Changes in Arabidopsis thaliana Gene Expression in Response to

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Changes in Arabidopsis thaliana gene expression in response to silver nanoparticles and silver ions Rashid Kaveh, Yue-Sheng Li, Sibia Ranjbar, Rouzbeh Tehrani, Christopher L. Brueck, and Benoit Van Aken Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es402209w • Publication Date (Web): 20 Aug 2013 Downloaded from http://pubs.acs.org on August 25, 2013

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Environmental Science & Technology

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Changes in Arabidopsis thaliana gene expression in response to silver nanoparticles and

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silver ions

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Rashid Kaveh†, Yue-Sheng Li‡, Sibia Ranjbar†, Rouzbeh Tehrani†, Christopher L. Brueck†,

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Benoit Van Aken†,*

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†

Pennsylvania 19122, United States

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Department of Civil and Environmental Engineering, Temple University, Philadelphia,

‡

Expression Microarray, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, United States

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* Corresponding author phone: 215-204-7087; fax: 215-204-4696; e-mail: [email protected]

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Abstract: The release of silver nanoparticles (AgNPs) in the environment has raised concerns

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about their effects on living organisms, including plants. In this study, changes in gene

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expression in Arabidopsis thaliana exposed to polyvinylpyrrolidone-coated AgNPs and silver

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ions (Ag+) were analyzed using Affymetrix expression microarrays. Exposure to 5 mg AgNPs L-

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reference to non-exposed plants. Exposure to 5 mg Ag+ L-1 for 10 days resulted in up-regulation

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of 84 genes and down-regulation of 53 genes by reference to non-exposed plants. Many genes

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differentially expressed by AgNPs and Ag+ were found to be involved in plant response to

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various stresses: up-regulated genes were primarily associated with response to metals and

(20 nm) for 10 days resulted in up-regulation of 286 genes and down-regulation of 81 genes by

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ACS Paragon Plus Environment


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oxidative stress (e.g., vacuolar cation/proton exchanger, superoxide dismutase, cytochrome P-

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450-dependent oxidase, and peroxidase), while down-regulated genes were more associated with

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response to pathogens and hormonal stimuli (e.g., auxin-regulated gene involved in organ size

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(ARGOS), ethylene signaling pathway, and systemic acquired resistance (SAR) against fungi

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and bacteria). A significant overlap was observed between genes differentially expressed in

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response to AgNPs and Ag+ (13% and 21% of total up- and down-regulated genes, respectively),

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suggesting that AgNP-induced stress originates partly from silver toxicity and partly from

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nanoparticle-specific effects. Three highly up-regulated genes in the presence of AgNPs, but not

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Ag+, belong to the thalianol biosynthetic pathway, which is thought to be involved in plant

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defense system. Results from this study provide insights into the molecular mechanisms of plant

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response to AgNPs and Ag+.

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Keywords: Silver nanoparticles, silver, Arabidopsis thaliana, gene expression, microarray

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TOC/Abstract Art

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Introduction

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Engineered nanoparticles (ENPs) are utilized in an increasing number of products, including

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textiles, electronics, pharmaceuticals, cosmetics, and water treatment reagents.1 ENPs have

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recently raised environmental concerns because they are likely to be released into the

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environment as a consequence of their widespread utilization and because they have been shown

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to exert particle-size specific toxic effects on most living organisms.2,3 Effective regulation of the

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release of ENPs in the environment has been slow to emerge, because of the relatively recent

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recognition of their environmental effects, the broad range of their application (e.g., industry,

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cosmetics, medicine), and the lack of characterization of their toxic effects.4,5 In the U.S., the

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utilization and potential release of ENPs are regulated through the Environmental Protection

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Agency (EPA) Toxic Substances Control Act (TSCA). Although EPA is currently developing a

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Significant New Use Rule (SNUR) to ensure that nanoscale materials receive appropriate

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regulatory review, it appears that there is currently no regulation specific to the utilization,

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release, and maximum acceptable levels of ENPs in the environment. There is therefore a critical

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need to collect more experimental data about the ecotoxicity of different kinds of ENPs to

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support further regulatory efforts from federal agencies. Silver nanoparticles (AgNPs) constitute

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the most widely used ENPs. Primarily because of their antimicrobial properties, AgNPs have

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been used in a wide variety of products, including textiles, bandages, deodorants, baby products,

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toothpaste, air filters, and house appliances.6,7 The ecotoxicology of AgNPs is complex because

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it may be related simultaneously to silver-specific and nanoparticle-specific biological effects.8,9

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Both AgNPs and dissolved silver have been shown to be toxic for bacteria, algae, aquatic

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organisms, plants, and humans.10,11,12 As plants constitute the basis of the terrestrial food chain,

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their exposure to AgNPs has potential implications for the agriculture and human health.10

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AgNPs released from consumer products are likely to enter wastewater. In wastewater treatment

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plants, AgNPs partition between biosolids and treated water, which can be applied on

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agricultural fields through fertilization or irrigation processes.13,14 Potential uptake of AgNPs by

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agricultural plants has therefore raised concerns about contamination of the food chain, including

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humans. As observed with other kinds of ENPs, prior studies focusing on the exposure of plants

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to AgNPs have reported both positive effects (hormeosis) and negative effects (toxicity).6,7,8,9,15

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Most toxicological studies on the effects of AgNPs have been conducted through acute toxicity

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testing (short-time exposure to high dose) although environmental effects are more adequately

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assessed by chronic toxicity testing (long-time exposure to low dose). Although chronic toxicity

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is typically difficult to observe in the laboratory, molecular studies (e.g., proteomic or

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transcriptomic methods) may provide useful information about the potential long-term effects of

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exposure to environmental contaminants.16 However, the molecular bases of the toxicity and/or

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growth promoting effects of AgNPs in higher plants have received little attention. Recently,

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changes in gene expression in plants exposed to various environmental contaminants have been

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studied using the microarray technology, providing better understanding of the molecular

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mechanisms of plant response.17-20 Following a similar strategy, the objective of this study is to

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provide new insights on the transcriptional response of the model plant, Arabidopsis thaliana,

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exposed to AgNPs and Ag+ through the use of whole-genome cDNA microarrays.

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Experimental

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Chemicals. Silver nanoparticles (silver nanopowder, 99.99%, 20 nm, CAS 7440-22-4) were

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obtained from U.S. Research Nanomaterials (Houston, TX). Silver nitrate (99.9%) was obtained

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from Sigma-Aldrich (St-Louis, MO). Polyvinylpyrrolidone (molecular weight 40,000 Dalton,

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PVP40) was obtained from Sigma-Aldrich. Phytoagar was obtained from Plant Media (Dublin,

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OH). Murashige and Skoog (MS) salt base was obtained from Carolina (Burlington, NC). Other

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chemicals were of analytical grade, solvents were of HPLC grade, and they were obtained from

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Acros Chemicals (Geel, Belgium), Fischer Scientific (Pittsburgh, PA), or Sigma-Aldrich.

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Nanoparticle characterization: For AgNP characterization, fresh particle suspensions were

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prepared in 0.5-strength Murashige and Skoog (MS) medium (at concentration of 5 to 20 mg L-1

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with an equal concentration of PVP40) and dispersed by sonication for 30 min in a water bath

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(150 W). Particle suspensions were analyzed immediately after preparation and after 24 h of

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agitation at 55 °C, 150 rpm. Particle suspensions were characterized by visible spectrometry,

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dynamic light scattering (DLS), and transmission electron microscopy (TEM). Visible spectra

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(300 to 800 nm) of particle suspensions were recorded using an Agilent 8453 spectrophotometer

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(Agilent, Santa Clara, CA). The particle size distribution and zeta potential of the suspensions

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were determined by DLS using a Zetasizer Nano (Malvern, Worcester, MA) with the following

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parameters: wavelength 632.8 nm, angle 173 °, temperature 25 °C. The particle size and

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morphology were characterized by TEM using a JEM-1400 (JEOL, Peabody, MA) with a HT

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voltage of 120 kV and a beam current of 66 mA. Samples were prepared by applying and air

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drying 4 µL of particle suspension onto a 400-mesh copper grid covered with ultrathin carbon

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film on Holey carbon support film (Ted Pella, Redding, CA).

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Plant species and culture conditions. Arabidopsis thaliana, ecotype Columbia (Col-0/Redei-

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L211497), was obtained from the Arabidopsis Biological Resource Center (Ohio State

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University, Columbus, OH). Seeds were kept on a wet filter paper at 4 ºC in the dark for 24 h.

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Seeds were then surface-sterilized by immersion successively in DI water for 1 h, in 95% ethanol

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for 5 min, and 0.6% sodium hypochloride for 5 min, and were rinsed 3 times in sterile DI water.

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Seeds were germinated under sterile conditions in 10 × 10-cm Magenta boxes (5 seeds per box)

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closed with vented lids and filled with 100 mL of semi-solid nutrient medium. The medium

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consisted of 0.5-strength MS nutrient solution supplemented with 0.3% sucrose and 0.7%

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phytoagar (pH 7.2), and it was sterilized by autoclaving (121 °C, 15 min). The boxes were

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incubated at 25 ºC under white (cool) fluorescent light (0.38 ± 0.02 W ft-2) with a 16-h light/8-h

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dark photoperiod.

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Growth inhibition experiments. The inhibitory effect of AgNPs and Ag+ was tested by cultivating

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Arabidopsis plants in the presence of increasing concentrations of the toxicants (1.0, 2.5, 5.0, 10,

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and 20 mg L-1) added to the nutrient medium. After sterilization, the medium (prepared as

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described above) was cooled to 55 ºC and supplemented with the AgNPs or Ag+ (formulated as

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aqueous stock solutions). AgNP stock solution was prepared by mixing 1.0 g AgNPs L-1 (0.1%

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w/v) and 1.0 g PVP40 L-1 (0.1% w/v) with DI water and dispersing AgNPs by sonication for 30

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min in a water bath (150 W). Ag+ stock solution was prepared by dissolving 1.575 g AgNO3 L-1

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(0.1% w/v Ag) and 1.0 g PVP40 L-1 (0.1% w/v) in DI water. Two sets of non-exposed control

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plants were grown in nutrient medium (MS) only and in nutrient medium supplemented with 20

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mg PVP40 L-1 (i.e., equivalent to the highest concentration of PVP40 applied in the experiments

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with AgNPs). After 10 days of growth, plants were removed from the medium, washed with

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water to remove the excess of medium, dried by blotting, and weighted. Twenty plants were used

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for each treatment. The significance of differences between treatments was evaluated using one-

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way ANOVA (Prim 6.0, GraphPad, La Jolla, CA) followed by Tukey's multiple comparison tests

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at 95% confidence level (p < 0.05).

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Analysis of silver in plant tissues: For the determination of silver content, plants were grown and

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exposed to AgNPs and Ag+ as described above. At harvesting time, plants were washed with

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deionized water to remove silver that was neither adsorbed nor integrated in plant tissues.1 Plants

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were then separated into roots and leaves, dried at 70 °C for 24 h, and digested in 4:1

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concentrated HNO3:30% H2O2 at 70 °C for 8 h. After dilution with deionized water, the solution

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was filtrated through 0.2 µm and analyzed using an Agilent 7500i Benchtop ICP-MS (Santa

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Clara, CA). Fifteen plants were used for each treatment. Leaf or root tissues from 5 plants were

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pooled together to obtain three biological replicates that were analyzed separately. Statistical

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analyses were performed using one-way ANOVA followed by Tukey's multiple comparison

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tests.

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Gene expression analysis using microarrays. Plants were grown as described above in the

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presence of 5 mg AgNPs or Ag+ L-1 (and, in each case, 5 mg PVP40 L-1). Control plants were

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grown in the presence of 5 mg PVP40 L-1 only. After 10 days of growth, plants were removed

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from the medium, washed, immediately soaked in 4 mL of RNA Later™ (Ambion, Foster City,

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CA), and incubated at 4 ºC for 24 h following the manufacturer's guidelines. The plants were

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then dried by blotting and stored at -80 ºC until RNA extraction. RNA was extracted from whole

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plant tissues using TRIzol® Plus RNA Purification kit with on-column PureLink® DNAase

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treatment following the manufacturer's guidelines. An additional step of tissue homogenization

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was performed after addition of the TRIzol® reagent using bead beating (1-mm glass beads,

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4,200 rpm, 40 sec). Purified RNA was kept at -80 °C. RNA was quantified by the OD260 using a

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NanoDrop™ ND-2000 spectrophotometer (Vernon Hills, IL). The quality of RNA was assessed

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by the ratios OD260/OD280 and OD260/OD230 and using an Agilent 2100 Bioanalyzer (Santa Clara,

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CA). RNA samples used for microarray analysis had OD260/OD280 ratios of 2.12 – 2.17,

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OD260/OD230 ratios of 2.16 – 2.36, and RNA integrity numbers (RIN) of 8.3 – 9.1. RNA samples

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were labeled and hybridized to the Affymetrix Arabidopsis Gene 1.0 ST Arrays according to the

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manufacturer's instructions (Affymetrix, Santa Clara, CA). For each treatment, three biological

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replicate samples were used for microarray experiments. Scanned microarray images were

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analyzed using the Affymetrix Gene Expression Console with RMA (Robust Multi-array

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Average) normalization algorithm. Further statistical analyses were performed using BRB-

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ArrayTools developed by Dr. Richard Simon and BRB-ArrayTools Development Team.21 Gene

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classification into ontology categories (GO) was performed using BLAST2GO® version 2.6.4

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(Biobam Bioinformatics, Valencia, Spain).

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Reverse-transcription real-time PCR. Quantitative analysis of gene expression was performed

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for selected genes using reverse-transcription real-time PCR (RT-qPCR). Four genes

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significantly down-regulated (fold change < 0.25), 4 genes significantly up-regulated (fold

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change > 4.0), and 4 genes with moderate expression differences (fold change between 0.4 and

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2.0) upon exposure to both AgNPs and Ag+ were selected (Supplemental Information, Table S1).

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The internal standard was the housekeeping gene, mitogen-activated protein kinase 6 (MPK6)

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(AT2G43790).20 RT-qPCRs were conducted for three biological replicates per treatment using

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the same RNA as used for the microarray experiments. RNA was reverse-transcribed into cDNA

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using SuperScript® III First-Strand Synthesis system and oligo-dT primers (Invitrogen, Foster

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City, CA). Negative controls were generated by running the reactions without reverse-

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transcriptase. Gene sequences were obtained from the National Center for Biotechnology

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Information (NCBI) and used to design gene-specific real-time primers using PrimerQuest (IDT,

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Coralville, IA). When possible (for 10 of the 12 selected genes), primers were designed with one

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of the primer sequence spanning an exon-intron boundary (primer sequences are provided as

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Supplemental Information, Table S1). Real-time PCR quantification of cDNA was performed on

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a StepOnePlus™ Real-Time PCR System using SYBR® Green PCR Master Mix (Applied

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Biosystems, Foster City, CA). The amplification efficiency for each primer set was determined

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by using log10-dilutions of cDNA according to standard protocols. CT (cycle threshold) data were

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computed by the StepOnePlus™ Software (version 2.1; Applied Biosystems). The mean relative

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levels of amplification of the target genes and standard deviations were calculated based on CT

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values and amplification efficiencies using REST 2009 (version 2.0.13; Qiagen, Foster City,

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CA).22

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MIAME compliance. This article is written in compliance with the Minimum Information About

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a Microarray Experiment (MIAME) guidelines (http://www.mged.org/miame). Microarray data

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have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) with

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the accession number E-MEXP-3950.

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Results and Discussion

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Nanoparticle characterization: Particle suspensions were characterized in fresh preparation (MS

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medium) and after 24 h of agitation at 55 °C, 150 rpm to detect potential aggregation and/or

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change in properties. The spectrum of the fresh AgNP suspension (5 mg AgNPs L-1, 5 mg

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PVP40 L-1 in 0.5-strength MS medium) showed a single peak at 405 nm, which is consistent

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with uniform-sized nanoparticles with a diameter of approx. 20 nm. No significant change of the

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spectrum was observed after agitation of the suspension for 24 h at 55 °C, 150 rpm (spectra are

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presented in Supplemental Information, Figure S1). DLS analysis of the fresh AgNP suspension

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(20 mg L-1) showed a narrow size distribution with a hydrodynamic diameter of 27.1 ± 0.3 nm,

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which shifted to 29.6 ± 0.1 nm after 24 h of agitation at 55 °C. The zeta potential of the fresh

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suspension was -38.1 ± 0.3 V, which shifted to -30.9 ± 0.6 V after 24 h of agitation at 55 °C (size

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distribution diagrams are presented in Supplemental Information, Figure S2). TEM pictures of

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AgNP suspensions showed mostly spherical particles of uniform size (Figure 1). No observable

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change was recorded after agitation at 55 °C for 24 h.

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Effect of AgNPs and Ag+ on A. thaliana growth. In order to determine the potential toxic effect

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of AgNPs and Ag+ on A. thaliana, plantlets were grown in MS medium containing increasing

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concentration of both factors, ranging from 0.0 to 20 mg L-1. Figure 2 presents the average fresh

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biomass of plants exposed for 10 days to each treatment. No significant biomass difference was

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observed between the sets of non-exposed control plants (i.e., growing in MS medium only and

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in MS medium containing 20 mg PVP40 L-1). Exposure of plants to 1.0 and 2.5 mg AgNPs L-1

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for 10 days resulted in significant increase of the biomass, although exposure to higher

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concentrations (from 5.0 to 20 mg L-1) resulted in reduction of the biomass. On the other hand,

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although no significant effect was recorded in the presence of 1.0 and 2.5 mg Ag+ L-1, the

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biomass after 10 days was significantly reduced upon exposure to 5.0 mg L-1 and above. At low

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levels (1.0 and 2.5 mg L-1), exposure to AgNPs resulted in a significantly higher plant biomass

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than exposure to Ag+. No significant difference was recorded between the two treatments at

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higher levels (5.0, 10, and 20 mg L-1).

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Previous studies focusing on the impact of AgNPs on plants have reported both positive and

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negative effects. Investigating the impact of AgNPs of different sizes (from 2 to 20 nm) in barley

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(Hordeum vulgare), flax (Linum usitatissimum), and ryegrass (Lolium perenne), El-Temsah and

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Joner6 reported a significant reduction of the germination and growth rates, which were

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dependent on the plant species and concentration of AgNPs: e.g., 20-nm particles resulted in a

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noticeable reduction of the shoot length at 10 mg L-1 in flax and ryegrass, and at 20 mg L-1 in all

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three species. Another study reported a limited effect of AgNPs (approx. 30 nm) on cucumber

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(Cucumis sativus) and lettuce (Lactuca sativa) plants.15 Germination rates were reduced by 24%

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and 5% and root elongation rates were reduced by 15% and 2% after exposure to 100 mg AgNPs

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L-1 in cucumber and lettuce, respectively. Kumari et al.7 studied the cytotoxicity and

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genotoxicity of 100-nm AgNPs on onion root tip cells (Allium cepa): based on microscopic

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observations, cells exposed to increasing concentration in AgNPs (from 25 to 100 mg L-1)

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showed a decrease of the mitotic index (MI) from 60% to 28% and the occurrence chromosomal

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aberrations. Although previous studies reported mostly toxic effects of AgNPs toward plants, a

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recent report from Wang et al.9 showed that exposure of Arabidopsis and poplar (Populus

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deltoides × nigra DN34) plants to AgNPs produced beneficial effects at low concentration: e.g.,

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exposure to 25-nm AgNPs resulted in increased biomass and evapotranspiration in poplars at the

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concentration of 1.0 mg L-1, although exposure to 10 and 100 mg L-1 resulted in decrease of

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these parameters.

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Analysis of silver content in plant tissues: After 10 days of exposure to AgNPs and Ag+, plant

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tissues were dried, acid-digested, and the silver content was determined by ICP-MS. At the same

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level of exposure, the Ag concentration was significantly higher in plants dosed with Ag+ than in

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plants dosed with AgNPs. The Ag concentration was also significantly higher in exposed root

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tissues than in exposed leave tissues. A positive relationship was observed between the exposure

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dose and Ag content in root tissues, although this relationship was less apparent in exposed leaf

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tissues. Overall, these results are consistent with previous studies described in the literature. As

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an example, Wang et al.9 recently reported a higher Ag content in A. thaliana leaves exposed to

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Ag+ (approx. 8 µg g-1) than exposed to AgNPs (approx. 2 µg g-1). As observed in our study, the

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Ag concentration detected in roots was higher than in aerial parts of the plants: 68% of total Ag+

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and 24% of total AgNPs were found in root tissues, while only 2% of total Ag+ and 1% of total

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AgNPs were found in aerial parts.

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Gene expression microarrays. The transcriptional response of plants exposed to AgNPs and Ag+

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was investigated using Affymetrix whole-transcript expression microarrays. AgNP and Ag+

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concentration of 5 mg L-1, which resulted in moderate reduction of the plant biomass, was

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chosen for microarray experiments. After filtering out the genes with low-quality signals and

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conducting univariate t-tests (p < 0.001; BRB-ArrayTools), 446 and 405 genes showed

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consistent expression levels after exposure to AgNPs and Ag+, respectively. Of these, 375 and

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141 genes were expressed at significantly different levels (change fold < 0.5 or > 2.0) by

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exposure to AgNPs and Ag+, respectively: 286 genes (78%) were up-regulated and 81 genes

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(22%) were down-regulated at significant levels by exposure to AgNPs; 84 genes (60%) were

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up-regulated and 53 genes (40%) were down-regulated at significant levels by exposure to Ag+.

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A significant overlap of differentially-expressed genes was observed upon exposure to AgNPs

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and Ag+: 15 genes were up-regulated and 29 genes were down-regulated in response to both

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AgNPs and Ag+ (representing 13% and 21% of the total up- and down-regulated genes,

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respectively). This observation suggests that some of the AgNP effects on gene expression

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originate from Ag+ released by AgNPs, which was previously observed or proposed by other

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authors.9,25 As in several other studies9,23,24, we chose to expose the plants to the same

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concentrations of AgNPs and Ag+ (expressed as mg Ag L-1). This approach was motivated by the

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lack of information about the nature of the toxic effects and cellular targets of both Ag forms.

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Soluble Ag is known to be cytotoxic and AgNP toxicity likely originates partly from the release

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of soluble Ag. Besides, nanoparticles are known to exert specific 'particulate' effects that may not

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be related to Ag+ toxicity. Dimkpa et al.25 reported that exposure of sand-grown wheat plants

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(Triticum aestivum) to both 10-nm AgNPs and soluble Ag (Ag+) (2.5 mg kg-1) resulted in

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comparable biomass reduction. Interestingly, exposure to Ag+ equivalent to soluble Ag released

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from AgNPs (63 µg kg-1) did not result in observable effect. Moreover, using TEM, the authors

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observed accumulation of AgNPs inside plant tissues, suggesting that AgNPs exert toxic effects

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that are, at least in part, unrelated to the release of soluble Ag. In order to determine the

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respective effect of AgNPs and Ag+, Stampoulis et al.8 exposed zucchini plants (Cucurbita pepo)

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to bulk (powder) Ag, AgNPs (100 nm), and Ag+ (both supernatant of AgNP suspension and

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AgNO3). The authors reported that exposure to bulk Ag at 1,000 mg L-1 did not significantly

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affect the plant biomass, while exposure to 1,000 mg L-1 AgNPs reduced the biomass by approx.

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75%. On the other hand, AgNP supernatant and Ag+ (AgNO3) at concentration as low as 1.0 mg

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L-1 reduced the biomass by approx. 25%, suggesting that about half the observed phytotoxicity

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originated from the elemental nanoparticles themselves. Few studies have been conducted on the

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transcriptional response of organisms exposed to AgNPs. Analyzing gene expression in human

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cells (HeLa) exposed to AgNPs and Ag+, Xu et al.12 reported a higher number of genes up-

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regulated (62%) than down-regulated (38%) with Ag nanoparticle exposure, which is consistent

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with our results. The authors also observed that a large number of genes were differentially

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expressed in response to both AgNPs and Ag+ (85% of up-regulated genes and 68% of down-

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regulated genes). The complete list of Arabidopsis genes up- and down-regulated by exposure to

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AgNPs and Ag+ is provided as Supplemental Information (Tables S2 and S3).

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Reverse-transcription real-time PCR. In order to validate the microarray results, quantitative

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analysis of gene expression was performed on selected genes using RT-qPCR. Figure 4 shows

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the plots of the expression levels of the selected genes as recorded using microarrays against

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their expression levels recorded using RT-qPCR. Correlations were generally satisfactory with

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Pearson's correlation coefficient of 0.96 and 0.97 for exposure to AgNPs and Ag+, respectively.

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The RT-qPCR amplification levels were corrected for the amplification efficiencies of different

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primer sets (ranging from 97.9% to 109.9%, R2 = 0.99 to 1.0) using REST 2009.

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Functional categories of differentially expressed genes. Differentially expressed genes were

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classified in gene ontology (GO) categories using the software BLAST2GO®. Distribution into

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major process and functional categories (GO level 2) showed little difference between genes up-

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and down-regulated by exposure to AgNPs and Ag+: most represented process categories for

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both up- and down-regulated genes were metabolic process, cellular process, response to

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stimulus, and biological regulation (Figure 5); most represented functional categories for both

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up- and down-regulated genes were catalytic activity, binding, nucleic acid binding transcription

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factor activity, and transporter activity (Figure 6). Two remarkable exceptions are the signaling

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process category involving only down-regulated genes (25% and 30% of total down-regulated

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genes by exposure to AgNPs and Ag+, respectively) and the electron carrier functional category

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involving (almost) only up-regulated genes (11% and 16% of total up-regulated genes by

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exposure to AgNPs and Ag+, respectively). Genes in the signaling category are mostly involved

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in hormone signaling pathways and cellular response to hormone stimuli, which may be related

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to the reduction of plant growth in response to the toxicity of AgNPs and Ag+.26 On the other

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hand, heavy metals are known to interact with electron carriers. Up-regulation of genes in this

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category may reflect the response of the plant to the decreased electron transport efficiency in the

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presence of AgNPs and Ag+.27

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A significant proportion of genes differentially expressed by exposure to AgNPs and/or Ag+ is

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involved in response to stimuli (GO level 2) (49% of total up-regulated genes and 68% of total

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down-regulated genes). Among them, most up-regulated genes (63% and 78% of genes in this

331

category up-regulated by AgNPs and Ag+, respectively) are involved in response to abiotic

332

stimuli (GO level 5), including metal ions, salts, light, starvation, oxidative stress, osmotic stress,

333

and radiation. On the other hand, most down-regulated genes (74% and 62% of genes in this

334

category down-regulated by AgNPs and Ag+, respectively) are involved in response to pathogens

335

and hormonal stimuli (GO level 5), including abscisic acid, auxine, cytokinin, ethylene,

336

gibberellin, jasmonic acid, and steroid hormones (Figure 6). Up-regulation of genes related to

337

abiotic stimuli likely reflects the response of the plant to AgNP/Ag+-induced stress. On the other

338

hand, down-regulation of genes related to hormones and biological stimuli can be seen as a plant

339

strategy to prioritize the response to AgNP and Ag+-induced stress.26 Alternatively, hormones are

15



340

involved primarily in the regulation of plant development and down-regulation of hormone-

341

responsive genes may simply reflect attempts of the plant to limit the growth under toxic

342

conditions. In addition, beside their role in plant development, hormones are known to be

343

involved in response to biological and abiotic stresses. For instance, the down-regulation of

344

abscisic acid and auxin signaling pathways has been shown to play a role in various plant

345

defense responses.26

346 347

Differential expression of remarkable genes. Remarkable genes discussed in this section were

348

differentially expressed in response to both AgNPs and Ag+ (reflecting the effects of Ag+) or in

349

response to AgNPs only (reflecting nanoparticle-specific effects). Genes highly up-regulated

350

(fold change > 4.0) in response to both AgNPs and Ag+ are involved in response to metal and

351

oxidative stresses. These genes encode the following proteins: a vacuolar cation/proton

352

exchanger involved in root development under metal stress (AT5G01490), a miraculin-like

353

protein (MLP) (AT2G01520), two copper/zinc superoxide dismutases (AT2G28190 and

354

AT1G08830), two cytochrome P-450-dependent monooxygenases (AT5G42590 and

355

AT3G28740), and a peroxidase (AT3G21770). MLPs have been suggested to be involved in

356

response to wounding and pathogen infection in other plant species.28 Superoxide dismutases and

357

peroxidases are involved in protection against reactive oxygen species (ROS), which are

358

frequently associated with metal and ENP toxicity.1 Although they are involved in many

359

physiological processes, cytochrome P-450 genes have been reported to be induced by metal

360

stress in Arabidopsis.29 Genes most down-regulated in response to both AgNPs and Ag+ (fold

361

change ≤ 0.25) include a gene encoding an ARGOS (auxin-regulated gene involved in organ

362

size) protein (AT3G59900), three genes involved in the ethylene signaling pathway

16


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(AT3G16770, AT5G25350, and AT2G40940), and two genes involved in systemic acquired

364

resistance (SAR) against fungi (AT4G12470) and bacteria (AT5G46330). The ARGOS gene (the

365

most down-regulated gene by both AgNP and Ag+, 0.08- and 0.09-fold change, respectively)

366

regulates the size of lateral organs and its down-regulation likely reflects attempts of the plant to

367

limit its expansion under stressed conditions.26 As suggested above, down-regulation of genes

368

involved in ethylene signaling pathway and SAR can be understood as a prioritization of plant

369

defense mechanisms under AgNP and Ag+-induced stresses.26 Genes differentially expressed in

370

response to both AgNPs and Ag+ are likely to be implicated in response to Ag+, either directly

371

added or released from AgNPs.

372 373

On the other hand, a number of genes were differentially expressed in response to AgNPs only,

374

which reflects their involvement in nanoparticle-specific responses. The most remarkable genes

375

up-regulated (fold change > 4.0) specifically by AgNPs include two genes involved in salt stress

376

(AT3G28220 and AT1G52000), a gene encoding a myrosinase-binding protein involved in

377

defense against insects and pathogens (AT1G52040), three genes involved in the thalianol

378

biosynthetic pathway (AT5G48010, AT5G48000, and AT5G47990), and a gene encoding a MLP

379

involved in response to wounding (AT2G01520). The most up-regulated gene in our study (28.6-

380

fold change) encodes a TRAF (tumor necrosis factor receptor-associated factor)-like protein

381

involved in salt stress response. Although the relationship between salt and AgNP-induced stress

382

is not readily apparent, similar functional genes were found to be induced in Arabidopsis

383

exposed to other kinds of ENPs.20 The induction of genes responsive to pathogens and wounding

384

may be related to mechanical damages caused by AgNPs to plant tissues.3 The three genes of the

385

thalianol pathway belong to a cluster of four genes, which constitutes a rare case of gene

17



386

clustering in higher plants.30 Although gene clusters are common in bacteria (i.e., operons), they

387

are less frequent in eukaryotes and were thought until recently to be restricted to paralogs

388

originating from repeated tandem genes. Only a few clusters of non-homologous, functionally-

389

related genes have been detected in fungi and plants. In plants, these clusters are all involved in

390

biosynthesis of stress-induced secondary metabolites that are (or are believed to be) required for

391

survival under specific conditions, such as the exploitation of new environments.31

392 393

Interestingly, the most down-regulated gene by exposure to both AgNP and Ag+ in our study

394

(ARGOS) was also reported down-regulated in Arabidopsis plants exposed to zinc oxide and

395

fullerene nanoparticles.20 Other remarkable genes up-regulated by both AgNPs (our study) and

396

zinc oxide nanoparticles20 are genes encoding a superoxide dismutase (AT1G08830) and two

397

peroxidases, which are involved in response to oxidative stress (AT3G21770, AT2G18150), and

398

a phytosulfokine-beta growth factor involved in response to wounding (AT3G49780).

399 400

This article presents the first whole-genome expression microarray experiment focusing on A.

401

thaliana plants exposed to AgNPs and Ag+. Results from this study are believed to provide new

402

insights into the molecular mechanisms of plant response to AgNPs and Ag+.

403 404

Acknowledgments

405

This research was supported by a U.S. Department of Agriculture (USDA) - National Institute of

406

Food and Agriculture (NIFA) grant (Award number: 2012-67009-19982).

407 408

Supplemental Information

18


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409

The genes selected for the RT-qPCR validation of microarray results and the corresponding

410

primer sequences are listed in Table S1. The complete list of Arabidopsis genes significantly up-

411

regulated (> 2.0) and down-regulated (< 0.5) by exposure to AgNPs and Ag+ is provided in

412

Tables S2 and S3. Visible absorption spectra and DLS size-distribution diagrams of AgNP

413

suspensions are presented in Figure S1 and Figure S2, respectively. This information is available

414

free of charge via the Internet at http://pubs.acs.org/.

415 416

References

417

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30. Field, B.; Osbourn, A.E. Metabolic diversification - Independent assembly of operon-like gene clusters in different plants. Science 2008, 320, 543-547. 31. Osbourn, A. Gene Clusters for secondary metabolic pathways: An emerging theme in plant biology. Plant Physiol. 2010, 154, 531-535.

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Figure Captions

505 506

Figure 1: Transmission electron micrograph of 20-nm silver nanoparticle (AgNP) suspensions

507

prepared in 0.5-strength Murashige and Skoog (MS) medium with polyvinylpyrrolidone

508

(molecular weight 40,000 Dalton, PVP40).

509 510

Figure 2: Relative biomass of A. thaliana plants exposed for 10 days to various concentrations

511

of silver nanoparticles (AgNPs) and silver ions (Ag+). Controls are non-exposed plants growing

512

in nutrient medium (MS) only and in nutrient medium containing PVP40 (PVP). Error bars

513

represent standard deviations between 20 biological replicates. Treatments resulting in

514

significantly different biomass (based on one-way ANOVA (p < 0.05) followed by Tukey's

515

multiple comparison tests) are shown by different letters.

516 517

Figure 3: Silver content measured in tissues of A. thaliana exposed to various concentrations of

518

silver nanoparticles (AgNPs) and silver ions (Ag+). Panel A: Leaves. Panel B: Roots. Controls

519

are non-exposed plants growing in nutrient medium (MS) only and in nutrient medium

520

containing PVP40 (PVP). Error bars represent standard deviations between 3 sets of pooled

521

biological replicates. Treatments resulting in significantly different biomass (based on one-way

522

ANOVA (p < 0.05) followed by Tukey's multiple comparison tests) are shown by different

523

letters.

524 525

Figure 4: Evaluation of microarray expression levels using RT-qPCR. Panel A: Log2 microarray

526

relative expression levels versus log2 RT-qPCR relative expression levels for exposure to silver

24


Page 24 of 31

Page 25 of 31


527

nanoparticles (AgNPs). Panel B: Log2 microarray relative expression levels versus log2 RT-

528

qPCR relative expression levels for exposure to silver ions (Ag+). Error bars represent the

529

standard deviations between three biological replicates.

530 531

Figure 5: Major gene ontology (GO) process categories of genes up- (fold change > 2.0) and

532

down-regulated (fold change < 0.5) in A. thaliana plants exposed for 10 days to silver

533

nanoparticles (AgNPs) and silver ions (Ag+). Distribution of genes into GO categories was

534

performed using BLAST2GO® (GO level 2). Only categories containing at least 5% of the total

535

number of genes significantly expressed in response to the treatments are shown. Panel A:

536

Genes differentially expressed by exposure to silver nanoparticles (AgNPs). Panel B: Genes

537

differentially expressed by exposure to Ag+.

538 539

Figure 6: Major gene ontology (GO) functional categories of genes up- (fold change > 2.0) and

540

down-regulated (fold change < 0.5) in A. thaliana plants exposed for 10 days to silver

541

nanoparticles (AgNPs) and silver ions (Ag+). Distribution of genes in GO categories was

542

performed using BLAST2GO® (GO level 2). Only categories containing at least 5% of the total

543

number of genes significantly expressed in response to the treatments are shown. Panel A:

544

Genes differentially expressed by exposure to silver nanoparticles (AgNPs). Panel B: Genes

545

differentially expressed by exposure to Ag+.

546

25



547

Figure 1

548

549 550 551

26


Page 26 of 31

on t C rol on M S t A gN rol P P 1. VP A gN 0 P mg / 2 A gN .5 L m P g A 5.0 /L gN m P g/ L 1 A gN 0 m P g 20 /L A g+ m 1 g/L A .0 m g+ 2 g/L A .5 m g+ 5. g/L A 0m g+ g /L 1 A 0m g+ g 20 /L m g/ L

C

Relative Biomass (%)

Page 27 of 31

552


Figure 2

553

554

555

556

27


557

25

A. Leaf tissues

10 a

5 a a a a a

a

C on t C rol o M A ntro S gN lP P VP 1 A gN .0 P mg 2 / A gN .5 L P mg A 5.0 /L gN m g P 10 /L A gN m P g/ L 2 A 0m g+ 1. g/L A 0m g+ 2. g/L A 5m g+ 5. g/L 0 A g + mg 10 /L A g + mg 20 /L m g/ L

C on t C rol on M tr A S gN ol P P V 1 P A gN .0 P mg 2 / A gN .5 L P mg A 5.0 /L gN m P g/ L 1 A gN 0 m P g/ L 2 A 0m g+ g 1. /L A 0m g+ g /L 2. A 5m g+ g / 5. 0 L A g + mg 10 /L A g + mg 20 /L m g/ L


b

200

20

a a

50

a a

0 a a

Treatment

559

560

561

28


Page 28 of 31

Figure 3

558

B. Root tissues

c b

f

150

15 100 e d

d c

b a

Treatment

e

b a b a d b

0 a

Page 29 of 31

562


Figure 4

563

B. AG

A. SNP

6

Log2(Signal) - RT-qPCR

Log2(Signal) - RT-qPCR

6 4 2 0 -2 -4

4 2 0 -2 -4 -6

-6 -5

-4

-3

-2

-1

0

1

2

3

-5

4

-4

-3

-2

-1

0

1

2

Log2(Signal) - Microarray

Log2(Signal) - Microarray

564

565

566

29


3

4


567

Figure 5

568

A

B

569 570

30


Page 30 of 31

Page 31 of 31

571


Figure 6

572

573

574

31


Changes in Arabidopsis thaliana Gene Expression in Response to

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