Round-shape gold nanoparticles: effect of particle size and concentration on Arabidopsis thaliana root growth
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Nowadays, due to a wide range of applications of nanoparticles (NPs) in many industrial areas, accumulations of those entities in environment pose a great risk. Owing to their inertness, noble metal NPs may remain in contaminated soils nearly unchanged for long time. Within this context, size-, shape-, and concentration-dependent uptake of particles by plants belongs to unexplored area. In this work, we present water solutions of biologically friendly synthesized spherical AuNPs with pretty narrow size distribution in size range from 10 to 18 nm. Their thorough characterization by atomic absorption spectroscopy, mass spectroscopy-equipped inductively coupled plasma, dynamic light scattering (DLS), and TEM methods was followed by the study of their effect on the growth of Arabidopsis thaliana (primary and lateral roots), in particle size- and concentration-dependent manner. Due to strictly round-shape form of AuNPs and absence of particle agglomeration, DLS-derived size and size distribution were in good concordance with those obtained from TEM. The length and number of A. thaliana lateral roots were significantly affected by all types of AuNPs. Smallest AuNPs at highest concentration inhibited length of primary roots and, in contrast, enhanced hair root growth.
KeywordsGold nanoparticles Size Concentration Arabidopsis thaliana Root growth
Atomic absorption spectroscopy
Dynamic light scattering
Mass spectroscopy equipped inductively coupled plasma
Murashige and Skoog
Noble metal nanoparticles
Transmission electron microscopy
Nowadays, modern chemistry and engineering churn out huge amounts of nanoobjects in order to improve utility properties of matter not only in special applications but increasingly in products of everyday consumption. Nanostructured materials, i.e., nanoparticles [1, 2], nanorods , nanotubes , nonwoven nanotextiles , as they stand or attached to different kinds of supports, significantly increase yields in almost all areas of industrial applications ranging from cosmetics  and health care , over bioengineering [8, 9], up to energy conversion applications , and catalysts . While the inclusion of nanomaterials in these products can enhance their performance, their breakdown at the end of their useful lifetime provides several key points of entry for synthetic NPs into the environment. Especially engineered NPs, which has been extensively used in catalysts, UV-protective dye stabilizers, antimicrobial agents in textile industry, or health care products and cosmetics (in particular that of high chemical inertness such as Au, Ag, Pt, and Pd) must be given special attention, since they may accumulate in the environment almost unchanged for many years, triggering so far unknown processes upon their plant uptake. Concerning noble metal nanoparticles (NMNPs), some pioneering works has been published on the effect of silver nanoparticles (AgNPs) on seedlings of thale cress (Arabidopsis thaliana), indicating that very low concentrations of AgNPs (< 1 ppm) could be toxic to the seedlings . AgNPs of 20 to 80 nm clearly stunted the growth, and their phytotoxicity is concentration and particle size dependent. The root tip (cap and columella) were observed to turn light brown when primary roots were exposed to AgNPs. The brown tip was attributed to the adsorption of AgNPs either itself or in conjunction with cell wall materials or secondary metabolites produced by root tips. However, the exact mechanism has not been elucidated yet.
Even though there have been some studies addressing the role of NPs in the environment , those targeting on the gold nanoparticles (AuNPs) are still rare . If available, the majority of the published data on nanotoxicology have focused on mammalian cytotoxicity [14, 15, 16] or impacts to animals and bacteria [17, 18, 19, 20], and only a few studies have considered the toxicity of engineered NPs to plants. Additionally, the interaction of NMNPs with plants and other organisms that share similarities with plant cells, such as algae, have been poorly studied so far, implying that the general consequences of NMNPs exposure for plant cells still remains unclear . The lack of these data leads to a defective understanding of how NMNPs are transferred and accumulated in the various food chain levels.
In this work, we report on the effect of gold nanoparticles on plant growth, particularly on the development of primary and lateral roots of A. thaliana in presence of different size particles. AuNPs were synthesized by wet method under biologically friendly protocol using no stabilizers, producing spherical nanoparticles with precise control over their size and size distribution. Before the plant treatment, AuNPs were thoroughly characterized by a broad spectrum of analytical methods (AAS, ICP-MS, DLS, and TEM).
Materials, apparatus, and procedures
Gold nanoparticles were synthesized by a slightly adapted procedure published by Batús et al. . Briefly, 149 mL of water was heated in a 250 mL two-necked round-bottomed flask until it starts to reflux. Then, 1 mL of 0.33 M sodium citrate and 0.945 mL of 10 mg/mL of potassium tetrachloroaurate(III) in water were subsequently added. After 30 min, heating was stopped and the reaction mixture was left to get cold. In all preparation experiments, Milli-Q water (18.2 MΩ at 25 °C) was used.
For root assays of A. thaliana, synthesized AuNPs of three different sizes (10, 14, and 18 nm) were centrifuged at 5000g for 1 h to increase particle concentration up to limit value of 2000 mg/L.
A. thaliana Columbia (Col-0) seeds (obtained from Lehle seeds, USA) were surface sterilized with 30% (v/v) bleach solution for 10 min and rinsed five times with sterile water. Sterile seeds were sown on agar plates containing ½ Murashige-Skoog (MS) medium and 1% plant agar (pH 5.8). To synchronize seed germination, the agar plates were kept at 4 °C for 2 days. A. thaliana plants were grown for 5 days in vertically oriented plates in a growth chamber at 22 °C with 100 μmol m− 2 s− 1 light intensity under long day conditions (16 h/8 h light/dark cycle).
Five-day-old seedlings of similar size were transferred to agar plates (20 plants per plate) containing 1/16 MS medium, different concentrations of AuNPs (0, 1, 10, and 100 mg/L) and 1% plant agar (pH 5.8). AuNPs were added to the medium after autoclaving. As a control, the effect of sodium citrate buffer was also investigated. The length of the root was marked, and seedlings were grown for the next 5 days. Both, increment of the primary root and lateral roots length were measured using JMicroVision 1.2.7 software.
Prepared solutions of AuNPs were characterized by Atomic Absorption Spectroscopy (AAS), mass spectroscopy-equipped inductively coupled plasma (ICP-MS), dynamic light scattering (DLS), and transmission electron microscopy (TEM).
Concentrations of prepared NPs were determined by means of AAS by a VarianAA880 device (Varian Inc., USA) using a flame atomizer at 242.8 nm wavelength. Typical uncertainty of concentration determined by this method is less than 3%.
Inductively coupled plasma with mass spectroscopy detector (ICP-MS) was used to determine the concentration of Au ions originating from unreacted Au source chemical, using Agilent 8800 triple-quadrupole spectrometer (Agilent Technologies, Japan) connected to an auto-sampler. AuNPs colloid solution was pipetted into 1.5 mL hydrophobic microtubes and centrifuged at 30000g on Eppendorf 5430 centrifuge for 1 h. After the centrifugation, 0.3 mL of supernatant was carefully removed using pipette and ICP-MS analyzed. Sample nebulization was performed using a MicroMist device equipped with a peristaltic pump. Pure buffer solution (2.2 mM sodium citrate) was used as a blank sample. The uncertainty of the measurement was less than 3%.
TEM images were measured using JEOL JEM-1010 (JEOL Ltd., Japan) operated at 400 kV. Drop of colloidal solution was placed on a copper grid coated with a thin amorphous carbon film on a filter paper. The excess of solvent was removed. Samples were air dried and kept under vacuum in a desiccator before placing them on a specimen holder. Particle size was measured from the TEM micrographs and calculated by taking into account at least 500 particles.
The particle size distribution was determined by Zetasizer ZS90 (Malvern Instruments Ltd., England) in the DLS regime for particle size distribution, equipped with an avalanche photodiode for signal detection. Diode-pumped solid state laser (50 mW, 532 nm) was used as a light source. The measurements were performed in polystyrene cuvettes at room temperature.
Results and discussion
The size and size distribution of AuNPs obtained by TEM and DLS analysis together with concentrations of AuNPs (AAS) and Au ions (ICP-MS)
NP size (nm)
Concentration of AuNPs/Au ions (μg/L)
AuNPs (× 10−3)
11.8 ± 2.7
9.6 ± 3.7
30.2 ± 0.9
339.8 ± 27.4
1.6 ± 0.3
15.1 ± 3.8
14.3 ± 3.9
29.8 ± 0.8
328.6 ± 24.1
2.1 ± 0.5
19.6 ± 3.2
18.1 ± 4.1
29.8 ± 0.8
314.9 ± 29.9
1.8 ± 0.4
Wang et al.  found that during a 3-day hydroponic exposure of A. thaliana to either Ag+ and AgNPs (5 nm) at the same concentration, the Ag concentration decreased faster in the Ag+-treated solution than in the AgNP one, indicating faster uptake of Ag+ ions. Therefore, we took special attention to minimize the possible effect of Au ions in result distortion. As-synthesized AuNPs were centrifuged up to limit concentration of 2000 mg/L and diluted with MS medium to required concentrations (1, 10, and 100 mg/L). After this procedure, the concentration of residual Au ions in solutions containing 100 mg/L of AuNPs was determined by ICP-MS (see Table 1). Apparently, the centrifugation had positive effect on both the presence of residual Au ions which concentrations were reduced by two orders of magnitude compared to as-synthesized solutions (Table 1, AAS) and the content of citrate buffer itself.
Adaptation of nanoparticle solutions to meet plant biology conditions
Due to strong aggregation tendency of AuNPs in plant growth media (MS medium), commonly used for plant growth in vitro , we had to optimize mutual ratio of these two components to avoid NPs aggregation while preserving acceptable conditions for plant growth. Different dilutions of MS were tested. Aggregation of AuNPs was easily visible by the change of the color (transition from red to purple). Due to low initial concentration of as-synthesized AuNPs (around 30 mg/L, see Table 1), insufficient for biological experiments, it was necessary to increase the NP concentration by centrifugation. By this procedure, we increased particle concentration up to the limit value of 2000 mg/L. Such NPs solutions were then diluted to final concentrations, which also decreased the concentration of the citrate buffer in our experiments. For growing experiments of A. thaliana, we diluted AuNPs in 1/16 MS medium. Minimal NPs aggregation was detectable in this medium and NPs were much more stable in comparison to 1/2 MS, 1/4 MS, and 1/8 MS with nearly unaltered plant growth. Concentration of AuNPs in final solutions was determined by AAS. Since plant growth experiments were performed in in vitro (in sterile conditions), the effect of sterilization procedure on NPs was studied too. Commonly used autoclaving (121 °C, 20 min) of prepared growth media caused complete aggregation of investigated NPs. Therefore, this procedure was not suitable for our experiments. Addition of NPs into about 60 °C autoclaved agar medium was finally used as an alternative procedure in which no NP aggregation was detected and sterilizing process was still effective.
Effect of NPs on the root growth of Arabidopsis thaliana in vitro
We have successfully prepared gold nanoparticles by gentle two-component (sodium citrate–potassium tetrachloroaurate) reduction in water environment providing round-shaped, narrow-distributed particles with excellent control over their resulting size. Post-synthesis centrifugation allowed to reach the desired NPs concentrations and eliminated the influence of ions and citrate buffer on results distortion in plant experiments. The effect of AuNPs of different sizes (10, 14, and 18 nm in diameter) and concentrations (1, 10, and 100 mg/L) on root growth of A. thaliana was investigated. The number and length of lateral roots were significantly decreased after the treatment with NPs solutions of higher particles concentrations, regardless of their specific size. A negative effect on primary root growth was observed in the case of 10 nm AuNPs. Surprisingly, the smallest AuNPs (10 nm) clearly induced root hair growth. Overall, this study showed that the direct exposure of plants to AuNPs significantly contributed to phytotoxicity and underscores the need for eco-responsible disposal of wastes and sludge containing Au nanoparticles.
The financial support of this work from the GACR projects nos. 17-10907S and 108/12/G108 (VS) is gratefully acknowledged.
JS conducted NP analyses and wrote the manuscript. KZ accomplished the synthesis of AuNPs. VS contributed to AuNP analysis evaluations. KK and JM designed and conducted all A. thaliana experiments. LB designed experiments and critically read the manuscript. All the authors read and approved the final manuscript.
JS is a senior researcher at the Department of Solid State Engineering, holding a Ph.D. from material engineering. KZ is a senior researcher at the Department of Analytical Chemistry, holding a Ph.D. from analytical chemistry. VS is the head of the Department of Solid State Engineering and a professor in the field of material engineering. JM and LB are senior researchers at the Institute of Experimental Botany of the Czech Academy of Sciences. JM holds his Ph.D. from plant physiology. LB holds Ph.D. from plant protection. KK is a young researcher at the Laboratory of Signal Transduction of the same institute.
The authors declare that they have no competing interests.
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