Uptake, Distribution, and Transformation of Zerovalent Iron

Aug 5, 2018 - Division of Environmental Science and Engineering, Pohang University of Science and Technology (POSTECH) , Pohang 37673 , Republic of ...
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Ecotoxicology and Human Environmental Health

Uptake, distribution and transformation of zerovalent iron nanoparticles in the edible plant Cucumis sativus Amarendra Dhar Dwivedi, Hakwon Yoon, Jitendra Pal Singh, Keun Hwa Chae, Sangchul Rho, Dong Soo Hwang, and Yoon-Seok Chang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01960 • Publication Date (Web): 05 Aug 2018 Downloaded from http://pubs.acs.org on August 6, 2018

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Uptake, distribution and transformation of zerovalent iron

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nanoparticles in the edible plant Cucumis sativus

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Amarendra Dhar Dwivedi1,2†‡, Hakwon Yoon1†, Jitendra Pal Singh3, Keun Hwa Chae3,

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Sang-chul Rho1,2, Dong Soo Hwang1,2, and Yoon-Seok Chang1*

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Division of Environmental Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea

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Division of Integrative Bioscience and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea

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Advanced Analysis Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea

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*

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(Y.-S. Chang) E-mail: [email protected]; Tel.: +82 54 279 2281; Fax: +82 54 279 8299

Corresponding authors contact information:

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These two authors contributed equally to this work.



Current address: Department of Chemistry, Y.N. College (J.P. University), Chapra, Bihar, India

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TABLE OF CONTENTS

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ABSTRACT

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Here, we investigated the fate of nanoscale zerovalent iron (nZVI) on the Cucumis sativus under

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both hydroponic and soil conditions. Seedlings were exposed to 0, 250 and 1000 mg/L (or mg/kg

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soil) nZVI during 6–9 weeks of a growth period. Ionic controls were prepared by using Fe-

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EDTA. None of the nZVI treatments affected the plant biomass. Based on the total iron contents

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and the superparamagnetic property of nZVI-exposed roots, there was no evidence of pristine

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nZVI translocation from the roots to shoots. Electron microscopy revealed that the transformed

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iron nanoparticles are stored in the root cell membrane and the vacuoles of the leaf parenchymal

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cells. X-ray absorption spectroscopy identified ferric citrate (41%) and iron (oxyhydr)oxides

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(59%) as the main transformed products in the roots. The shoot samples indicated a larger

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proportion of ferric citrate (60%) compared to iron (oxyhydr)oxides (40%). The 1.8-fold higher

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expression of the CsHA1 gene indicated that the plant-promoted transformation of nZVI was

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driven by protons released from the root layers. The current data provide a basis for two potential

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nZVI transformation pathways in Cucumis sativus: (1) interaction with low molecular weight

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organic acid ligands, and (2) dissolution-precipitation of the mineral products.

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INTRODUCTION

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Nanoscale zerovalent iron (nZVI) is a commercially used engineered nanoparticle (ENP) used

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for environmental remediation1-2. Although ample literature exists regarding the superb

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application prospects of nZVI for the removal of pollutants such as halogenated hydrocarbons,

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dyes, heavy metals, nitrate, and so on3-6, predicting the fate and risks associated with nZVI in the

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ecosystem is only in its infancy7. Notably, nZVI is the sole ENP that has been injected into the

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ground in large quantities and disperse with groundwater flow in unconfined aquifers, possibly

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exposing many aquatic and terrestrial organisms8. One concern in particular is that nZVI is a

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highly reactive reducing agent, and the toxicity of this agent may change upon its

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transformation9.

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The nature and degree of transformation, specifically, the biochemical alteration of chemical

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compounds within a living system, are reflected in the physicochemical properties,

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macromolecular interaction, and biologically mediated pathways10-12. Recently, there has been a

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growing consensus that bioavailable transformed species of ENPs may be taken up by biota,

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causing serious concerns for bioaccumulation and biomagnification in food chains and

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eventually impact on humans13. In groundwater, released iron oxide NPs find pathways, whether

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inadvertently or intentionally, into environmental matrices, where their high colloidal stability

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results in potential bioavailability and increased residence time8,

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environmental impact of nZVI on plants is still poorly understood, and formidable challenges

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have largely been ignored.

14-15

. However, the

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Plants are the vital primary producer in an ecological unit, supplying food to different consumer

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levels and concomitantly balancing ecological stress. Commonly, there are two conceivable

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pathways proposed to explain iron acquisition in plants: (1) reductive and proton-promoted

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processes and (2) secretion of plant-borne chelate transporters (phytosiderophores) with a high

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affinity for Fe(III)16. The reduction-based strategy is well documented in many nongraminaceous

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species. As an example, in response to iron deficiency in Arabidopsis thaliana, protons are

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released into the rhizosphere by AHA H+-ATPase activity in the epidermis. This release of

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protons typically lowers the pH of the surrounding media, increasing the solubility of the iron

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compounds17. Several dicotyledonous plants, such as Arabidopsis thaliana, Cucumis sativus and

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Solanum lycopersicum, produce organic ligands to enhance the iron solubility and uptake when

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iron is limited16. Low molecular weight organic acids (LMWOAs) are of particular importance

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among the plant root exudates due to their ability to mobilize insoluble iron by acidifying the

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rhizosphere and by providing carbon-rich environment, which support microbial growth and

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community structure18-20. Our plant of interest — cucumber (Cucumis sativus) — uses this

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acquisition strategy. However, the differences in NP uptake may arise due to the abiotic and

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biotic interactions in natural systems and plant-specific interactions, such as root exudates and

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environmental matrix conditions12. For example, Unrine et al. observed biotic and abiotic

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interactions between Ag nanoparticles and plants in aquatic microcosms to assess the fate and

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toxicity of NP21. However, the current awareness of such issues is rather limited.

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In the past few years, various researchers have employed synchrotron radiation-based advanced

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techniques to understand plant-ENP interactions at the atomic level. For example, specific

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studies of ENP behavior and their fate, examining Fe3O4 (ryegrass and pumpkin), CeO2

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(cucumber), ZnO (soybean), TiO2 (cucumber), and CuO (rice), are a few of the recent valuable

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contributions to this field22-26. However, such information from nZVI research is still lacking.

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Thus, to advance the field and awareness of the fate of nZVI in plants, this study relied on a

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detailed understanding of nZVI coordination chemistry (local electronic/atomic properties and

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their effect on plants intracellularly) and in turn characterized the distinctive transformed

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products by integrating near edge X-ray absorption fine structure (NEXAFS) and extended X-ray

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absorption fine structure (EXAFS) spectroscopy with high-resolution electron microscopy and

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related characterization techniques. Investigation of the behavior and fate of nZVI within the

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edible plant, via root or shoot, is imperative for understanding the risk of potential exposure

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and/or uptake by biota. To our knowledge, this is the first comprehensive report of nZVI

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transformation in plant research. Our finding reveals the transformation of nZVI by an edible

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cucumber plant in the natural environment, which should be considered in predicting the fate and

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risk concerns of nZVI for environmental remediation.

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MATERIALS AND METHODS

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Iron Nanoparticles. To create conditions relevant to the surrounding environment of a real

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remediation site, NANOFER STAR (NANOIRON, the Czech Republic), a commercially

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available nZVI, was used in this experiment instead of nZVI synthesized in the laboratory. The

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BET surface area of nZVI was 27.5 ± 2 m2 g-1, and the weight percent of Fe0 in NANOFER was

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65 - 80% based on the previous study27. The morphology of nZVI was analyzed using high-

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resolution transmission electron microscopy (HR-TEM, JEM-2010, JEOL Ltd., Japan).

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Hydrodynamic diameter and ζ-potential measurements of nZVI dispersed in media were

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performed on a Zetasizer (Nano ZS90, Malvern Instruments, UK). The structural and

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compositional characteristics of the NPs were investigated by X-ray diffraction (XRD, Rigaku

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Corporation, Japan) using monochromatic Cu Kα radiation (40 kV, 100 mA) over a range of 10 –

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90°.

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Plant Growth and nZVI Application. Cucumber seeds were sterilized and arranged in petri

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dishes with moist filter papers and placed in a dark growth chamber at 24°C. After five days of

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incubation, uniform seedlings were selected, and each seedling was anchored by a sponge and

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transferred into a 250 mL magenta box containing 200 mL of 1/4 strength Hoagland solution

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(Sigma-Aldrich, St. Louis, MO). The cucumber seedlings were cultivated in the plant growth

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chamber (DS-330DHL, Daewon Sci., Korea) at a controlled temperature (24°/22°C day/night

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cycle) and 60% relative humidity set to a 16-hour photoperiod for six weeks before treatment.

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Seedlings were grown under the Hoagland’s solution for 6 weeks, and exposed to 250 and 1000

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mg/L (or mg/kg soil) nZVI during the 6-9 week growth periods. The nZVI concentrations were

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chosen under the worst assumption that nZVI will spread to soil and groundwater at high

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concentrations. Control (non-nZVI treated) and ionic treatments using Fe-EDTA (2.5, 10, 50 and

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250 mg L-1) were prepared in both hydroponic and soil conditions in addition to the nZVI

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treatments in order to provide mechanistic information of iron uptake into the plant. The essence

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of the approach to preparing the experimental samples is described schematically in Figure 1.

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Each treatment consisted of eight plants (replicates), representing one set. Individual plant

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samples were combined in equal mass and homogenized. Each homogenate sample was then

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analyzed three times for the individual endpoint measurement.

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In the hydroponic culture, the nZVI slurry was mixed with autoclaved 1/4 strength Hoagland

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solution at pH 5.8 (adjusted with 0.1 N KOH, Sigma-Aldrich) and poured into a Magenta box at

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0, 250, and 1000 mg L-1 concentrations. The seedlings exposed to nZVI were placed in the

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growth chamber and allowed to grow for three weeks. The solution in each beaker was

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replenished with fresh nutrient solution twice per week to maintain a constant volume.

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In the soil culture, 0.2 kg of test soil (purchased from Hungnong Co., Korea) and nZVI slurry

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were mixed together in a 250 mL Magenta box to prepare for the soil treatment. The

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concentrations of nZVI in soil were 0, 250 and 1000 mg kg-1. The physiochemical property of

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test soil including pH, electrical conductivity, cation exchange capacity, organic matter levels

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and other nutrient analyses are described in the Supporting Information section (Table S1). The

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water content of the soil was maintained at 60-70%. Growth conditions remained the same as

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those of the hydroponic culture.

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Determination of Iron and Other Mineral Nutrients (Mg and Mn). Root tissues were washed

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in three successive baths of tap water (x3), 10 mM CaCl2 solution (x3), and DI water (x3) to

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remove adhered surface-bound iron particles. The sample preparation for shoot tissues followed

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the same procedure, excluding the CaCl2 treatment. Then, the plant samples were dried at 70°C

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for 3 d. Dried samples were dissolved in 60% HNO3 and 30% H2O2 at 105°C overnight. After

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diluting the nitric acid mixture, the elemental contents were measured with an inductively

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coupled plasma mass spectrometer (iCAP6300 DUO ICP-OES; Thermo Scientific). The total

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iron and other mineral nutrient contents including Mg and Mn were calculated using the USEPA

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SW-846 method28. QA/QC of ICP-OES analysis is provided in Supporting Information.

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Magnetic Property Measurement. Roots and shoots of washed plants were freeze-dried

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(FreeZone Plus 6; Labconco), and their dry weight was measured. Reference standards (nZVI,

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ferric citrate, lepidocrocite, goethite, and maghemite) and dried roots and shoots from nZVI

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treatment and control groups were used to detect the presence of iron NPs and to estimate the NP

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uptake. Magnetic measurements were carried out at room temperature using a superconducting

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quantum interference device (SQUID) magnetometer (MPMS-5, Quantum Design).

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Microscopic Observation. Plant tissues (root, leaf) were collected from hydroponically grown

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Cucumis sativus exposed to nZVI. Root and leaf samples were characterized with field emission-

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scanning electron microscopy (FE-SEM) and high resolution-transmission electron microscopy

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(HR-TEM). A detailed description of how to prepare the sample is provided in Supporting

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Information.

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Synchrotron-based X-ray Absorption Spectroscopic Investigations. Hydroponically grown

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cucumber plants treated with 1000 mg L-1 nZVI were harvested after 21 days. Freeze-dried

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samples were ground with a mortar and pestle in liquid nitrogen. In the Pohang Accelerator

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Laboratory, South Korea, soft and hard X-ray beamlines (operated at 3.0 GeV energy with a

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maximum storage ring current of 320 mA) were used to probe interactions of iron materials and

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model compounds. Near-edge X-ray absorption fine structure (NEXAFS) measurements were

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performed with the soft X-ray 10D KIST-PAL beamline. Additionally, extended X-ray

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absorption fine structure (EXAFS) measurements of the Fe K-edge were measured using the hard

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X-ray 1D KIST-PAL beamline. A detailed discussion of analysis conditions is provided in the

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Supporting Information. The spectral features that were obtained were shell-fit using the

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ARTEMIS program29. Linear combination fitting (LCF) of Athena (version 0.9.18) was used to

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estimate the sample composition.

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Quantitative Real-Time PCR (qRT-PCR). To evaluate the expression of the two genes

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encoding the plasma membrane (PM) H+-ATPase, CsHA1 and CsHA2, real-time PCR was

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performed30-31. Total RNA was separately isolated from 9-week-old plant roots using an RNeasy

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Plant Mini kit (Qiagen). cDNA conversion was performed with a cDNA reverse transcription kit

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(PrimeScriptTM RT Master Mix, Takara). Quantitative real-time PCR (qRT-PCR) was carried out

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with SYBR Premix EXTM Taq II (Takara) to detect amplified DNA. The cucumber α-tubulin

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was employed as an endogenous control. A detailed description of the conditions for qRT-PCR

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analysis is provided in Supporting Information.

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RESULTS AND DISCUSSION

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Characterization of nZVI and Effects on Plant Growth. The morphology and size of nZVI

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were characterized using HR-TEM. Although the primary size of nZVI was approximately 60

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nm, the particles were composed of aggregates having 200-300 nm diameters in distilled water

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(Figure S1). nZVI was negatively charged in Hoagland’s solution, and the hydrodynamic

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diameter of nZVI aggregates approached 8500 nm in a concentration of 1000 mg L-1 nZVI

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(Table S2). Additionally, the hydrodynamic diameter increased as the concentration of nZVI

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increased.

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The root and shoot growth of control and nZVI-treated plants is shown in Figure S2. The results

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indicated that none of the nZVI treatments affected the biomass of plants in either hydroponic or

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soil systems. Only the 1000 mg L-1 nZVI-treated plant shoots grown under the hydroponic

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conditions exhibited increased biomass (approximately 15%) early in the growth cycle. However,

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high concentrations of iron ion (Fe-EDTA) treatment significantly reduced plant growth.

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Although iron is an essential nutrient for plants, its high accumulation within plants can be

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toxic32. nZVI did not affect the plant growth despite the use of high concentrations of nZVI in

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this study. Even though nZVI dissolution will occur in the acidic solution, the dissolved Fe(II)

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ions were not detectable at pH 5.8 in this study (data not shown), probably because the acid-

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assisted dissolution of NANOFER nZVI is active from the pH below 333.

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Intriguingly, we observed the chlorosis (yellowing of leaves) with the naked eye in the leaves of

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the control plants, but not in the plants treated with nZVI (Figure S3 (a)). Chlorosis (yellowing of

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leaves) is usually caused by iron and other mineral nutrient deficiencies in plants34. To clarify the

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reason for the chlorosis in our study, other mineral nutrients that relate to photosynthesis (Mg

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and Mn) were determined. The results showed that the total Mg and Mn contents in plants were

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not significantly affected by nZVI injection (Table S3). The control group used in our

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experiments was based on ¼-strength Hoagland solution that contains a low concentration of

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iron ion (1.33 mg/L) not only as a minimum amount for plant growth but also to minimize the

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effect of other factors in this experiment. In addition, we did not observe the chlorosis because

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the soil contains a sufficient amount of iron for plant growth (Figure S3 (b)). Thus, iron

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deficiency was observed in the control group grown in hydroponics but was reduced by the

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additional iron uptake in plants treated with nZVI.

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Iron Accumulation in the Plant. Iron accumulation in the plant tissues was analyzed to

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identify the bioavailability of nZVI. The total iron content in the roots and shoots of cucumber

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treated with nZVI was higher than in the control, with most of the iron in the plants accumulating

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in the roots (Figure 2). However, in the group treated with Fe-EDTA, total iron content in the

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roots was not significantly different from the total iron content of the control, while a large

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amount of iron was translocated into the shoot. In addition, the total iron contents are relatively

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independent of the dosage of nZVI. As a result, the translocation factor (Cshoot / Croot; C = total

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iron content) of nZVI-treated plants was lower than the translocation factor of the control at both

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concentrations of nZVI (250 and 1000 mg L-1) (Table S4). In other words, nZVI taken up by the

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root was poorly translocated to the aerial part of the plant. Our results are in good agreement

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with the previous study showing translocation factors of Zn in the ryegrass was much lower

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under ZnO NP exposure than Zn2+ treatments, implying that few ZnO nanoparticles could

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translocate to the shoots35. The total iron content in the soil-grown plants showed a tendency

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similar to the plants cultivated by hydroponics, but the degree of the tendency was decreased

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(Figure S4). One possible reason for this tendency would be the reduced mobility of nZVI in soil

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compared to water owing to its enhanced aggregation, absorption of soil particles and/or

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interaction with natural organic matters. It was impossible to distinguish whether the results

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analyzed by ICP-OES were obtained from the ionic form or the NP itself. Presumably, Fe ions

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dissolved out of nZVI could also affect to the total iron content27.

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To overcome this disadvantage, SQUID measurements were performed to observe the

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movement of magnetic particles in the plant22, 36. Figure 3 depicts the magnetic behavior in plant

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tissues and reference compounds of possible transformed products of nZVI in the cucumber

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(detailed discussion is provided in a later section). Most samples displayed a diamagnetic

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property (straight line) that is commonly observed in the biological tissues. Interestingly, only

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the plant roots treated with nZVI showed a weak superparamagnetic behavior similar to the

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behavior recorded for nZVI or iron (oxyhydr)oxide, indicating that more magnetic material, not

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ions, accumulated in roots. The same phenomenon was also found in the soil-grown plants

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(Figure S5). Combined with the total iron content results, this phenomenon also suggests that

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pristine nZVI was not transported from the root to the aerial part of the plant under the

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experimental conditions studied.

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Distribution of nZVI in Cucumber Plant. In Figure S6, FE-SEM images show large numbers

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of suspended particles adsorbed on the entire surfaces of the roots. For a better assessment of NP

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distribution in the tissue, roots were thoroughly washed with CaCl2 solution before analyzing the

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iron accumulation, distribution and speciation. Aliquots were subjected to CaCl2 extraction to

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obtain the exchangeable fraction of CaCl237. Figures S6 and S7 show the efficiency of the

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washing process to remove iron NPs from root surfaces. EDS analysis showed that CaCl2

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solution removed >92% of the adhering iron from root surfaces compared to the unwashed

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sample. Therefore, CaCl2 washings minimized interference with the experiment results.

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To investigate whether nZVI could penetrate through the cell wall in the root and internalize in

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plant cells, HR-TEM analyses were performed on root tissue (Figures 4(a)-(c) and S8). The

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images of the sample treated with nZVI containing iron NPs are clearly visible in the cytoplasm

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and surrounding membranes. Individual particles in the aggregate were very close in size to

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nZVI (~60 nm), and the observed NP aggregates were almost 2000 nm in size. These

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observations suggest that nZVI could penetrate the membrane and locate in the root cells.

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However, the aggregates similar to those observed in the root cells were not seen in the leaves.

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We hypothesize that it was difficult for nZVI aggregates to translocate to the shoots because of

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their larger hydrodynamic diameters, and this hypothesis is well matched with SQUID analysis.

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Instead, aggregates with chain-like structures were deposited in the vacuoles of leaf parenchymal

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cells. HR-TEM-coupled EDS analyses identified the dark aggregates found in leaf cells as iron

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with a single particle measuring ~10 nm (Figures 4(d)-(f)). These particles appeared to be

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different from the particles found in the roots, probably due to the biomineralization of iron.

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Biomineralization is a process by which living organisms produce iron minerals in their tissues, a

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widespread phenomenon in algae, diatoms, bacteria, and plants38-39. When excess iron ions are

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concentrated in plant tissues, the absorbed ions are remineralized, depending on the ambient

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conditions of the cell40. Even though studies have reported the presence of mineral structures

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associated with the tissues of higher plants, it is not clear whether the iron biominerals are

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formed within their internal structures.

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Speciation of nZVI in the Cucumber Plant. To elucidate iron nanoparticles found in both the

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root and the shoot, the normalized Fe K-edge NEXAFS and their first derivatives of cucumber

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root and shoot with reference compounds were analyzed (Figure 5). Ferrous oxide (FeO), ferrous

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oxalate, magnetite (Fe3O4), maghemite (γ-Fe2O3), goethite (α-FeOOH), lepidocrocite (γ-FeOOH),

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ferric citrate, and commercial NANOFER (nZVI) were selected as reference compounds because

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of the possible transformed products, speciation and coordination structures of iron in plants41-42.

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The distinct differences with respect to the intensities and positions of the pre-edge and main

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edge as well as the absorption ramp were investigated among the set of the root and shoot

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samples. The absorption edge of nZVI occurred at a lower energy with a distinct shape due to the

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nanoparticulate iron (Fe0), which was significantly different from the absorption edge of the

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treated samples. Similarly, different from the sample peaks, ferrous oxide and ferrous oxalate

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indicated the distinct edge and oscillation patterns of the Fe(II) species. We know that NEXAFS

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at the Fe K-edge is highly sensitive to the coordination environment and redox changes of iron at

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the atomic level43. Hence, the presence of Fe(III) could be clearly discriminated from that of Fe0

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and Fe(II) in samples. In contrast, as shown in Figure S9, the normalized Fe K-edge NEXAFS

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spectra of the cucumber growth medium fed with nZVI (from week 1 to week 3) and nZVI

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reference spectra showed that nZVI precipitated and suspended in the growth medium was not

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transformed much more than nZVI absorbed on the root. To reaffirm the oxidation state of nZVI,

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samples of precipitated nZVI were retrieved from the bottom of the medium container and

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analyzed using XRD. The patterns of the pristine and precipitated nZVI are shown in Figure S10.

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The results reveal that the intensity of the Fe0 peak was slightly reduced, but the main

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composition of the precipitated nZVI was still metallic iron (Fe0) at peak position 2θ of 44.6°.

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While the underlying mechanism requires further investigation, we attribute the rapid

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transformation of the adsorbed iron particles of plant roots to the release of root exudates

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(organic acids, protons, and metabolites) in the rhizosphere, which promote dissolution and

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oxidation/reduction of the nanoparticles44. Ma et al. attributed the enhanced dissolution of ceria

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nanoparticles on the surface of cucumber root to the exudates. They deduced that interactions

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between the root exudates and the nanoparticles is necessary for the transformation of ceria

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nanoparticles in plants45.

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LCFs of the NEXAFS spectra were used to estimate the sample compositions, and the results

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are presented in Figure 6. Based on our study design and spectral edge features (shown as dotted

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lines in Figure 5), the 4 best components (ferric citrate, lepidocrocite, goethite and maghemite)

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were used for the LCF fits that corresponded to the number of iron products present in the

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samples46-47. The uncertainties are below the 10% level for all test samples (see SI, Table S5).

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Notably, adding more components can improve the fit but is an ineffective use of the LCF

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approach if their contribution is close to the method uncertainty48. These data revealed that the

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major iron proportions in the root are 41% and 25% for ferric citrate (hydroxyl-carboxylate

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product) and lepidocrocite ((oxyhydr)oxide product), respectively. The data also showed that

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similar oxidation states (III) of the iron species and other (oxyhydr)oxide — goethite and

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maghemite — compositions were comparatively low (33%). In contrast, shoot samples indicated

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a larger proportion of ferric citrate (60%) compared to the (oxyhydr)oxide phases (approximately

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40%), indicating that organic acid was possibly the functional center in the translocation of iron

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(from root to shoot in the cucumber). Notably, nongraminaceous plants solubilize iron by

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extruding protons and couple to the production of citrate that deposits in the xylem49. Hence, the

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root acidification of the rhizosphere could allow the formation of stable Fe-organic complexes,

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with translocation within the plant. Additionally, ferric citrate complex is less toxic than the

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ferrous iron but stable only at low pH50. The pH changes in the xylem and leaf cells promoted

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the dissociation of the complex and led to the precipitation as (oxyhydr)oxides of iron51. These

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results are in good agreement with previous studies showing biomineralized iron in the plant

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epidermal cells, and different iron minerals such as lepidocrocite and goethite have been

313

reported52-53. The LCF results also agree well with the oxidation products of nZVI in oxygenated

314

water, i.e., crystalline structures from nZVI identified as (oxyhydr)oxide, notably lepidocrocite41-

315

42

.

316

Local Molecular Environment Investigations through EXAFS. The k3-weighted (k-range =

317

3 to 10 Å-1) and Fourier-transformed EXAFS spectra along with reference compounds were

318

examined to investigate the local atomic structures of the cucumber root. The oscillation patterns

319

in k-space and R-space (Figures S11(a) and (b)) demonstrated resemblance of the root

320

predominantly with citric acid. These observations were consistent with the Fe K-edge NEXAFS

321

and the LCF fit results. Thus, the standard model (ferric citrate) was selected to represent the

322

unknown root-bearing structures because of the ability to extract accurate EXAFS parameters.

323

The simulated Fourier-transform EXAFS spectra for citric acid and cucumber root are shown in

324

Figures S11(c) and (d), respectively. The main peaks occurred at 1.51 Å and 1.56 Å for ferric

325

citrate and the root specimen, respectively, which corresponded to the Fe-O interatomic distances.

326

Moreover, second strong peaks were observed at the Fe-Fe bond distances of 2.65 Å and 2.63 Å

327

for ferric citrate and treated roots, respectively. These results were consistent with the iron

328

atomic level investigations of previous studies43. The oscillation at 0.83 Å in the root (marked as

329

↓) could indicate interactions of iron with biochemical constituents except for the contributions

330

of ferric citrate, and as anticipated, this oscillation was absent from the control model. The fit

331

results provided further iron structural information with nearest neighboring atoms in the

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coordination shells. As in Table S6, four parameters were varied in the analysis of each sample:

333

coordination number (CN), interatomic distance/bond length (R), Debye-Waller factor (σ2), and

334

correction to main edge energy (∆E). The goodness-of-fit of the data was evaluated by the R-

335

factor. One result of particular importance was from the shell-fitting analysis of nZVI-treated

336

roots that indicated that iron had 6.7 Fe-O bonds (i.e., distorted octahedral Fe(III) structure) with

337

an average distance of 2.01 Å, which is in fair agreement with Fe(III) complexes in organic

338

ligands54. Presumably, CN was reduced in the root (6.7) compared to the control model (8.4), so

339

Fe-O complexes could also form by other iron phases in addition to the coordination

340

environments of the ferric citrate, therefore indicating local disorders in the shell of the complex

341

root networks. Additionally, considering the Fe-Fe path, 12.7 iron atoms coordinated at average

342

distances of 3.05 Å. These Fe-Fe distances were coherent with those in complexes between

343

Fe(III) and small organic ligands (2.99-3.42 Å) in organic soil55. The higher CN value of Fe-Fe

344

(12.7) compared to the CN value of the control model (2.8) could be explained by additional

345

crosslinked iron within the plant tissue network, signifying that all contributions were not only

346

from ferric citrate but also from iron (oxyhydr)oxides (aforementioned discussion). We further

347

attempted data processing and analyses of shoot specimens, and although NEXAFS data were

348

obtained, we did not receive good data/signals that could simulate EXAFS spectra (12 scans

349

were performed and then averaged to improve the signal/noise ratio). We believe that low beam

350

flux and/or specimen iron content could explain why there were weak signals.

351

Collectively, the current data provide a good basis for two mechanistic pathways taken together

352

for nZVI transformation by the cucumber plant: (1) interaction with low molecular weight

353

organic ligands (most likely the hydroxyl-carboxylate-citrate end products), and (2) dissolution-

354

precipitation of the mineral (e.g., (oxyhydr)oxide products due to iron oxophilicity) after 21 days

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of the experiment. Our findings suggest that citrate, an important iron chelator and ligand, could

356

mobilize and transport nZVI as ferric citrate in plants. Another possibility is the formation of

357

mineral phase accumulated in the tissue caused by surface-controlled iron dissolution on the root

358

surface.

359

Response of PM H+-ATPase As mentioned earlier, when sparingly soluble metal ions such as

360

iron are present, dicotyledonous plants follow a strategy that releases protons into the rhizosphere

361

by PM H+-ATPase in the epidermis16. As anticipated, acidification of the root was correlated

362

with an increase in H+-ATPase activity. Additionally, we have stated in a previous report that

363

nZVI reduced the iron availability in the rhizosphere, which enabled the operation of a proton

364

pump in the plant56. To elucidate the mechanism of plant-promoted transformation of nZVI,

365

quantitative real-time polymerase chain reaction was used to analyze the levels of CsHA1 and

366

CsHA2 (two different major H+-ATPase isoforms known to be present in Cucumis sativus) at the

367

transcriptional level30-31. The increased expression of CsHA1 in the roots of plant exposed to

368

nZVI (Figure S12) clearly supports the hypothesis that nZVI induces the proton extrusion to the

369

rhizosphere. The expression of CsHA1 and CsHA2 in cucumber showed that these isoforms

370

respond differently to nZVI application, because CsHA1 was upregulated under iron deficiency

371

while CsHA2 was unaffected by iron status and constitutively expressed as the housekeeping

372

gene57-58. Therefore, proton extrusion is predominantly mediated by CsHA1, and the

373

transcriptional regulation is at least in part responsible for the different activity of H+-ATPase

374

isoforms under the different environmental conditions for cucumber.

375

This research reveals that the nZVI with high reactivity can undergo biotransformation in the

376

cucumber plant. Further investigation for life cycle assessment needs to be conducted to achieve

377

nanotechnology safety and to protect the environment. Information obtained from this study may

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help to garner fundamental clues and enhance our understanding of the nZVI transformation

379

mechanisms.

380 381 382

SUPPORTING INFORMATION

383

The additional experimental section (growth conditions, QA/QC for ICP-OES analysis,

384

microscopic observation, XAS investigation, qRT-PCR and statistical analyses); TEM image of

385

nZVI (Figure S1); biomass results (Figure S2); photographs of plants and leaves (Figure S3); Fe

386

contents (Figure S4) and magnetization curve (Figure S5) in soil-grown plants; SEM images

387

(Figure S6) and elemental mapping (Figure S7) of root surface; additional TEM images of root

388

cells (Figure S8); normalized Fe K-edge NEXAFS spectra (Figure S9) and XRD patterns (Figure

389

S10) of nZVI precipitated in the container and pristine ZVI; oscillation patterns in k-space and

390

R-space and the simulated Fourier-transform EXAFS spectra (Figure S11); relative expression

391

levels of PM H+-ATPase genes (Figure S12); Physicochemical characterization of the test soil

392

(Table S1) and nZVI (Table S2); levels of Mg and Mn in plants (Table S3); Translocation factors

393

(Table S4); Linear combination fit reports of NEXAFS data (Table S5); Simulated parameters

394

from EXAFS (Table S6);

395 396 397

ACKNOWLEDGMENTS

398

This research was mainly supported by the National Research Foundation of Korea (NRF) grant

399

funded by the Korea government (MSIP) (NRF-2017R1A2B3012681). In addition, this research

400

was partially granted by No. NRF-2017R1A2B3006354 and “The GAIA Project” by the Korea

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Ministry of Environment (RE201402059). All the synchrotron-based X-ray absorption

402

experiments were performed at Pohang Accelerator Laboratory, the third generation Pohang

403

Light Source II (PLS II), Republic of Korea.

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CsHA1 gene and of the new isolated ferric reductase CsFRO1 and iron transporter CsIRT1 genes

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in cucumber plants. Plant Physiol. Bioch. 2007, 45 (5), 293-301.

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Figure 1. Schematic diagrams showing the preparation of experimental cucumber samples (root

566

and shoot) in the present work.

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567 568

(b)

569 570

Figure 2. Fe content in (a) roots and (b) shoots grown in the hydroponics under the different

571

treatments and exposure times. Result are shown as the means ± SD (n = 3). The unit is ppm.

572

Different letters represent significant differences among the different groups (LSD).

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(a)

573 574

(b)

575 576

Figure 3. Magnetization curves from SQUID of (a) reference standard of iron materials, and (b)

577

cucumber root and shoot samples grown hydroponically. The inserts of (a) and (b) are higher

578

magnification of the rectangular regions.

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Figure 4. HR-TEM images of cucumber (a) root and (d) leaf after exposure to 1000 mg L-1 nZVI

581

for 3 weeks. (b) and (e) are higher magnifications of the rectangular areas from (a) and (d),

582

respectively. The clusters in (b) and (e) were analyzed with EDS, and the spectra are presented in

583

(c) and (f), respectively.

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Figure 5. For cucumber samples (shoot and root) (a) normalized Fe K-edge NEXAFS spectra

586

(transition from 1s core state to 3d empty state shows the origin of the associated pre-edge

587

structure), and (b) first derivative spectra along with reference compounds.

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Figure 6. Linear combination fitting results of (a) cucumber root and (b) cucumber shoot

590

obtained from Athena (version 0.9.18) using reference compounds. Dotted lines (green color)

591

show simulation range for both spectra. Estimated percentages of reference compounds are

592

shown using the pie charts for (c) root and (d) shoot.

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