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Molecular Response of Crop Plants to Engineered Nanomaterials Luca Pagano, Alia D. Servin, Roberto De La Torre Roche, Arnab Mukherjee, Sanghamitra Majumdar, Joseph Hawthorne, Marta Marmiroli, Elena Maestri, Robert E. Marra, Susan M. Isch, Om Parkash Dhankher, Jason C. White, and Nelson Marmiroli Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01816 • Publication Date (Web): 15 Jun 2016 Downloaded from http://pubs.acs.org on June 19, 2016
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Environmental Science & Technology
Molecular Response of Crop Plants to Engineered Nanomaterials
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Luca Pagano1,2,3, Alia D. Servin3, Roberto De La Torre-Roche3, Arnab Mukherjee3, Sanghamitra
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Majumdar3, Joseph Hawthorne3, Marta Marmiroli1, Elena Maestri1, Robert E. Marra3, Susan M.
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Isch4, Om Parkash Dhankher2, Jason C. White3,*, Nelson Marmiroli1,*
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1, Dept. of Life Sciences, University of Parma, Parma, Italy.
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2, Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA, USA.
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3, The Connecticut Agricultural Experiment Station, New Haven, CT, USA.
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4, Dr. Katherine A. Kelley State Public Health Laboratory, Rocky Hill, CT, USA.
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Abstract
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Functional toxicology has enabled the identification of genes involved in conferring tolerance
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and sensitivity to Engineered Nanomaterial (ENM) exposure in the model plant Arabidopsis
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thaliana (L.) Heynh. Several genes were found to be involved in metabolic functions, stress
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response, transport, protein synthesis and DNA repair. Consequently, analysis of physiological
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parameters, metal content (through ICP-MS quantification) and gene expression (by RT-qPCR)
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of A. thaliana orthologue genes were performed across different plant species of agronomic
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interest in order to highlight putative biomarkers of exposure and effect related to ENMs. This
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approach led to the identification of molecular markers in Solanum lycopersicum L. and
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Cucurbita pepo L. (tomato and zucchini) that might not only indicate exposure to ENMs (CuO,
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CeO2, La2O3), but also provide mechanistic insight into response to these materials. Through
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Gene Ontology (GO) analysis, the target genes were mapped in complex interatomic networks
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representing molecular pathways, cellular components and biological processes involved in
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ENM response. The transcriptional response of 38 (out of 204) candidate genes studied varied
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according to particle type, size and plant species. Importantly, some of the genes studied showed
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potential as biomarkers of ENM exposure/effect and may be useful for risk assessment in foods
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and in the environment.
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Keywords: Nanoecotoxicology, Functional genomics, Food safety, Biomarkers of exposure.
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Corresponding authors:
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Jason C. White. E-mail:
[email protected] Phone: +1 (203) 974-8523. Address: The
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Connecticut Agricultural Experiment Station, 123 Huntington St. 06504 New Haven, CT. Fax:
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+1 (203) 974-8502.
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Nelson Marmiroli. E-mail:
[email protected] Phone: +39 0521905606. Address: Dept.
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Life Sciences, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma (IT). Fax: +39
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0521906222.
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Introduction
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The application of nanotechnology has occurred across many sectors: health/medicine,
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communications/electronics,
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production/agriculture; the increases have been exponential in the last 10 years, with even greater
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use predicted in the future.1 There is a general consensus in the scientific community that our
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understanding of the fate and effects of these materials in the environment has lagged behind and
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is not adequate for accurate risk assessment. Important steps forward have been made in the last
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3-4 years, especially on the properties that determine the behavior and the distribution of
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Engineered Nanomaterials (ENMs) in the environment,2-4 as well as the particle size dependent
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process of bioaccumulation and the trophic transfer within food chains.5,6 The current state of
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knowledge regarding plants and ENMs interactions at both the physiological and molecular
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response level, including uptake, toxicity, and cellular compartmentalization, has been
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reviewed,7,8 and there is now some understanding of which plant organs, tissues, cells and
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organelles are involved in response, as well as of the molecular pathways associated with more
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general toxicity (e.g. DNA damage, ROS production, protein misfolding, etc.).9 However, given
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the wide range ENMs used (composition, size, shape, coating, etc.) and of their effects, it
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remains difficult to highlight consistent endpoints commonly shared in response to different
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classes of these materials. Unlike the situation for humans,10 biomarkers for exposure, effects
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and ENMs susceptibility are unknown in plants. Typical biomarkers of exposure often involve
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the measurement of metabolites or other physiological parameters that reflect the biological
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dose/effect, showing directly or indirectly the physiological implications of exposure.
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Biomarkers of effects show changes at cellular/molecular level and reflect the expression of
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genes or the abundance of proteins under experimentally controlled conditions. Considering this
energy
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potential, biomarkers of effects can be usefully applied as a tool for the assessment of toxicity.11
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Biomarkers of susceptibility indicate the constitutive responsiveness to contaminant exposure,
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such as through tolerance/resistance pathways as described for non-sensitive/hypersensitive
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phenotypes.12 Some or all of these categories of biomarkers comprehensively reflect the “whole”
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organism response to ENM exposure, and provide valuable knowledge for the determination of
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actual risk.13,14
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Whole-genome studies performed in Arabidopsis thaliana (L.) Heyhn15-17 revealed some 18
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of the main biological processes involved in the response to different ENMs. Marmiroli et al.
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screened A. thaliana mutants for tolerance to cadmium sulfide quantum dots (CdS QDs) and
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subsequently combined physiological/genetic characterization of the phenotypes with a genome-
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wide transcriptomic analysis. A systems biology approach led to identification in the wild type
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line (accession Ler-0) of approximately 200 impacted genes, including those involved in
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metabolic functions, detoxification/stress response, transport, protein synthesis and DNA repair.
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This approach also enabled a determination of the mechanistic basis of CdS QDs tolerance and
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the key genes involved in the plant’s response to exposure. Last, a comparison showed that the
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response to cadmium ions and to CdS QDs were clearly different at both the molecular and
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physiological level.
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Three different types of metal oxide nanoparticles (NPs) were used in the current study:
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copper oxide (CuO NPs), cerium oxide (CeO2 NPs) and lanthanum oxide (La2O3 NPs). In
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addition, corresponding bulk and ion controls were included. These particles were chosen as
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model analytes given their properties and potential for wide scale usage. CuO NPs are used in
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catalysis, superconducting materials, thermoelectric and sensing materials, propellant, glass, and
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ceramics.19 In addition, CuO NP interactions with plant species of agronomic interest have been
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initiated.20,21 CeO2 NPs are utilized in catalysis, electrolyte and electrode materials, UV and
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infrared absorbents, oxidation and heat resistant coatings.22 Studies performed with CeO2 NPs on
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A. thaliana and Phaseolus vulgaris L. showed variable effects on growth, physiological response
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and nutritional quality.23,24 La2O3 NPs are an emerging material used as a magnetic nanoparticle
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for electronic devices, in laser crystals and optics, catalysis, propellant and biosensors. Recent
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studies have also focused on the toxicity and trophic transfer of La2O3 NPs.25,26
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Data obtained from A. thaliana guided our comparative analysis in other plants, which
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can then provide important information required for assessing the environmental and public
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health risks related to ENM exposure. The primary aim of this work was the identification of
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biomarkers for exposure and effects in plants exposed to several ENMs. Previously identified
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candidate genes were tested in two species of agricultural interest, tomato and zucchini, whose
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genomes have been characterized.27,28 This approach enabled us to use a diverse set of ENMs
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and plants; the intent being to identify genes consistently modulated regardless of particle type
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and species. Following ortholog identification, a transcriptional approach was applied to validate
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the “plant-specific” targets found in A. thaliana, and to find genes commonly involved in
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response to ENMs; these genomic analyses were coupled with elemental and physiological
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analyses.
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Experimental section
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Plants and NP treatments
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Copper oxide (CuO) nanopowder (99% purity, 40nm particle size) and lanthanum oxide
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(La2O3) nanopowder (99.99% purity; 10-100 nm particle size range) were purchased from US
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Research Nanomaterials, Inc. (Houston, TX, USA). Cerium oxide (CeO2) nanopowder (
