Two-photon Microscopy and Spectroscopy Studies to Determine the

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Environmental Measurements Methods

Two-photon Microscopy and Spectroscopy Studies to Determine the Mechanism of Copper Oxide Nanoparticle Uptake by Sweetpotato Roots during Post-harvest Treatment Nestor J Bonilla-Bird, Aurelio Paez, Andres M Reyes, Jose A. HernandezViezcas, Chunqiang Li, Jose R Peralta-Videa, and Jorge L. Gardea-Torresdey Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02794 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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Environmental Science & Technology

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Two-photon Microscopy and Spectroscopy Studies to Determine the Mechanism of Copper

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Oxide Nanoparticle Uptake by Sweetpotato Roots during Post-harvest Treatment

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Bonilla-Bird, N.J.,† Paez, A.,‡ Reyes, A., ‡ Hernandez-Viezcas, J.A., ║,§ Li, C.,‡ Peralta-Videa, J.R.,

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†,║,§

Gardea-Torresdey, J.L.†,║,§*

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† Environmental Science and Engineering PhD program, The University of Texas at El Paso; 500

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West University Ave. El Paso, TX 79968 United States

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║

Department of Chemistry and Biochemistry, The University of Texas at El Paso; 500 West

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University Ave. El Paso, TX 79968 United States

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§

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Texas at El Paso, 500 West University Ave. El Paso, TX 79968 United States

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‡

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TX 79968 United States.

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*Corresponding author: J. Gardea ([email protected]); phone 915 747 5359; fax: 915 747-5748

UC Center for Environmental Implications of nanotechnology (UC CEIN), The University of

Department of Physics, The University of Texas at El Paso, 500 West University Ave. El Paso,

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17

ACS Paragon Plus Environment


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Abstract

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The interaction of engineered nanoparticles with plant tissues is still not well understood. There is

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a lack of information about the effects of curing (post-harvest treatment) and lignin content on

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copper uptake by sweetpotato roots exposed to copper-based nanopesticides. In this study,

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Beauregard -14 (lower lignin) and Covington (higher lignin) varieties were exposed to CuO

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nanoparticles (nCuO), bulk CuO (bCuO), and CuCl2 at 0, 25, 75 and 125 mg/L. Cured and uncured

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roots were submerged into copper suspensions/solutions for 30 min. Subsequently, root segments

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were sliced for imaging with a 2-photon microscope, while other root portions were severed into

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periderm, cortex, perimedulla, and medulla. They were individually digested and analyzed for Cu

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content by inductively coupled plasma-optical emission spectroscopy (ICP-OES). Microscope

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images showed higher fluorescence in periderm and cortex of roots exposed to nCuO, compared

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with bCuO. At 25 mg/L, only bCuO showed higher Cu concentration in the periderm and cortex of

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Beauregard -14 (2,049 mg/kg and 76 mg/kg before curing; 6,769 mg/kg and 354 mg/kg after

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curing, respectively) and in cortex of Covington (692 mg/kg before curing and 110 mg/kg after

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curing) compared with controls (p ≤ 0.05). In medulla, the most internal tissue, only Beauregard-

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14 exposed to 125 mg bCuO/L showed significantly (p ≤ 0.05) more Cu before curing (17 mg/kg)

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and after curing (28 mg/kg), compared with control. This research has shown that the 2-photon

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microscope can be used to determine CuO particles in non-dyed plant tissues. The lack of Cu

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increase in perimedulla and medulla, even in roots exposed to high CuO concentrations (25 mg/L),

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suggests that nCuO may represent a good alternative to protect and increase the shelf life of

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sweetpotato roots, without exposing consumers to excess Cu.

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Keywords: CuO nanoparticles, ICP-Ms, 2-photon microscope, Periderm, Medulla, Curing, Cu

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uptake


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INTRODUCTION

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The literature indicates a rapid increase in the use of nanomaterials (NMs) in agriculture industry.

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Currently, 9% of the nanoproducts targeted for agri/feed/food application are used in agriculture.1

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Moreover, there are several NMs in development for application as nanopesticides or

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nanofertilizers. Nano-compounds of zinc, iron, and copper have been shown to have great potential

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as nanofertilizers.2 In addition, copper-based NMs, whose global production for 2010 was

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estimated at 200 tons,3 may have additional applications, since Cu has been approved as a

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pesticide in organic agriculture.4 The fast development of agriculture-oriented nanoproducts would

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reduce the amount of Cu used in the production and shelf preservation of several types of produce.

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So far, several Cu-based nanoparticles (NPs) have been tested to control fungus and bacteria, with

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promising results. CuO NPs, (nCuO), have proven to be efficacious of controlling coliform

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bacteria in ultra-filtrated cheese.5 It was also reported that Cu-based NPs are able to control fungi

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at field level.6 These researchers reported that nCuO, at 150 mg/l (15 g/hl), controlled about 60%

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of Phytophthora infestans in field grown tomato (Solanum lycopersicum).7

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Sweetpotato (Ipomoea batatas L. Lam.) ranks seventh in the world in staple food production and

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fifth in developing countries. The world production is around 105 million tons/year, while the

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production in the US is around 23.8 billion tons/year.8 Sweetpotato plants have different types of

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roots including storage, pencil, fibrous, and string roots.9 Storage roots, the edible portion of sweet

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potato plants, are carefully cured and stored before they are exposed to the market. Curing implies

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the storage of roots under special temperature and ventilation for three to five days right after

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harvesting.10 During the curing, the outermost parenchyma cells wounded at harvest desiccate,

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while the internal parenchyma cells suberize.11 These changes heal wounds and protect the roots

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from future microbial infections throughout the storage period. Improper handling during a long



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curing period (one to four months)12 may lead to a loss of up to 65% due to a reduction of

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freshness or fungal infestation. Several fungicides have been used to control sweetpotato fungal

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root rots, dicloran being the most used. However, foods with dicloran residues are no longer

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accepted in many markets including infant food, organic, and export.13 Thus, other fungicides,

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including Cu have been tested.14 However, until now, only ionic Cu has been utilized; thus, new

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Cu products, including nano forms, may have great potential in preserving the quality of sweet

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potato roots, before they reach the market.

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To the authors’ knowledge, only one publication has described the interaction of metal oxide NPs

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with sweetpotato.15 These researchers cultivated sweetpotato slips in soil amended with either

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CeO2, CuO, or ZnO NP (100, 500, or 1000 mg/kg of the respective metal) and evaluated treatment

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effects on storage root production, metal concentration, and possible dietary intake of Ce, Cu, or

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Zn. The authors reported that the peel retained more Ce than Cu or Zn; 15 however, there is no

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information about the role of the peel components on the retention mechanism.

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Confocal microscopy has been used to observe the root uptake of nanoparticles.16 However, this

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technique requires the staining of non-fluorescent NPs. The Two-photon microscopy (TPM)

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offers the advantage of observing the uptake of NPs without staining. The capacity of TPM to

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obtain high depth of field images is greater than that of confocal microscopy. However, this is

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limited by the thickness of the samples17 because the quality of the image depends on the

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scattering and the absorption coefficients of the tissue, the type of fluorophore, and the objective

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lenses.18 CuO NPs may be good candidate to treat sweetpotato roots while they are cured.

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However, there is a lack of information concerning the penetration of the particles into the edible

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portion of the roots. It is hypothesized that the peel constitution will affect the retention of the NPs.


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Thus, the aim of this study was to determine the role of peel tissues and the curing process on the

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retention of CuO NPs and the possible penetration of NPs into the root.

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Variation in the constitution of sweetpotato root peel may have differential effects on the

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interaction with metal oxide NPs. Thus, the aim of this study was to determine the role of peel

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tissues and the curing process on the retention of CuO NPs and the possible penetration of NPs

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into the root. The roots of two sweetpotato varieties, with different lignin content, were exposed to

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CuO NPs, bulk CuO, and CuCl2. Subsequently, the samples of roots were screened with a two-

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photon microscopy and analyzed with inductively coupled plasma- optical emission spectroscopy

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to determine the retention of CuO and the absorption of Cu. Two-photon microscope has been

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used to track cell mobility in zebrafish.19 2-photon microscope, combined with nanoparticle-

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assisted near-infrared has been used for in vivo image of fluorescent nanoparticles in mice ear.20

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To the authors’ knowledge, this is the first time that a two-photon excited fluorescence and

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second-harmonic generation (SHG) microscope has been used to image metallic particles into the

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edible tissue.

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MATERIAL AND METHODS

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Characteristics of the Cu products and suspension/solution preparations. Commercial nano

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CuO (nCuO), bulk copper oxide, (bCuO), and reagent grade salt CuCl2 (Sigma Aldrich, St. Louis,

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MO) were used in this research. These materials were supplied by the UC Center for

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Environmental Implications of Nanotechnology. The physicochemical properties of both the nCuO

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and bCuO were previously determined.21, 22 Table S1 in Supporting Information (SI) shows major

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physicochemical properties of the particles used in this study. The nCuO had 50 nm, rhombus

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irregular, hydrodynamic diameter of 280 ± 15 nm and zeta potential of -34.4 ± 0.5 mV. bCuO had

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particles smaller than 1 µm with diverse morphology, hydrodynamic diameter of 376 ± 26 nm, and



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zeta potential of -42.7 ± 0.153 mV.22 Suspensions/solutions of Cu NP or compounds were prepared

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at 0, 25, 75, 125 mg/L in Millipore water (MPW) and homogenized by sonication in a water bath

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(Crest Ultrasonics, Trenton, N) at 25 ºC for 30 min and 180 watts. The 25 mg/L treatment was

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selected because the average Cu concentration in soil of the United States is 20 mg/kg,23 while 75

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mg/L was selected to mimic the highest- Cu concentration reported in agricultural soils amended

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with sewage sludge (75 mg/kg).24 The 125 mg/L treatment represents 83% of the CuO NPs used to

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control fungi at field level.6, 24

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Preparation of root samples and Cu treatment applications. Storage roots of Covington and

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Beauregard-14 sweetpotato varieties varying in peel lignin content were used in this study.

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Beauregard-14 has 156 g/kg and Covington 178 g/kg lignin in the peel; they are significantly

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different at p ≤0.05.25 Both varieties were procured from Mississippi State University, Pontotoc

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Experiment Station. Enough roots of both varieties were separated in two sets, one set was cured

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and the other one was uncured. For curing, roots were set at 29 ºC and 80% relative humidity for

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10 days in a growth chamber (Environmental Growth Chamber, Chagrin Falls, OH). Uncured roots

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were kept in the laboratory at ~24 ºC and 47% relative humidity. At the end of the curing process,

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uncured and cured roots were individually tied up with a cotton thread and immersed in the Cu

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suspensions/solutions for 30 min under continuous stirring. The exposure time of 30 min was

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selected because the treatment period before storing in the curing facility is very short. Roots were

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kept in the laboratory for 72 h after treatment and processed for microscopy imaging and elemental

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analyses. Each Cu treatment had three replicates and one root was used as a replicate. Three

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replicate samples of uncured and cured roots were used as control (no Cu application).

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Sample preparation for Cu determination. For element determination, roots were washed gently

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with Millipore water (MPW) to remove the debris adhered to the periderm of the storage roots.


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The washing procedure was also supposed to remove most of the CuO particles adhered to the

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roots surface. The roots were not washed with acid solution to avoid deterioration of the flesh

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tissue. After washing, the layers of tissues from each root were pulled apart with a bistouri

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(scalpel) in the following order: periderm, cortex, perimedulla, and medulla. Each tissue was put in

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a paper envelop, oven dried at 70 ºC for 72 h, and manually ground using a ceramic mortar and

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pestle. Dried samples of 0.2 g were acid digested in a block digestion system (DigiPREP MS, SCP

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Science. Canada) with 3 mL of 30% plasma pure HNO3. Digests were adjusted to 50 mL with

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MPW and analyzed for Cu content by using an inductively coupled plasma-optical emission

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spectrometer (ICP-OES, Perkin-Elmer optima 4300 DV, Shelton, CT). To evaluate the digestion

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and analytical methods, (QC/QA), spiked samples and the standard reference material 1547 from

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the National Institute of Standards and Technology were treated and analyzed in a similar way as

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the root samples. The recovery rate was 99%. The detection limit for copper was determined by

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reading eight replicas of the blank. The mean plus three standard deviations (µ± 3SD) was in the

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range of 50 µg/L.

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Sample preparation for microscopy imaging. For microscopy observation with the 2-photon

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microscope, fresh transversal thin slides from plants exposed to bCuO and nCuO after curing were

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mounted in water-immersion objective lens (Olympus LUM Plan FLN) and observed in a

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nonlinear optical microscope, which can detect both 2-photon excited fluorescence. The light

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source is a femtosecond Ti: Sapphire laser (Spectra-Physics, Mai-Tai HP). Our experiment was set

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at the Mai Tai at 710 nm, 200 Watts. Its pulse duration was about 100 fs width at a repetition rate

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of 80 MHz. The wavelength of the laser was tuned from 690 to 1040 nm, with the maximum

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power up to 2.5 Watts. The fluorescence signal from the sample was deflected with the 665 nm

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long-pass dichroic mirror. A second long-pass dichroic beam splitter was used to separate the blue



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and green/red fluorescence signal. The blue signal was deflected by dichroic beam splitter and

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transmitted through a 417-477 nm band-pass filter. The green signal was transmitted through a

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500-550 nm band-pass filter, while the red signal through a 570-616 nm band-pass filter. The

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outputs of the three signals were individually detected by photomultiplier tubes set at 0.9V, 2.20V,

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and 2.10 V for the red, green, and blue channels, respectively. The outputs of these three signals

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were fed into red/green/blue channels of a frame grabber installed on a computer. The images in X-

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Y plane were acquired through a home built software program.26 Covington variety was selected

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because its higher lignin content in periderm could limit the Cu transport to cortex.25

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Statistics analysis. The analysis of variance (ANOVA) of the interaction between the main factors

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(curing, varieties, treatments, and root tissues) and the Cu concentrations was performed using

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univariate analysis (SPSS 19.0 package, Chicago, IL). The Eta-squared (kind of t-test family

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similar to r2) was used to assign the portion of variation to each one of the factors. The differences

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between averages of triplicate data per treatment and control was calculated with the Tukey HSD

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multiple comparisons test. The data presented in tables and figures are means ± the standard error

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(SE).

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RESULT AND DISCUSIONS

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Figure 1 shows a description of the distribution of tissues in the sweetpotato storage root. As seen

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in this figure, the periderm has three layers of tissues, which contain lignin. Lignin is an organic

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polymer that increases during the curing process, which has been proven to bind metals.12

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Microscopy analysis

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Figure 2 shows 2-photon microscopy images of the control (A) and the CuCl2 treated Covington

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variety roots (B). The samples were from roots exposed to the CuCl2 after curing. The figures

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show the periderm in the left part and the cortex in the right part. The autofluorescence shown in


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the control sample is due to the suberization of the cell walls, which gives firmness and resistance

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to excoriation to the roots.23The insert in Figure 2B shows a lack of fluorescence from the CuCl2

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solution. Figure 3 shows the 2-photon images of Covington variety roots exposed after curing to

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75 mg/L nCuO (A) or bCuO (B) as shown in Figure 3A, less fluorescence points were observe in

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the cortex of nCuO treated roots, compared to bCuO (Figure 3B). The inserts in both figures show

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the fluorescence of the CuO suspensions. The areas in squares show CuO particles in the cortex

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tissues. The 2-photon microscope images of nano CuO and bulk CuO treated samples showed

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differences, compared with control. For instance, the root exposed to 75 mg/L of nCuO (Figure

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3A) shows fluorescent points inside of the cortex tissue, which is located below the periderm. Cell

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walls in the periderm show higher bright intensities than the inside cells. In the cortex tissue,

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fluorescence from several nanoparticles stuck in intercellular spaces were observed (arrow). Due

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to the fast flash moment of the excitation of the nanoparticles, only a few 2-photon fluorescences

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were obtained in the image. The internal part of the root cortex (Figure 3A) shows an increase in

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the number of 2-photon fluorescences from different bulk particles. Images of roots exposed to

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nCuO at 75 mg/L also showed 2-photon fluorescence (Supporting Information, Figure S1),

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indicating that these particles also penetrated the tissues, the cortex tissue in (S1-A and S1-B). In

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addition Figure S1 (C) shows the surface graph to detect the nCuO between the cell walls. The

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fact that the 2-photon fluorescences were observed between the cells suggests the photochemical

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decomposition, which occurs when the particles are excited by the laser beam, allows their

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localization within the root tissues. The molecular constitution of each material has a different

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reaction when they are impacted by the laser beam, creating varied ranges of fluorescence.27 The

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signal/noise ratio for nCuO were 26/25, 21/168, and 27/122 for red, green, and blue, respectively.

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For bCuO were 22/27, 20/181, and 26/122 for red, green and blue, respectively.



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Effect of factors on Cu accumulation in roots. The statistical analysis of Cu concentration in

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roots showed effects of single factors variety, tissue, and Cu treatments (Table S2, SI). In addition,

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the double, triple, and quadruple interactions between curing × tissue, curing × variety × Cu

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treatments, variety × tissue × Cu treatment, and curing × variety × tissue × Cu treatments were also

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statistically different. The Eta-squared statistics showed that the higher variance was due to the

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differences between tissues and the interaction tissue × Cu treatment, with 50% and 18%,

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respectively (p ≤ 0.001). Table S2, SI, shows the Tukey test for the main factors curing, varieties,

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and tissues. As seen in this table, the curing, as a whole, had no statistical effects; however, there

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were effects for varieties and tissues. Beauregard-14, the variety with less lignin.25 had lower Cu

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concentration (720 ± 108 mg/kg), with differences statistically significant at p ≤ 0.05, while

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Covington, the variety with higher lignin, 25 had 1003 ± 148 mg/kg, with differences statistically

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significant at p ≤ 0.005. It has been demonstrated that lignin binds metals through ion-exchange.

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The binding strength of Cu to lignin is one the strongest.28

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Copper concentration in individual tissues

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The Cu concentration was analyzed from the outermost tissue, the periderm, to the innermost one,

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the medulla. Figure 5 shows the Cu concentration in cortex, and Figure S2, SI the periderm of

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uncured and cured storage roots of variety Beauregard-14. Before curing, at 25 mg/L, only roots

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exposed to bCuO had significantly more Cu, 2,049 mg/kg in the periderm (the most external

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layer). While at 75 and 125 mg/L, all treatments showed significantly more Cu. At 125 mg/L, the

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increases from bCuO and nCuO were 6,105 mg/kg and 2,635 mg/kg. After curing, similar results

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were observed at 25 mg/L, but not at 75 and 125 mg/L. At 75 mg/L, there was no significant

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increase in Cu concentration, while at 125 mg/L, bCuO and nCuO showed 5,437 mg/kg and 4,147

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mg/kg more Cu, respectively, compared with control. It is possible that the small differences in


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hydrodynamic dimeter caused the similarity in Cu accumulation. Hong et al.29 demonstrated that

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the removal of CeO2 NPs from cucumber leaves washed with water was lower, compared to the

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washing with CaCl2 or HNO3. As expected, less Cu was found in the cortex, the tissue beneath

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periderm (Table 1 and Table S3). As shown in Figure 6, variety Covington before curing, Cu

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concentration in cortex followed a similar trend than in periderm Figure S2. After curing, no

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effects were observed at 75 mg/L, while at 25 mg/L and 125 mg/L, bCuO increased (354 mg/kg

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and 144 mg/kg) and nCuO increased (142 mg/kg and 208 mg/kg) Cu in cortex (p ≤ 0.001). The

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difference in Cu accumulation between uncured and cured roots was attribute to the suberization

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and lignification of the internal parenchyma cells.11

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Figure 6 shows data for cortex and Figure S3 shows data for periderm in Covington variety (higher

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lignin content). As seen in this figure, before curing, exposure to 25 mg/L of the three Cu-based

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compounds did not increase Cu in periderm, compared with control. While after curing, at 25 mg

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bCuO/L increased Cu in periderm by 8,311 mg/kg. In addition, before curing, at 75 mg/L, only

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nCuO increased Cu in periderm (7,803 mg/kg), and 125 mg/L, both bCuO and nCuO showed more

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Cu in periderm (7,052 mg/kg and 9,235 mg/kg). In cortex, before curing, all the treatments

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increased Cu. After curing, all bCuO concentrations increased Cu in periderm, and at 25 mg/L and

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75 mg/L increased Cu in cortex- Figure 7- (110 mg/kg and 126 mg/kg, respectively). On the other

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hand, at 75 and 125 mg/L, nCuO increased Cu in periderm (4,279 mg/kg and 7,055 mg/kg,

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respectively) and only at 125 mg/L, increase Cu in cortex (77 mg/kg). CuCl2, at any

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concentrations, increased Cu in periderm and cortex, before and after curing. The fact that Cu

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concentration in cortex from particulate compounds were higher than in CuCl2 treatments may be

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due to both the uptake of aggregates that contain a high proportion of Cu and to the high atomic

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percentage Cu in the CuO. The lack of Cu increase in uncured roots exposed to 25 mg/L could be



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due to the lower lignin content,11 or the natural Cu accumulation from soil,28 while after curing, the

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Cu increase was attributed to the increment in lignin, which occurs during the curing process.11

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There were not much differences in the most internal tissues, perimedulla and medulla (Figures 8

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and 9). Before curing, the perimedulla of Covington and medulla of Beauregard-14 showed

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significantly higher Cu concentration, respect to control, in roots exposed to 125 mg/L of bCuO

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After curing, none of the treatments increased Cu in perimedulla or medulla of both varieties,

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except bCuO, which at 25 mg/L increased Cu in perimedulla of Beauregard-14 (p ≤ 0.001. The

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fact that only bCuO showed significantly more Cu in the internal tissues might be a results of the

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lower dissolution of bCuO, compared with nCuO.30 bCuO aggregates could remain in the tissues

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resulting in high total Cu concentration. Thus, some of the bCuO particles that penetrated into

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cortex dissolved, allowing the movement of the Cu ions into deeper tissues. In addition, it is

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assumed that Cu did not increase in perimedulla and medulla of cured roots because Cu was

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retained in suberized and lignified external tissues. Kulik et al. demonstrated that lignin in decay of

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wood strongly bind Cu through the COO- binding group.31,32 Unfortunately, there are no previous

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reports about the Cu uptake by non-growing plant/tissue exposed to nCuO; thus, it is difficult to

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make comparisons. The uptake of Cu by the roots of growing plants exposed to nCuO, bCuO, and

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CuCl2 has shown different results. For instance, Rawat et al.30 reported that the roots of bell pepper

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cultivated in soil amended with 250 mg/kg of bCuO, nCuO, or CuCl2 had similar Cu

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concentrations. Conversely, the roots of rice (Oryza sativa L.) plants grown in soil amended with

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1000 mg/kg of nCuO had significantly more Cu than the roots of plants exposed to the same

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concentration of bCuO.33 This suggest that the Cu uptake of non-growing tissues cannot be

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compared with that of growing tissues.


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A comparison between Covington and Beauregard-14 shows some differences in Cu accumulation

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(Table 1). The data suggests and interaction variety × treatment × tissues (Table S1). In

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Beauregard-14, the variety with less lignin content (156 g/kg), before curing, all the compounds, at

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the highest concentration, showed significant Cu concentration in periderm and cortex ,while after

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curing, at 125 mg/L, both bCuO and nCuO, showed significantly more Cu in periderm and cortex,

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compared with control. However, in uncured roots of Covington, the variety with higher lignin

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content (178 g/kg), both bCuO and nCuO, at 125 mg/L, significantly increased Cu in the periderm

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(p ≤ 0.001), but had no effect in cortex. While in cure roots, the same compounds, at the same

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concentration, increased Cu in periderm but not in cortex. A comparison of the total Cu

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contraptions, before and after curing (Table S3), shows that in Beauregard-14, only the periderm

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showed more Cu after curing than before curing, while converse data was found in cortex,

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perimedulla and medulla. The difference between uncured and cured roots was statistically

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significant (p ≤ 0.05) only in medulla (Table S3); the data showed that the difference was caused

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by bCuO (Figure 6). On the other hand, in Covington, the Cu concentrations in all tissues where

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higher after curing; however, the differences did not show statistical significance. The data

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suggest that the curing, lignin content, tissue, and compound influence the Cu uptake in

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sweetpotato roots. Suberization during curing increases lignin,11 which has shown to bind Cu

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ions;33 this could explain the reduction of Cu concentration in the ionic treatment. On the other

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hand, Figures 2-4 show that bCuO and nCuO penetrated the tissues through the apoplast, which

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explains the Cu increase in roots treated with such compounds.

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CONCLUSIONS

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This research has shown, for the first time, the use of the 2-photon microscope for the observation

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of fluorescence from particulate CuO in sweetpotato root tissues. The fluorescence of the particles



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was observed in the periderm and cortex. The data showed that the nCuO and bCuO are able to

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penetrate the tissues through the fluid uptake into the xylem of the sweetpotato roots. Results also

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showed that lignin content has a strong effect in retaining the NPs in the exterior layers. As a

296

whole, the curing process seems to have no effect in the absorption of the copper products and the

297

copper content in internal tissues, except for bCuO treatment that at 25 mg/L caused and effect in

298

Beauregard-14, the variety with less lignin content. The Cu concentration in internal tissues of

299

roots treated with nCuO was similar than control roots, which suggests there should be no risk of

300

Cu contamination in peeled roots. This also suggests that CuO nanoparticles may be used as an

301

alternative to preserve and increase the shelf life of sweetpotato roots before they reach the market,

302

without an overload of the copper content.

303


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15 304

ACKNOWLEDGEMENTS

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This material is based upon work supported by the National Science Foundation and the

306

Environmental Protection Agency under Cooperative Agreement Number DBI-1266377. Any

307

opinions, findings, and conclusions or recommendations expressed in this material are those of the

308

author(s) and do not necessarily reflect the views of the National Science Foundation or the

309

Environmental Protection Agency. This work has not been subjected to EPA review and no official

310

endorsement should be inferred. Authors also acknowledge the USDA grant 2016-67021-24985

311

and the NSF Grants EEC-1449500, CHE-0840525 and DBI-1429708. Partial funding was

312

provided by the NSF ERC on Nanotechnology-Enabled Water Treatment (EEC-1449500). This

313

work was also supported by the grant 2G12MD007592 from the National Institutes on Minority

314

Health and Health Disparities (NIMHD), a component of the National Institutes of Health (NIH)

315

and by the grant 1000001931 from the ConTex program. J.L. Gardea-Torresdey acknowledges the

316

Dudley family for the Endowed Research Professorship, the Academy of Applied Science/US

317

Army Research Office, Research and Engineering Apprenticeship program (REAP) at UTEP, grant

318

no. W11NF-10-2-0076, sub-grant 13-7, and the LERR and STARs programs of the University of

319

Texas System.

320 321

Supporting Information

322 323 324 325 326

The Supporting Information is available free of charge on the ACS Publications website. Two-photon microscopy images of the Covington variety root; copper concentration in periderm; Major physicochemical properties of the particles used in this study; tables of ANOVA

327 328



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Figure legends

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Figure 1. The positions of the tissues as seen in transverse section on sweetpotato storage root. The outermost the periderm, followed by cortex, perimedulla, and medulla or edible part. Figure 2. (A) Two-photon microscopy image of control root of the Covington variety showing the fluorescence of lignin in cell walls of periderm. The arrows are pointing the cell structures in the periderm and cortex. The opaque segment correspond to the cortex. (B) Two-photon microscopy image of the Covington variety root exposed after curing to 75 mg/L of copper chloride. The insert picture corresponds to the copper chloride solution in DI water. There was no fluorescent points. The signals were obtained at a wavelength of 710 nm and a power of 200 mW. Figure 3. (A). Two-photon microscopy image of the Covington variety root exposed to 75 mg/L of CuO nanoparticles showing the fluorescence of lignin in the periderm. The arrows are pointing out the fluorescence from the CuO nanoparticles inside the square in cortex. The insert corresponds to the nCuO suspension in DI water, the fluorescence is from the nCuO particles (B) 2-photon microscopy image of the Covington variety root exposed to 75 mg/L of bCuO showing the fluorescence of lignin in the periderm and the fluorescence of the bCuO particles. The arrow is pointing out the fluorescence from bCuO particles inside the square in cortex. The insert corresponds to the bCuO suspension in DI water, the fluorescence is from the bCuO particles. The fluorescence signals were obtained at a wavelength of 710 nm and a power of 200 mW. Figure 4. (A) Two-photon microscopy image of the Covington variety root exposed to 75 mg/L of nCuO particles showing the fluorescence (square) of the nCuO particles in the cortex tissue. The particles are located between the intercellular spaces (B) Image of the Covington variety root exposed to 75 mg/L of bCuO (square) of the bCuO particles inside of the vascular tissue. The square shows the bCuO particles between the cells walls. The signals were obtained at a wavelength of 710 nm and a power of 200 mW. Figure 5. Copper concentration (mg/kg dwt) in cortex of the Beauregard-14 roots exposed to nCuO, bCuO, and CuCl2 at 0, 25, 75, and 125 mg/L, before and after curing. Data are average of three replicates ± SE. Different letters stand for statistical differences at p ≤ 0.05, (n = 3). Figure 6. Copper concentration (mg/kg dwt) in cortex of the Covington roots exposed to nCuO, bCuO, and CuCl2 at 0, 25, 75, and 125 mg/L, before and after curing. Data are average of three replicates ± SE. Different letters stand for statistical differences at p ≤ 0.05, (n = 3); (ns) stands for not significant. Figure 7. Copper concentration (mg/kg dwt) in perimedulla and medulla of the Beauregard-14 roots exposed to nCuO, bCuO, and CuCl2 at 0, 25, 75, and 125 mg/L, before and after curing. Data are average of three replicates ± SE. Different letters stand for statistical differences at p ≤ 0.05, (n = 3); (ns) stands for not significant. Figure 8. Copper concentration (mg/kg dwt) in perimedulla and medulla of the Covington roots exposed to nCuO, bCuO, and CuCl2 at 0, 25, 75, and 125 mg/L, before and after curing. Data are



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average of three replicates ± SE. Different letters stand for statistical differences at p ≤ 0.05, (n = 3); (ns) stands for not significant.

473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490


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Table 1. Tukey HSD – Post Hoc test for the main factors. The sweetpotato roots were exposed to nCuO, bCuO, and CuCl2 at 0, 25, 75, and 125 mg/L.

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Factors 1. Curing Before curing After curing

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Copper concentration (mg/kg) 841 ± 176 882 ± 186

2.Variety Beauregard-14 Covington

720 ± 108* 1003± 148**

3.Tissues Perimederm Cortex Perimedulla Medulla

3,326 ± 261*** 99.0 ±18** 15.0±2** 6.3±1**

*significant at p

Two-photon Microscopy and Spectroscopy Studies to Determine the

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