Cytokinin response in pepper plants (Capsicum ...

Environmental Research 156 (2017) 10–18

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Cytokinin response in pepper plants (Capsicum annuum L.) exposed to silver nanoparticles

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Tomislav Vinkovića, Ondrej Novákb, Miroslav Strnadb, Walter Goesslerc, Darija ⁎ Domazet Jurašind, Nada Parađikovića, Ivana Vinković Vrčeke, a

Faculty of Agriculture in Osijek, J.J. Strossmayer University of Osijek, Trg Svetog Trojstva 3, 31000 Osijek, Croatia Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University & Institute of Experimental Botany AS CR, Šlechtitelů 27, 78371 Olomouc, Czechia c Institute for Chemistry, University of Graz, Universitätsplatz 1, 8010 Graz, Austria d Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, 10 000 Zagreb, Croatia e Institute for Medical Research and Occupational Health, Ksaverska cesta 2, 10001 Zagreb, Croatia b

A R T I C L E I N F O

A BS T RAC T

Keywords: Abiotic stress Cytokinin response Silver nanoparticles Pepper Uptake Biodistribution

The increasing development of different nanomaterials, such as silver nanoparticles (AgNPs), and their practical use in agriculture and biotechnology has created a strong need for elucidations of biological effects and risk assessments of AgNPs in plants. This study was aimed to investigate AgNPs effects on metal uptake and their biodistribution in pepper plants as well as on morphological parameters and hormonal responses of the isoprenoid cytokinin (CK) family. In addition, the comparison of effects silver form, nanoparticles vs. ionic, has also been examined. To the best of our knowledge, this is the first study describing CK responses in plants exposed to metallic NPs. The obtained results indicate that both AgNPs and Ag+ ions significantly increased total content of Ag+ in pepper tissues in a dose-dependent manner and affected on plant development by decreasing both plant height and biomass in a similar way. This study evidenced for the first time the role of CKs in abiotic stress in plants caused by AgNPs. The hormonal analysis, conducted by an ultra-high performance liquid chromatographyelectrospray tandem mass spectrometry, revealed a significant increase in total CKs in the leaves and also highlighted the importance of cis-zeatin type CKs in plants treated with AgNPs. Our observations suggest potential risks of AgNPs on plant ecosystems upon their release into the environment.

1. Introduction Engineered nanomaterials (ENMs) have been developed and expanded for improving many industrial sectors, including consumer products, pharmaceutics, biomedicine, water purification, wastewater treatment, food processing, packaging, energy, etc. (EC, 2013). ENMs are also being increasingly developed to provide new solutions to problems in agriculture such as plant disease control, slow pesticide decay or novel diagnostic tools (González-Melendi et al., 2008; Monica and Crenomini, 2009; Le et al., 2014). Among the various types of ENMs, silver nanoparticles (AgNPs) have been extensively studied owing to their biocidal activity (EC, 2014; Eckhardt et al., 2013). In agriculture, AgNPs have been investigated as plant-growth stimulators (Steinitz and Bilavendran, 2011; Monica and Crenomini, 2009), fungicides to prevent rot, mold and other fungal diseases (Alavi and Dehpour, 2009), or agents for enhancement of fruit ripening and

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abscission and oil recovery (Seif Sahandi et al., 2011). In order to properly evaluate benefits of nanotechnological applications in agriculture, the first step should be to analyze the biological effects of NPs on plant growth and development. Other possible ways for terrestrial plant's exposure of to NPs include wastewater effluent discharge, leaching from different nanoproducts, use of ENMs for environmental remediation, irrigation using contaminated surface water, land application of contaminated biosolids and many others (Pokhrel and Dubey, 2013). There is now extensive debate about the risks and benefits of the ENM on ecosystems and human health (EC, 2014). Despite the abundance of published toxicological studies on AgNPs, information about the biological effects of AgNPs on higher plants is still lacking. Most studies evaluated the uptake and accumulation of AgNPs (Pokhrel and Dubey, 2013; Gardea-Torresdey et al., 2003; Lin and Xing, 2008; Judy et al., 2011; Rico et al., 2011; Yin et al., 2011), their biodistribution (Lin and Xing, 2008), their effects on plant

Corresponding author. E-mail address: [email protected] (I.V. Vrček).

http://dx.doi.org/10.1016/j.envres.2017.03.015 Received 10 October 2016; Received in revised form 9 March 2017; Accepted 10 March 2017 0013-9351/ © 2017 Elsevier Inc. All rights reserved.


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measured at 25 °C by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) methods using a Zetasizer Nano ZS (Malvern, UK) equipped with a 532 nm “green” laser (Vinković Vrček et al., 2015). All data processing was done by Zetasizer software 6.32 (Malvern instruments). DLS measurements were performed at a backscatter angle of 173° in order to reduce the extent of multiple scattering as well as the effects of dust. The hydrodynamic diameters (dH) and the size distributions of silver nanoparticles were obtained from the volume size distributions. The results are reported as an average value of 10 measurements. The AgNPs charge was evaluated by the ELS method. Results on the electrophoretic ζ potential of AgNPs are reported as an average value of 5 measurements. In addition, particles were visualized using a transmission electron microscope (TEM, Zeiss 902 A). The microscope was operated in bright field mode at an accelerating voltage of 80 kV. Images were recorded with a Canon PowerShot S50 camera attached to the microscope. TEM samples were prepared by depositing a drop of the sample suspension on a Formvar®coated copper grid. Samples were air-dried at room temperature. The silver dissolution in UPW and TWW was set by determining the dissolved silver ions using centrifugal ultrafiltration (Millipore Amicon Ultra-4 3K) through a membrane with a nominal molecular weight limit (NMWL) of 3 kDa. Suspensions were centrifuged for 30 min at 15000g (Eppendorf Microcentrifuge 5417R, Eppendorf AG, Hamburg, Germany). The concentration of the dissolved Ag+ in the filtrate, and the total Ag concentration before ultrafiltration were determined by ICPMSto calculate the extent of AgNPs dissolution.

phenotype (e.g., root/shoot length, biomass) (Pokhrel and Dubey, 2013; Lin and Xing, 2007; Rico et al., 2011), seed germination (Pokhrel and Dubey, 2013), and DNA damage (Atha et al., 2012). Recent studies have also revealed that AgNPs damage root cells, impair plant growth and seed germination, and affect leaf transpiration, root elongation, and plant biomass (Dimpka et al., 2013). It is well-known that AgNPs release Ag+ ions which contribute to their biological effects (Dimpka et al., 2013). Metal (Cd, Hg, Pb, Cr, As, Zn, Cu, and Ni) toxic effects has been well demonstrated in plants (Hossain et al., 2012). Exposure to heavy metals is one of the major abiotic stresses altering major plant physiological and metabolic processes, like photosynthesis and respiration, leading to reduced plant growth and yields (Hossain et al., 2012; Sharma and Dietz, 2009). Plants have various efficient response systems to cope with environmental stress signals or to adapt to new conditions. The key components of these defence and adaptation mechanisms are plant hormones (O’Brien and Benková, 2013). In recent years, exhaustive research has been focused on plant hormone-mediated tolerance to stress (O’Brien and Benková, 2013). The plant hormones cytokinins (CKs), essential for plant growth and development (Ha et al., 2012; Macková et al., 2013; Vescovi et al., 2012), have also been recognized to play an important role during plant acclimation to stress conditions at various levels (Ha et al., 2012; O’Brien and Benková, 2013; Macková et al., 2013). The levels of CKs in plants are controlled by de novo synthesis, conversion between free bases, nucleosides, and nucleotides, and by their inactivation, degradation, and translocation (Cedzich et al., 2008). Comparative investigations of the biological impacts of AgNPs vs. Ag+ application on pepper plants have never been carried out. The objective of this research was to investigate the effect of the nanoparticulate and ionic form of silver on (a) growth and morphological parameters of pepper plants, (b) uptake and biodistribution of silver, and (c) endogenous CKs levels in pepper plants. All natural cytokinin groups have been examined including free bases, ribosides, nucleotides, and glucosides of isopentenyladenine (iP), trans-zeatin (tZ), dihydrozeatin (DHZ), and cis-zeatin (cZ). Pepper (Capsicum annuum L.) was selected as a member of the Solanaceae family due to its similarity to Lycopersicon esculentum Mill., a recommended by US EPA test plant (USEPA, 1996). To the best of our knowledge, this is the first study describing CK responses in plants exposed to metallic NPs.

2.2. Plant material and growing conditions Seeds of sweet pepper (Capsicum annuum L.) hybrid Vedrana F1 were purchased from Enza Zaden Beheer B.V. (Enkhuizen, Netherland) and kept in the dark at 4 °C until use. The experiments were carried out in the greenhouse conditions without heating during 2013 year in Osijek, Croatia. The seeds were sown to commercial substrate Brill Typ 3 (Gebr. Brill Substrate GmbH & Co) in polystyrene containers. According to the manufacturer, the composition of substrate is 65% white and 35% black peat, and it is intended for the production of pepper and tomato transplants. It is characterized by pH of 5.5 – 6.0 and contains 500 g of NPK fertilizer/m3. The total salt content of substrate is 0.3–0.8 g/L. The substrate in each container was saturated with TWW before plant sowing. Each variant consisted of 30 pepper plants grown in the containers with three replications of each experiment. During the first 15 days after sowing (DAS), the containers were watered with TWW daily. The treatments started on the 15th DAS when the plants emerged and formed roots big enough to reach the bottom of the container. At this time, each container was placed into a separate vessel containing 2 L of well water (control plants) and/or 2 L of AgNPs or AgNO3 solutions (in TWW) at five different concentrations (0.01, 0.05, 0.1, 0.5 and 1 mg/L). Thus, the experiment consisted of eleven different variants. The fresh solutions of water were prepared every day for treatment of plants grown in the containers in order to diminish the effects of sedimentation, sorption to containers, agglomeration, and dissolution behaviour of AgNPs during experiment. The pepper plants were grown until they developed 6–7 true leaves and formed the first flower buds. On the 47th DAS, the fresh biomass of overground part of plants was cut at the substrate surface, identified, sorted and weighed. The roots were taken out of the soil substrate, carefully washed with distilled water, surface dried with a filter paper and their fresh biomass determined. At first, the 3rd and 4th leaves from each of the plants per variant were collected and separated in 3 discrete samples (from each container). Each sample was immediately frozen in liquid nitrogen, ground and lyophilized. Samples were stored at −70 °C until extraction and analysis of endogenous cytokinins. The rest of plant biomass (leaves, stems and roots) were separated in 3 discrete samples for each replicate of each variant, placed into paper bags, then oven dried at 80 °C for 48 h and then dry biomass was

2. Materials and methods 2.1. Synthesis and characterisation of AgNPs Citrate-coated AgNPs were synthesized according to Dementéva et al. (2008). Briefly, 90 mg of AgNO3 were dissolved in 500 ml of ultrapure water (UPW). The solution was brought to a boil under vigorous stirring. Afterwards, 7.5 ml of 10% (w/v) aqueous trisodium citrate solution was quickly added into one portion to the 500 ml of boiled AgNO3 solution. The reaction mixture was boiled for 4 h at 80 °C. The final molar ratio of AgNO3/Na3C6H5O7*2H2O was determined to be 1 mM: 5 mM. Immediately after synthesis, freshly prepared NP suspensions were washed twice with UPW after centrifugation at 15.790g for 20 min. The washed AgNPs were resuspended in UPW using ultrasound, and stored in the dark at 4 °C until use. Total silver concentration in AgNPs colloidal suspension was determined in solutions acidified with 10% (v/v) HNO3 using an Agilent Technologies 7.500cx inductively coupled plasma mass spectrometer (ICP-MS) (Waldbronn, Germany) as described elsewhere (Vinković Vrček et al., 2015). Careful characterisation of AgNPs was conducted at concentrations of 1 mg Ag/L in both ultrapure water (UPW) and sterilized tap water used for the plant watering (TWW) - to check whether there is a timedependent agglomeration of NPs in two different media, since the properties of agglomerated and individual NPs may differ significantly (Vinković Vrček et al., 2015). The size and charge of NPs were 11


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ribosides, N-glucosides, 0.5 pmol of CK O-glucosides and nucleotides per sample added (Novák et al., 2008). The extracts were purified using two solid phase extraction columns, the C18 octadecyl silica-based column (500 mg of sorbent, Applied Separations) and after that the Oasis MCX column (30 mg/L ml, Waters). Analytes were eluted by two-step elution using a 0.35 M NH4OH aqueous solution and 0.35 M NH4OH in 60% v/v CH3OH solution. The samples were evaporated to dryness, dissolved in 30 µl of the mobile phase and analysed by an ultra-high performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS) using ACQUITY UPLC® System (Waters, Milford, MA, USA) linked to a Xevo™ TQ-S triple-quadrupole mass spectrometer (Waters, Manchester, UK). Samples were injected on a C18 reverse-phase column (Waters ACQUITY UPLC® BEH C18, 1.7 µm, 2.1×50 mm), and elution was performed with a methanolic gradient composed of 100% methanol (A) and 15 mM formic acid (B) adjusted to pH 4.0 with ammonium (Svačinová et al., 2012). Determination of the different cytokinins was performed in multireaction monitoring mode with optimized conditions (cone voltages, collision energies in the collision cell, dwell times) corresponding to the exact diagnostic transition for each cytokinin. Quantification was performed by Masslynx software using stable isotope-labelled internal standards as a reference (Novák et al., 2008). Analyses of three independent biological replicates were performed.

weighed. These dry samples were used for analysis of the total Ag concentrations as described in the next section. 2.3. Quantification of silver in pepper plant samples The total Ag content in the dried leaves, stems and roots of control and treated pepper plants was measured by inductively coupled plasma mass spectrometry (ICPMS) after microwave assisted acid digestion (Vinković Vrček et al., 2015). An Agilent Technologies 7500cx ICPMS system (Agilent, Waldbronn, Germany) equipped with an integrated auto-sampler, a Scott Quartz spray chamber and a MicroMist nebulizer (Glass expansion, Australia) were used. The interface cones were the standard items, made of Ni. Operating conditions were normal for general and high matrix analysis, recommended by Agilent Technologies. The instrument settings and measurement parameters along with a complete listing of the scanned isotopes are detailed in Table S1 of Supplementary Materials. The instrument was operated in an air-conditioned laboratory (20–22 °C) equipped with a filter to remove dust particles. The instrument was tuned daily with an ICP-MS tuning solution (Agilent Technologies, Japan) containing 10 μg/L of lithium, yttrium, cerium, thallium and cobalt in 2% (w/v) HNO3 to achieve higher or compromised intensities and lower yields for both oxide ions and doubly charged ones. Calibration standards were prepared daily from stock elemental standard solutions of 1000 mg/L from Merck (Darmstadt, Germany). The ICP-MS system was calibrated by the method of external standards with Rh and Lu as the internal standards. Both samples and standards were enriched with the ‘internal standard stock solution’ to a final concentration of 10 μg/L. For the purpose of contamination control, each series of measurements included a reagent blank. Each calibration curve was constructed linearly through zero after subtraction of the reagent blank. Standards and blanks were subjected to the same treatment as the pepper samples. Verification of the accuracy of the proposed method was performed using the following Standard Reference Materials (SRMs): NIST 1573a (Tomato leaves) from the National Institute of Standards and Technology (NIST, U.S.) and Certified Reference Material No.9 (Sargasso) from the National Institute for Environmental Studies (NIES, Japan). Digestion of pepper samples and SRMs was performed in closed vessels with the microwave UltraCLAVE IV Milestone digestion device (MLS GmbH Mikrowellen-Laborsysteme, Leutkirch, Germany) by addition of 5 ml of Suprapur 65% HNO3 (Merck, Darmstadt, Germany) to accurately weighed 0.25 g of pepper samples in a quartz digestion vessels and the mixture was irradiated at 800 W and 120 °C for 10 min, followed by irradiation at 1600 W and 180 °C for 30 min. The method resulted in total and simultaneous dissolution of pepper samples to colourless solutions. A set of digestion blanks was also prepared and subjected to the same microwave procedure. After the vessels had cooled, deionised water was added to each vessel to a final volume of 50 ml. The overall dilution was 200 v/m and the final solution for the ICP-MS analysis contained 10% v/v HNO3. Obtained values for total Ag content in dry matter of the leaves, stems and roots of pepper plants were used to calculate bioaccumulation factor (BF) and translocation factor (TF). The BF was calculated as % of applied Ag found in dry matter of plant organs, while the TF was determined as the ratio between Ag content in upper parts of plants (leaves and stems) and Ag content in roots.

2.5. Statistical analysis The data are reported in tables and figures as means with standard deviations in parenthesis and error bars, respectively. Statistical analysis was performed using the SAS 9.0 statistical package (SAS Institute, Cary NC). To determine the difference between the treatments, two-way analysis of variance (ANOVA) with interaction was used (main factors: concentration (C) and silver form (F), with interaction concentration (C) × silver form (F)), followed by the Tukey post hoc HSD test. Differences were considered statistically significant at P < 0.05. Since one control group was measured for both treatments (corresponding to the concentration of 0 mg Ag/L), control group was not included in the factorial ANOVA analysis, as it would not be possible to estimate the interaction between the factors of group and concentration. Thus, to determine the differences between control and the treatments, t-test was used with Bonferroni correction for multiple comparisons (P < 0.005). 3. Results and discussion 3.1. Characterisation of AgNPs The careful characterisation and stability evaluation of NPs are critical for an interpretation of experimental results involving NPs as well as for understanding their behaviour in biological systems. Depending on their characteristics and the characteristics of the media (i.e. pH, ionic strength, concentration of biomolecules and redox conditions), NPs will either diffuse or aggregate within certain biological media (Vinković Vrček et al., 2015; Auffan et al., 2009). The aggregation behaviour of AgNPs will modify the effective doses leading to specific surfaces and concentrations that are usually very different from those of the original AgNP dispersion. Thus, the characterisation and stability evaluation of NPs has to be the first step in their evaluation, which is then followed by their biological testing and risk assessment. Table 1 shows the hydrodynamic diameter (dH), ζ potential and polydispersity index (PdI) of citrate-coated AgNPs used in this study after 24 h upon dispersion in UPW and TWW. DLS measurements of AgNPs in UPW showed that the volume size distribution was bimodal, with smaller particles (12.9 ± 9.1 nm), being dominant (90%), while only 9% of the whole AgNP population consisted of bigger particles (87.6 ± 41.7 nm).

2.4. Quantification and identification of endogenous cytokinins Pepper leave samples were analysed for free endogenous cytokinins using a modified protocol described by Svačinová et al. (2012). Samples (20 mg FW) were homogenized and extracted in 1 ml of modified Bieleski buffer containing 60% CH3OH, 10% HCOOH and 30% H2O, together with a cocktail of stable isotope-labelled internal standards (Olchemim Ltd., Czech Republic) containing 0.25 pmol of CK bases, 12


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gated AgNPs in TWW (Fig. 1b). However, individual particles invisible under DLS measurement were observed. In order to estimate the stability of AgNPs regarding dissolution, the release of Ag+ in both UPW and TWW was evaluated using centrifugal ultrafiltration and ICPMS measurements. Dissolution experiments showed that free Ag+ was lower than 1.5% of the total Ag in citrate-coated AgNPs suspended in UPW after 24 h, while total Ag was even lower than 0.5% in TWW. This implies that Ag+ release was not occurred in TWW. However, we were not able to determine Ag+ in plants treated with AgNPs. The dissolution of AgNPs in TWW determined in this study is comparable to our previously published data (Milić et al., 2015; Vinković Vrček et al., 2015), and lower than results reported by Yin et al. (2011) and Dimpka et al. (2013).

Table 1 Hydrodynamic diameter (dH), zeta potential (ζ) and polydispersity index (PdI) of citratecoated silver nanoparticles in ultrapure water (UPW) and well water used for pepper plant (TWW). dH (nm) (% of mean volume)

ζ (mV)

PdI

UPW

12.9 ± 9.1 (90%) 87.6 ± 41.7 (9%)

−16.9 ± 0.6

0.3

TWW

74.2 ± 19.1 (14%) # 328.2 ± 156.3 (78%)

−0.8 ± 0.1

0.6

#

#

#

Note The values are given as mean ± SD. If values in the TWW are marked with #, the characteristic of nanoparticles in the TWW is significantly different corresponding parameter in the UPW according to the t-test (P < 0.005).

3.2. Uptake and biodistribution of total Ag in pepper plants Main goal of the biological experiments was to investigate the effect of citrate-coated AgNPs on pepper plants grown in substrate under greenhouse condition. The biological effect of the ionic Ag in the form of AgNO3 was evaluated for comparison. Considering that the predicted environmental concentrations of AgNPs in different environmental conditions are in the range between 5 ng/kg to 10 mg/kg, but do not exceed 10 mg/kg (Fabrega et al., 2011), this study tested AgNPs and Ag+ concentrations in the range 0.01–1 mg/L. Fig. 2 shows total Ag contents in leaves, stems and roots of pepper plants treated with increasing concentrations of AgNPs and Ag+(in the form of AgNO3), respectively. Both treatments increased total Ag concentration in pepper tissues in a dose-dependent manner. There was no significant difference of Ag uptake between treatments with the two Ag forms up to the concentration of 0.1 mg/L. The present results clearly show that both Ag forms can lead to the accumulation of Ag in different pepper tissues in a similar manner. The calculated bioaccumulation factors (BF), i.e. % of applied Ag found in dry matter of plant organs, were highest, as proposed, for roots of treated pepper plants (Fig. 2d). When pepper plants were treated with 0.5 and 1 mg/L of Ag+, the silver content in leaves, stems and roots became almost twice as high compared to AgNPs-treated plants (Fig. 2a-c). This difference could be explained by either the binding of AgNPs to roots or the aggregation behaviour of AgNPs. AgNPs aggregated probably more easily and faster with increased concentrations leading to slowing their transport. The calculated translocation factor (TF), determined as ratio between Ag levels in upper parts of plants (leaves and stems) and Ag levels in roots, was also dose-dependent (Fig. 2d). TF was lowered with increasing AgNPs and Ag+ concentration, indicating that higher Ag level, both in the nano or ionic form, inhibited Ag uptake. More interestingly, TF was twice as high for peppers treated with 0.01 mg/L AgNPs in comparison to treatment with 0.01 mg/L Ag+. This is in contrast to studies on maize, which showed significantly higher Ag+ uptake compared to AgNPs (Pokhrel et al., 2013). The reason for such differences could be due to several orders of magnitude higher AgNPs and AgNO3 concentrations used in the experiment with maize compared to our study. The results obtained here are in accordance with recently published effects of AgNPs on common ryegrass (Lolium multiflorum) (Yin et al., 2011) and zucchini (Cucurbita pepo L.) (Stampoulis et al., 2009), which clearly indicated that plants can easily uptake and accumulate silver NPs.

Fig. 1. Transmission electron micrographs (TEM) of citrate-coated silver nanoparticles in (a) ultrapure water (UPW) and (b) water used for pepper plant watering (TWW). Scale bars are in 100 nm.

TEM analysis (Fig. 1a) confirmed these results and revealed the presence of non-uniformly shaped NPs. The ζ potential value equal to −16.9 ± 0.6 mV indicated electrostatic stabilization of AgNPs due to the ionization of the polar citrate carboxyl groups on the surface of the NPs. Upon suspension in TWW, the substantial aggregation of the AgNPs occurred (Table 1). Smaller AgNPs became less populated in TWW, while the dH of larger AgNPs increased to the value equal to 328.2 ± 156.3 nm after 24 h. In the case of electrostatically stabilized citrate-coated AgNPs, higher ionic strength of TWW affected both the collapse of the diffuse layer of citrate anions around the particle and the frequency of particle-to-particle interactions leading to an aggregation. ELS results also supported this conclusion (Table 1). It is clear that the absolute value of ζ potential for AgNPs dispersed in TWW significantly decreased compared to UPW. When the ζ potential approaches zero, interparticle repulsion decreases as well as the stability of the AgNPs dispersion (Fornes, 1985). TEM consistently showed the presence of flocculated and aggre-

3.3. The effects of AgNPs and Ag+ on pepper plant growth For most metallic NPs, relatively high concentrations are needed to cause observable toxicity on plants (Lin and Xing, 2007; Lee et al., 2008). For AgNPs, reported studies have consistently showed that they affect plant growth harmfully (Yin et al., 2012; Pokhrel and Dubey, 2013; Stampoulis et al., 2009). However, the applied AgNPs concentrations (total Ag applied) were far above the predicted AgNPs environmental concentrations (Fabrega et al., 2011). 13


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Fig. 2. Uptake and distribution pattern of silver in tissues of pepper plants treated with AgNPs and Ag+. Silver concentrations, given in mg Ag/g of dry weight (DW), in (a) roots, (b) stems, and (c) leaves of control and treated pepper plants. Note. The values are given as mean ± SD. If values are marked with *, AgNP treatment is statistically different compared to the same concentrations of Ag+ treatment according to the Tukey's HSD test. If values are marked with #, the treatment is significantly different than control according to the t-test (P < 0.005). Bioaccumulation and translocation factors (d) are given in %. Bioaccumulation factors (BF) for roots, stems and leaves were calculated as ratio between found Ag levels in a plant organ and used Ag concentrations in a particular treatment. Translocation factors (TF) for leaves were calculated as ratio between Ag levels in leaves/stems and Ag levels in roots. Table 2 Effect of silver nanoparticles (AgNP) and ionic silver (Ag+) concentrations on plant height and fresh weights (FW) of leaves, stems and roots of pepper transplants. Variant

Ag

FW (g/plant)

Plant height (cm)

(mg/L)

Root

Stem

Leaves

Control

0.0

2.28 ± 0.08

2.43 ± 0.25

3.73 ± 0.66

15.83 ± 0.16

AgNPs

0.01 0.05 0.1 0.5 1

1.73 ± 0.15# 1.29 ± 0.09#* 1.72 ± 0.14# 1.74 ± 0.16# 2.01 ± 0.04

2.03 ± 0.27 1.39 ± 0.15# 1.55 ± 0.06#* 1.69 ± 0.10#* 1.44 ± 0.13#

3.18 ± 0.40 2.26 ± 0.10# 2.75 ± 0.24 2.84 ± 0.12* 2.53 ± 0.12#

14.56 ± 1.34 12.14 ± 1.10# 12.25 ± 0.50#* 13.73 ± 0.49* 12.06 ± 0.69#

Ag+

0.01 0.05 0.1 0.5 1

2.11 ± 0.34 1.64 ± 0.05# 1.51 ± 0.32# 1.49 ± 0.24# 1.67 ± 0.11#

1.53 ± 0.12# 1.65 ± 0.16# 1.29 ± 0.03# 1.23 ± 0.08# 1.54 ± 0.01#

2.67 ± 0.29# 2.61 ± 0.33# 2.28 ± 0.07# 2.22 ± 0.18# 2.56 ± 0.30#

11.43 ± 0.68# 13.31 ± 0.49# 10.96 ± 0.38# 10.99 ± 0.56# 12.65 ± 0.34#

Note. The values are given as mean ± SD. If values are marked with *, AgNPs treatment is statistically different compared to the same concentrations of Ag+ treatment according to the Tukey's HSD test. If values are marked with #, the treatment is significantly different than control according to the t-test (P < 0.005).

clear dose-dependency, probably due to the very low doses of AgNPs and Ag+ applied in this study. Recent plant studies applied quite high AgNP doses accounting more than 5 mg/L of AgNPs for common grass, (Yin et al., 2011), more than 100 mg/L of AgNPs for sorghum and rice (Lee et al., 2012; Mazumdar et al., 2011), 20–100 mg/L of AgNPs for Lolium perenne and barley, respectively (El-Temsah et al., 2012). Consequently, our results shed new light on NP toxicity showing that vascular plants are susceptible also to very low doses of AgNPs.

This study shows that exposure to Ag used in concentration of 0.05 mg/L or lower levels of AgNPs and Ag+ significantly decreased pepper plant biomass in a dose dependent manner (Table 2). Leaves were more sensitive to treatments than roots, as their biomass decreased by 29–42%, whereas roots decreased by 18–38% compared to control. However, the roots of the treated plants were brownish, while the colour of the control roots remained white. Interestingly, there was no significant difference between AgNPs and Ag+ treatments at the highest dose of 1 mg Ag/L indicating that the toxicity thresholds were identical (Table 2). A consistent finding was that the AgNPs and Ag+ significantly altered plant height (Table 2) decreasing it by at least 15% compared to control. The decrease in plant height and plant biomass did not show a

3.4. The effect of AgNPs and Ag+ on endogenous cytokinin levels in pepper leaves Different growth and uptake assays give valuable information but 14


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AgNPs treatments and control were observed only for cZ and iP types of CKs (Fig. 4). The cZ9G and iP9G were significantly higher in leaves of all treated peppers compared to controls, the higher levels were observed for lower AgNPs and Ag+ treatment concentrations (Tables S1-S4, Supporting materials). Total DHZ was higher in treated than in control lants, but not significantly increased for each AgNP or Ag+ concentration (Fig. 4d). Only the tZ type of N-glucosides did not show any significant changes or trend upon treatment (Fig. 4b). The concentrations of the physiologically active free bases in pepper leaves followed the order: iP > tZ > cZ > DHZ (Fig. 5). The contribution of free bases to the CK pool in the pepper leaves was rather low, below 7 pmol/g of FW (Fig. 5). Similarly to the behaviour of CK types of N-glucosides, cZ and DHZ free bases significantly changed in the treated pepper leaves compared to controls. It is interesting that AgNPs treatments induced a significant increase of only tZ free bases in treated compared to control pepper leaves, while no significant changes in the levels of cZ and iP free bases were observed between treated and control plants (Fig. 5). Only the highest AgNPs amount (1 mg/L) increased DHZ free bases (Fig. 5d). It should be emphasized here that tZ free bases were the only form of tZ CK types for which a significant increase was observed in the treated peppers compared to control (Fig. 5a). However, their contribution to the total pool of tZ types of CKs is rather small (Table S2, Supporting materials). The ribosides of cZ (cZR) and iP (iPR) types were observed at 80and 20-times higher concentrations, respectively, than the corresponding free bases. The tZ ribosides (tZR) were found to be below 1.6 pmol/ g of FW, while DHZ ribosides were not detectable. Levels of tZR, cZR and iPR did not follow dose-response trend after AgNPs or Ag+ treatment (Fig. 6). For cZR, increased levels were found in AgNPs treatment, but significant only for 0.05 and 1 mg/L of AgNPs (Fig. 6b). Treatments with Ag+ induced changes in iP-R levels (Fig. 6c). The overall trend implies a significant increase in total cZ and iP CK levels and no change in total tZ types in the leaves of peppers exposed to AgNPs compared to control (Fig. 4). Total DHZ did not show a dose response, but significant increase only for the lowest and the highest AgNPs concentrations applied (Fig. 4d). For Ag+ treatments, levels of total tZ types also did not change, whereas a significant increase for total cZ, DHZ and iP types was observed only for 0.01 and 0.05 mg/L of Ag (Fig. 4). This difference in the response of different CK types to AgNPs vs. Ag+ treatment may imply a different mode of action of the nanoparticle and ionic form of silver in plants. Interestingly, the ratio of the various isoprenoid CK types changed upon treatment with AgNPs and Ag+. The most abundant and similar levels of tZ and iP types were found in control pepper leaves with a twice as low amount of total cZ CKs. This ratio changed in peppers exposed to AgNPs and Ag+, where concentrations of total tZ decreased in favour of total cZ content. The iP type of CKs remained at a low concentration. The response of CKs to stress conditions is regulated by CK metabolism in a highly dynamic way (Ha et al., 2012). Transient elevation of CK levels may occur after short-term and mild stress, while

are poor indicators of the biological effects and mechanisms of action of NPs in living organisms. Toxicity indicators based on biological markers could offer novel mechanistic insights (Rico et al., 2011). Plant hormones, as versatile regulators of plant growth and development, present an unexplored field in plant-NPs interaction studies. Examining the relevant literature, we found only a few papers on the analysis of plant hormone responses in plants treated with NPs (Shukla et al., 2014; Le et al., 2014). There are no data on cytokinin responses in plants treated with NPs. It is known that abiotic stress tolerance in plants may be positively or negatively affected by CKs (Zwack and Rashotte, 2015). Research studying interactions between CK signalling and abiotic stress responses has published recently (Zwack and Rashotte, 2015; Stirk et al., 2012; O’Brien and Benková, 2013; Macková et al., 2013); however, this is the first study investigating CK responses in plants treated with metallic NPs. Cytokinin structure is characterized by the nucleobase adenine substituted at N6-position with an aliphatic side chain. Variations in the structural substituents on different position of the adenine moiety yields a plethora/wide range of CK forms including free bases, ribosides, nucleotides, glycosides, while variations in the aliphatic side chain and stereoisomeric configuration on N6-position divide isoprenoid CKs into isopentenyladenine (iP), trans-zeatin (tZ), dihydrozeatin (DHZ), and cis-zeatin (cZ) types (Antoniadi et al., 2015). Free bases and corresponding ribosides and nucleotides of CKs can be interconverted (Tarkowská et al., 2014). It is generally considered that CK free bases are more bioactive than the corresponding ribosides, while the biological activity of cytokinin nucleotides is still unclear (Tarkowská et al., 2014). Recently, it has been postulated that cZ-types of CKs are more abundant in tissues exposed to various stresses (drought, heat or biotic stress), while tZ-type of CKs predominate in unstressed tissues (Havlová et al., 2008; Pertry et al., 2009; Vyroubalová et al., 2009; Dobra et al., 2010). Analysis of CK content in leaves of control pepper plants and plants treated with AgNPs and Ag+ revealed that free bases, ribosides and Nglucosides of iP-, tZ-, cZ-, and DHZ-types of CKs were present at detectable amounts, whereas levels of CK nucleotides (iPRMP, tZRMP cZRMP and DHZRMP) and CK O-glucosides (tZOG, tZROG, cZOG cZROG, DHZOG and DHZROG) were below the detection limits (Tables S1-S4, Supporting materials). In addition, DHZR was also not found at detectable amounts. The most abundant isoprenoid CKs were N-glucosides (iP9G > tZ7G > cZ9G > iP7G), for which the endogenous concentrations were found to be above 100 pmol/g of FW (Tables S1-S4, Supporting materials). Concentrations of the other detected CKs were below 12 pmol/g of FW. The levels of N-glucosides, representing irreversibly inactive CK forms, significantly increased in pepper leaves upon exposure to either AgNPs or Ag+(Fig. 3a). Interestingly, the highest increase in total CK N-glucosides was observed for treatments with 0.01 and 0.05 mg/L of Ag+(Fig. 3a). Significant changes between

Fig. 3. Effects of AgNPs and Ag+ treatments on levels of total cytokinin (a) N-glucosides (CK-NG), (b) bases (CK-B), and (c) ribosides (CK-R) in leaves of control and treated pepper plants. Note. Data, given in pmol/g of fresh weight (FW), are presented as mean ± SD. If values are marked with *, AgNP treatment is statistically different compared to the same concentrations of Ag+ treatment according to the Tukey's HSD test. If values are marked with #, the treatment is significantly different than control according to the t-test (P < 0.005).

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Fig. 4. Effects of AgNPs and Ag+ treatments on levels of total (a) cis-zeatin (cZ), (b) trans-zeatin (tZ), (c) isopentenyladenine (iP) and (d) dihydrozeatin (DHZ) type cytokinins in leaves of control and treated pepper plants. Note. Data, given in pmol/g of fresh weight (FW), are presented as mean ± SD. If values are marked with *, AgNP treatment is statistically different compared to the same concentrations of Ag+ treatment according to the Tukey's HSD test. If values are marked with #, the treatment is significantly different than control according to the t-test (P < 0.005).

Fig. 5. Effects of AgNPs and Ag+ treatments on free bases of (a) trans-zeatin (tZ), (b) cis-zeatin (cZ), (c) isopentyladenine (iP), and (d) dihydrozeatin (DHZ) type cytokinins in leaves of control and treated pepper plants. Note. Data, given in pmol/g of fresh weight (FW), are presented as mean ± SD. If values are marked with *, AgNP treatment is statistically different compared to the same concentrations of Ag+ treatment according to the Tukey's HSD test. If values are marked with #, the treatment is significantly different than control according to the t-test (P < 0.005).

more severe or prolonged stresses is associated with downregulation of active CK contents (Ha et al., 2012). It is assumed that CKs with low activity such as cZs are responsible for the maintenance of basal CK

activity necessary for plant survival (Gajdošová et al., 2011). The increase in cZ and iP forms of CKs in pepper leaves under nanostress is consistent with recent results on plant responses to other 16


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Fig. 6. Effects of AgNPs and Ag+ treatments on ribosides of (a) trans-zeatin (tZ), (b) cis-zeatin (cZ), and (c) isopentyladenine (iP) type cytokinins in leaves of control and treated pepper plants. Note. Data, given in pmol/g of fresh weight (FW), are presented as mean ± SD. If values are marked with *, AgNP treatment is statistically different compared to the same concentrations of Ag+ treatment according to the Tukey's HSD test. If values are marked with #, the treatment is significantly different than control according to the t-test (P < 0.005).

studies. Therefore, our observations suggest potential risks of AgNPs on plant ecosystems upon their release into the environment, be it intentional or unintentional.

types of abiotic stress (Havlová et al., 2008; Pertry et al., 2009; Vyroubalová et al., 2009; Dobra et al., 2010). Thus, we clearly confirmed the involvement of CKs in mediating abiotic stress response in plants and impact of CKs in the regulation of plant adaptation to environmental stresses, as it has been already postulated (Ha et al., 2012; Zwack and Rashotte, 2015). Moreover, our results emphasized and strengthened the role of the cZ type of CKs in abiotic stress, particularly stress caused by metallic NPs. As the treated pepper plants were inhibited in growth and development (Table 2 and Fig. 3), the higher CK content in the leaves may have alleviated the consequences of nanoparticle stress. Concerning the role of CKs in the development of the vascular system, in promoting leaf serrations, inhibiting senescence, and in chloroplast biogenesis (Kieber and Schaller, 2014), we may assume a possible phytotoxicity mechanism for AgNPs. The decreased biomasses of leaves and roots in peppers treated with AgNPs indicated an absolute requirement for CKs in order to promote development of normal leaf and root size. When leaf size is decreased, CKs may help in promoting a leaf photosynthetic performance. AgNPs could induce oxidative stress in plant cells interfering with photomorphogenesis. However, the inhibition of plant growth may not have been caused directly by the chemical phytotoxicity of AgNPs, but rather by the physical interactions between AgNPs and plant transport pathways. For example, AgNP may block the vascular system by clogging and thus decreasing water and nutrient uptake as already shown after application of TiO2 nanoparticles in maize (Asli and Neumann, 2009). All of these effects could cause numerous growth and functional inhibitions which in turn may trigger the up-regulation of CKs.

Funding This work was supported by the bilateral Austrian-Croatian project No. WTZ-Hr04/2012, by the Palacký University Olomouc, Czech Republic (IGA_PrF_2016_018), and the Ministry of Education, Youth and Sports of the Czech Republic (the National Program for Sustainability I, No. LO1204). Notes The authors declare no competing financial or any other interest. Acknowledgements This work was financially supported by the bilateral AustrianCroatian project No. WTZ-Hr04/2012. We are grateful for financial support from the Palacký University Olomouc, Czech Republic (IGA_PrF_2016_018), and the Ministry of Education, Youth and Sports of the Czech Republic (the National Program for Sustainability I, No. LO1204). We are grateful to Makso Herman, MA for help in language editing, and to our biostatistician Jelena Kovačić, mag. math. for recommendations and explanation of statistical analysis. Appendix A. Supporting material Supporting material associated with this article can be found in the online version at doi:10.1016/j.envres.2017.03.015.

4. Conclusion Both forms, nano and ionic, of silver significantly affected development of pepper (Capsicum annuum L.) grown in a commercial substrate. Comparison of AgNPs with Ag+ treatment showed that the phytotoxicity effects of nanoparticulate and ionic form of silver were observed at a similar concentration range being probably caused by an analogous mode of action. Both treatments increased total Ag in pepper tissues in a dose-dependent manner. Only the two highest Ag+ doses increased Ag tissue concentrations in comparison with AgNPs. Analysis of endogenous CK levels in leaves of control and treated pepper plants revealed a significant increase in levels of total cZ and iP type cytokinins in plants exposed to AgNPs compared to control. For Ag+ treatments, a significant increase of total cZ and iP types was observed only at lower Ag+ doses. Levels of total tZ types did not change in both treatments compared to control. This study established for the first time the role of CKs in abiotic stress in plants caused by metallic NPs indicating possible importance of cZ types of CKs in nanostressed plants. In addition, the concentrations of AgNPs and Ag+ tested in this study showed biological effects on pepper plants at several orders of magnitude lower compared to AgNPs concentrations used in other

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