanoparticles. Similar to the results of this study, Barrios et al. [57] reported that nanoparticles did not affect CAT activity in tomato.

Fig. 10.

Fig. 10

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Effect of treatment of zinc oxide nanoparticles on CAT activity in soybeans. Vertical bars indicate ± standard error

4 Conclusion

ZnO nanoparticles bio‐synthesised in soybeans reduced the chlorophyll content, carotenoids and proline, while increased the MDA, H2 O2, anthocyanin contents and PAL activity. The induction of anthocyanin content, low content of carotenoids and proline content by ZnO nanoparticles was not enough to detoxify ROS.

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