Biosynthesis of Ag nanoparticles and two‐dimensional element distribution in Arabidopsis

Abstract

Metallic nanoparticles can be synthesised in living plants, which provide a friendly approach. In this work, the authors aimed to study the synthesis of silver nanoparticles (AgNPs) in Arabidopsis and the two‐dimensional (2D) distribution of Ag and other elements (Ca, P, S, Mg, and CI) in the Arabidopsis plant tissues. The concentrations of Ag in the plant tissues were determined by inductively coupled plasma‐atomic emission spectrometer, showing that the majority of Ag was retained in the roots. Transmission electron micrographs showed the morphology of AgNPs and the location in plant cells. The distributions of Cl and Ag were consistent in plant tissues by 2D proton‐induced X‐ray emission. In conclusion, this is the first report of the AgNP synthesis in Arabidopsis living plants and its 2D distribution of important elements, which provide a new clue for further research.

1 Introduction

As an emerging field, nanotechnology has been receiving great attention from researchers over the last few decades [1, 2]. Due to its unique physical and chemical properties, nanomaterials with at least one dimension in the size of 1–100 nm have excellent optical, magnetic, mechanical, and catalytic properties in diverse fields [3, 4, 5, 6]. Among the metallic nanoparticles (NPs), the silver nanoparticles (AgNPs) are widely used because of their strong antimicrobial efficiency [4, 7, 8, 9], optical functions [10], catalytic activity [9, 11]. AgNPs have also been used in medical applications, such as drug carriers [12], diagnostics, biosensors, targeted drug therapy [13]. AgNPs are also found in consumer products [14].

The main methods of synthesising the metallic nanomaterials are divided into two ways, top‐down approach and bottom‐up approach. The top‐down approach mainly breaks down the bulk material into nanostructures by various physical and chemical methods. The common physical methods are evaporation and laser ablation [15, 16]. The drawback of this approach is high‐energy consumption, low yield and surface imperfection of the synthesised nanomaterials [17]. The bottom‐up approach produces the desired NPs by the assembly of molecules or atoms, where the chemical and biological methods mostly belong to this category. However, the chemical methods may use a toxic solvent to stabilise the NPs and avoid the aggregation of NPs, which limits the application of the nanomaterial and causes environmental problems [18]. In recent years, the green chemistry approach draws more attention. Compared to the physical and chemical methods, the biological methods are inexpensive, biocompatible, environmental friendly and mostly one‐step synthesis, which are consistent with the green chemistry approach [19, 20, 21].

The biological methods or the green chemistry approach uses the biological entities, such as plants and microorganisms (like bacteria, fungi, yeast) to synthesise NPs. [22]. The plant‐mediated synthesis approach is known as the best biological process for the production of NPs, which is more biocompatible and safer, and also more suitable for medicinal applications [23]. Several studies have illustrated that plants are able to synthesise NPs in their tissues. For example, gold NPs were first found in living vascular plant (alfalfa), which confirmed that alfalfa is able to uptake gold ions and synthesise NPs [24]. Brassica juncea (Indian mustard) was the second species having the capacity to synthesise gold NPs [25]. Brassica juncea was also reported to synthesise the NPs of metal alloys [26]. AgNPs can also be formed in living plants, Brassica juncea, Festuca rubra and Medicago sativa [27]. Besides, some other plants have the ability to synthesise metallic NPss [28]. In our research group, we first used living plant maize (Zea mays L.) to synthesise AgNPs [29]. Also, we used Euglena to synthesise AgNPs [30].

In this paper, we used the model plant Arabidopsis thaliana L. (Arabidopsis) to confirm the formation of AgNPs. Arabidopsis thaliana plays an important role in the field of plant biology. It has a small genome and the whole genome sequencing was completed in 2000. In addition, it has a short 6‐week lifespan, small size, high seed production, and easy to grow. Nowadays, Arabidopsis is widely used in plant genetics and molecular biology research. Plants are the predominant photosynthetic autotrophs and can be as sustainable and renewable resources to synthesise NPs. Arabidopsis can absorb metal irons and survive in metal contaminated soil. However, there are few reports about the interaction between Ag and Arabidopsis. Therefore, we decide to choose Arabidopsis as the research object to study the interaction between the silver ions and plants.

2 Materials and method

2.1 Silver uptake experiments

Arabidopsis thaliana L. (Col‐0) were surface sterilised by 75% ethanol for 10 min, and then put on the sterile filter paper. Seeds were sown onto sterilised agar plates containing half‐strength Murashige and Skoog (1/2 MS) using sterilised toothpicks and placed at 4°C for 3 days in the dark. Seeds were germinated and grown at 22°C under the long day growth conditions (16/8 light/dark). After 4 weeks, seedlings were harvested, with their root intact, and the growth solution was replaced with a range of silver nitrate solution (0, 0.6, 1, 2, 10, 50, and 100 mM) in petri dishes. Deionised water was added to control seedlings. Then seedlings were washed five times with deionised water. In order to examine the effect of exposure time, 4‐week‐old seedlings were dealt with 2 mM AgNO₃ for periods of 0, 5, 15, 24, or 48 h. All experiments were performed with 16/8 light/dark at 22°C. Each treatment was repeated three times.

2.2 Inductively coupled plasma‐atomic emission spectrometer (ICP‐AES) analysis

Seedlings were collected, and the root and aerial parts of seedlings were separated carefully and dried in an oven at 80°C for 24 h to a constant weight. Two millilitres of concentrated nitric acid solution (HNO₃) was added to the sample. Then the sample was heated until the complete carbonisation. The sample was ashed in a muffle furnace at 600°C for 4 h [31]. The ash was dissolved and stored in plastic bottles in the dark. The content of Ag of different plant tissues was analysed by an ICP‐AES (Hitachi Z‐5000).

2.3 Transmission electron microscopy (TEM)

The main root of the fresh plant was cut into about 1 cm long sections. The sections were prefixed with 2.5% glutaraldehyde in phosphate buffer (0.1 mol/l, pH 7.2) for 2 h and washed three times with 100 mM phosphate buffer. One per cent of osmium tetroxide fixed the sample again for 1 h and then the sections were washed three times with 100 mM phosphate buffer. The sample was dehydrated with ethanol, embedded in epoxy resin, and was sliced into 70 nm thick ultrathin sections without staining. The ultrathin sections were analysed by TEM (Tecnal G² F20 S‐Twin) equipped with CCD Camera, operating at 200 kV. Energy‐dispersive X‐ray spectroscopy (x‐Max T80) was used for analysing the element composition of biosynthetic NPs.

2.4 X‐ray diffraction (XRD)

For the XRD study, the root was dried in an oven at 80°C for 24 h and then grinded into powder. The crystalline nature of biosynthetic AgNPs was conducted on a BRUKER D2‐PHASER X‐ray diffractometer at a scanning rate of 4°/min over the 2θ range of 5–80°, employing Cu Kα radiation (l  = 0.15418 nm).

2.5 Two‐dimensional (2D) proton‐induced X‐ray emission (PIXE)

2.5.1 Sample preparation

After exposure, seedlings were harvested and washed 5 times. Five centimetre long main root and leaf were fixed in 4% paraformaldehyde for 4 h with the sample full immersion. Then, the sample was put into a 30% sucrose solution for dehydration and stored in 4°C until cryosectioning. After that, the sample was embedded in the optimal cutting temperature (OCT) compound and frozen for half an hour at −22°C. Then, the OCT embedded tissues were placed on the sample holder and were cut into 30 μm sections using a Cryostat Microtome (Leica CM1950, Germany). The cryosections were transferred to the mylar membrane.

2.5.2 2D PIXE analysis

PIXE is a highly sensitive technique for elemental mapping in two dimensions [32]. We used a Si (Li) detector (Sirius80, Gresham Ltd.) installed at 45°C to collect proton‐induced characteristic X‐rays. This detector has 80 mm² active area, energy resolution of 150 eV, and the size of the proton beam was nearly 20 × 20 µm². The data analysis was performed using the OMDAQ 2007 software.

3 Results and discussion

3.1 Silver concentration in plant tissues

When exposed to a silver nitrate solution, we could clearly observe the silver absorption by root tissues that result in fast progressive browning of roots. It denotes that Ag as a toxic and non‐essential metal which can be absorbed by plants and was harmful to plants to some extent. Despite the short‐time exposure, the silver was taken up quickly by the roots and was subsequently transported to other parts of the plant with an increasing exposure time [33]. From Fig. 1, it showed an increasing metal absorption with increasing silver concentration and exposure time. The content of Ag in plant tissues was determined by the ICP analysis.

Fig. 1.

Silver concentration in Arabidopsis plant tissues

(a) Ag contents in plant tissues treated with different silver nitrate solution for 24 h, (b) Ag contents in the presence of 2 mM silver nitrate solution for 0, 5, 24, and 48 h. The values were given as mean ± SD of triplicate samples

Fig. 1 a shows the Arabidopsis absorbed Ag depending on the silver ions concentration in the media. It may be due to the high ion driving force at high concentrations [34]. More Ag was existed in roots than in aerial parts from 0.6 to 100 mM AgNO₃ for 24 h. The maximum silver content in roots and in aerial parts was both appeared in exposing to 100 mM AgNO₃. The Ag content in roots exposed to 100 mM AgNO₃ was more than 1.39 times higher than that to 50 mM AgNO₃, 8 times higher than that to 10 and 2 mM AgNO₃, 12 times higher than that to 1 mM AgNO₃ and almost 18 times higher than that to 0.6 mM AgNO₃. At low concentrations (0.6, 1, 2, and 10 mM AgNO₃), only a small amount of silver in the plant was transferred into the aerial parts. At high concentrations (50 and 100 mM AgNO₃), it was seen that the silver content in aerial parts increased significantly compared to low concentrations. Yin et al. [35] have also reported that there was less Ag translocated into aerial parts. The results showed that the major Ag were retained by roots, which may be that a limited apoplastic translocation may be related to the upward translocation of silver to aerial parts [36].

In order to investigate the effects of exposure time, Arabidopsis was treated with 2 mM silver nitrate solution for 5, 15, 24, and 48 h. Fig. 1 b shows that the silver absorption of the plant tissues gradually increased within 24 h. More than 94% of the silver content was present in the roots and a small part of Ag was transported to the aerial parts as exposure time from 5 to 24 h. However, after 24 h, the silver content increased both in roots and in the aerial part and as the time increased, the silver was transported into the aerial parts gradually. The silver content in aerial parts reached a higher value, which was about half of the roots’ silver content exposed for 48 h. It was likely because of nutrient starvation [37]. Thus, it could be clearly seen that the content of the silver was significantly increased after 48 h.

Results showed that the Arabidopsis can absorb silver ions, transport silver to other plant tissues, and Ag content in plants increased significantly with increasing concentration of AgNO₃ and exposure time. In addition, the translocation from root to aerial parts was very low, suggesting the maximum accumulation of Ag in roots.

3.2 AgNPs formation in Arabidopsis

TEM analysis was used to analyse the existence of AgNPs in Arabidopsis. From Fig. 2, a number of black dots were observed (Fig. 2 a). It can also be seen that the black dots were either disperse or aggregated. The composition of the small black dots was determined as Ag using energy dispersive spectrometer (EDS) (Fig. 2 c). The presence of O, S, and Cl came from the plant, and Cu and C were due to the grids for section support. The morphology of synthesised AgNPs is mostly spherical with the particle size ranging from 1 to 22 nm (Figs. 2 b and d). This phenomenon indicated the formation of AgNPs in Arabidopsis (Fig. 3).

Fig. 2.

AgNPs formation in Arabidopsis plant

(a), (b) TEM image of the Arabidopsis root showing aggregates of AgNPs after exposing to 100 mM AgNO₃ for 24 h, (c) X‐ray EDS analyses confirming that the NPs are constituted by Ag, (d) Particle size distribution of AgNPs was recorded using Nano Measurer software 1.2

Fig. 3.

Localisation of AgNPs in the roots of Arabidopsis treated with 100 mM AgNO₃ for 24 h. Control group was shown in part a. AgNP, Ag nanoparticle; Cy, cytoplasm; Cw, cell wall

(a) Arabidopsis was treated with deionized water for 24 h, (b) – (d) AgNPs in Arabidopsis roots cells

In addition, TEM was used to further analyse the localisation of AgNPs in root cells. AgNPs were observed both extracellularly and intracellularly (Fig. 3). Some even aggregated into clusters. It was found that AgNPs were located at the cell wall, plasma membrane, and cytoplasmic vacuoles. Most of the AgNPs appeared in the plasma membrane (Fig. 3 b). Arabidopsis was able to synthesise AgNPs and the synthesised AgNPs were almost spherical with narrow size distribution. AgNPs have certain locations in the plant. TEM picture further observed the changes in the ultrastructure of the cells. Fig. 3 c also shows that the structural integrity of the cells was destroyed seriously, such as the detached plasma membrane from the cell wall, collapse of vacuoles, and cellular structural distortion.

The synthesis of AgNPs in living plants is a complicated process. Many papers have reported the biosynthesis of metal nanoparticles (MNPs), but the mechanism of synthesising NPs in plants is not explained in detail [38, 39]. Possibly that plants uptake silver ions and transform them into other forms, which is a way of detoxification of plants themselves. Some researchers have proposed other possible mechanisms. For example, Beattie and Haverkamp [40] have reported that the reduction of sugar (glucose and fructose) was responsible for the formation of AuNPs and AgNPs.

3.3 XRD analysis

The XRD pattern showed that the AgNPs are crystalline in nature. The characteristic diffraction peak values at 37.995°, 44.201°, and 64.313° corresponded to (111), (200), and (220) lattice planes of face‐centred cubic (FCC) structure of Ag crystals (JCPDS NO.04‐0738). In addition, peaks appearing at 27.682°, 32.277°, and 46.134° were indexed to FCC silver chloride crystals (111), (200), and (220) lattice faces (JCPDS No. 31‐1238), which indicated that this process had a small amount of silver chloride in Arabidopsis roots. Except this, some other peaks belonged to other components, which would require further research (Fig. 4).

Fig. 4.

XRD pattern of Ag in the roots of Arabidopsis after exposing to 100 mM AgNO₃ for 24 h

3.4 Distribution of elements in two dimensions in plant tissues

PIXE was used to study the distribution of elements in roots and leaves. Figs. 5, 6, 7, 8 show the 2D distribution maps of Ag and other elements (Ca, P, S, Mg, and Cl) in the Arabidopsis plant tissues. P and Ca are the main elements of the cell membrane, which could be used for reflecting the fundamental shape of the plant tissues of both control and treated plants.

Fig. 5.

2D distribution of Ag and Cl in Arabidopsis roots

(a1) –(a4) Control group, (b1) – (b4) Plants were treated with 2 mM AgNO₃ for 48 h

Parts (a1), (a2), (b1), (b2) are the distribution maps of Ag and Cl in the root longitudinal section, and parts (a3), (a4), (b3), (b4) are the distribution maps of Ag and Cl in the root cross‐sections. Scan size is 500 × 500 μm². In the colour bar, dark region represents the count of zero and red region represents the highest count

Fig. 6.

Elemental distribution in the root longitudinal section. Control group: the upper; the blow: the plants were treated with 2 mM AgNO₃ for 48 h. Scan size is 500 × 500 μm². In the colour bar, dark region represents the count of zero and red region represents the highest count

Fig. 7.

Elemental distribution in the root cross‐section. Control group: the upper; the blow: the plants were treated with 2 mM AgNO₃ for 48 h. Scan size is 500 × 500 μm². In the colour bar, dark region represents the count of zero and red region represents the highest count

Fig. 8.

Elemental distribution in the leaf cross‐section. Control group: the upper; the blow: the plants were treated with 2 mM AgNO₃ for 48 h. Scan size is 500 × 500 μm². In the colour bar, dark region represents the count of zero and red region represents the highest count

The Ag element of the treated plants in roots first showed that the preferred accumulation sites were the epidermis and endothelial layer, followed by the vascular cylinder. Ag was mainly localised in the root epidermis of the treated plants, which possibly suggested that the epidermis of the plant may be the main region stocking heavy metals. Very low Ag concentration was observed in the vascular cylinder. Roots absorb silver ions and then they are loaded into the xylem. Following the water stream, silver is transported to the aerial parts [41]. Therefore, it could be seen that there was a small amount of Ag in the vascular cylinder. In addition, from Fig. 5, it was seen that the epidermis had some aggregated Ag (Fig. 5, circle). From the control group it was observed that the trace element Cl was evenly distributed in the epidermis and vascular cylinder, but after AgNO₃ treatment, the Cl element was mainly distributed in the epidermis. Comparing Ag and Cl elements in treated groups (Fig. 5), it was found that the distribution of Ag and Cl was highly similar, and it was possible to form AgCl particle deposits. The XRD analysis had confirmed the formation of AgCl.

Furthermore, an increase in epidermal concentration of P, S, Cl, Ca, and Mg of AgNO₃ treated plants was observed compared to the control groups in roots (Figs. 6 and 7). It can be seen from the control group that the macroelements P and S were evenly distributed in the epidermis and vascular cylinders. After treating with AgNO₃, the distribution of P and S had changed obviously. P increased significantly in the cortex with a small amount of P in the vascular column in Ag‐treated plants. The S element was similar to that of the P element, but the change was not as obvious as the P element. In addition, the Ca element is one of the most important components for cell walls and the cell membrane of the plant. An increase of Ca concentration in the epidermis and a decrease of Ca in the vascular cylinder could be clearly seen in AgNO₃ ‐treated plants compared to the control group. The changes in P and Ca distribution may reflect that the root cell integrity was injured to some extent. In our study, we also found that the Ca and Ag distributions in treated roots were highly consistent in both the longitudinal section and the cross‐section (Figs. 6 and 7). There may be some correlation between the Ca element and the Ag element. The change of Mg element distribution was also obvious. From the control group, it could be seen that the Mg concentration in roots was extremely low, but after treating with AgNO₃, it was known that the Mg concentration changes in the cortex were obvious.

Fig. 8 shows the essential elements and metals (P, Ca, Cl, and Ag) in the Arabidopsis plant leaf issues. It can be observed that the macroelements P and Ca were evenly distributed in the leaf tissues. The change in P and Ca distributions was not obvious compared to the control group and the treated group. The biophysical function of the Cl element is to maintain the electrical charge differences across the membrane [42]. From the control group, the concentrations of Cl were even in the leaf of the plant. However, in the treated group, the Cl concentration was significantly lower. In addition, the distributions of Cl and Ag in leaf tissues were also similar, which was consistent with the distributions of Cl and Ag in roots. In the leaf (Fig. 8), the Ag distribution of different regions of leaf tissue was equally distributed on the whole. In summary, the influence on the elements in the root was obvious compared to the leaf tissues. The distribution of Ag and Cl was highly consistent, whether in the root or in the leaves. PIXE visually analyses the 2D distribution of Ag and other elements, providing new ideas for studying the interaction between metals and plants.

Our data provides a green one‐step, economical, and easy process for synthesising AgNPs. It is the first time being reported that Arabidopsis is used for the simple single‐step synthesis of AgNPs. Also, we further observe the distribution of the Ag element and other elements in Arabidopsis, which helps to study the interaction between plants and metals. Compared to the chemical methods, using plants in the recovery of Ag from Ag pollution will be the cost‐effective environmentally compatible method. This biological approach will be beneficial for the Ag recycling.

4 Conclusion

Our study suggests that Arabidopsis has the ability to absorb the silver ions and transform the silver ions into AgNPs. The TEM results show the localisation of AgNPs in plant cells, such as the cell wall, plasma membrane, and cytoplasmic vacuoles. The XRD pattern shows that the AgNPs are crystalline in nature. The 2D‐PIXE analysis investigated the distribution of Ag and other elements (Ca, P, S, Mg, and Cl) in the Arabidopsis plant tissues. It shows that the distributions of Cl and Ag are consistent both in roots and in leaves. Plants like Arabidopsis with a short span of the life cycle can be used for synthesising AgNPs. Our research provides a new and more friendly approach for synthesising the AgNPs.