Iron nanoparticle-induced activation of plasma membrane H(+)-ATPase promotes stomatal opening in Arabidopsis thaliana.

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Iron nanoparticle-induced activation of plasma membrane H +-ATPase promotes stomatal opening in Arabidopsis thaliana Jae-Hwan Kim, Youngjun Oh, Hakwon Yoon, Inhwan Hwang, and Yoon-Seok Chang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es504375t • Publication Date (Web): 12 Dec 2014 Downloaded from http://pubs.acs.org on December 19, 2014

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Environmental Science & Technology

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Iron nanoparticle-induced activation of plasma membrane

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H+-ATPase promotes stomatal opening in Arabidopsis thaliana

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Jae-Hwan Kim1, Youngjun Oh2, Hakwon Yoon1, Inhwan Hwang2, Yoon-Seok Chang1,*

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School of Environmental Science and Engineering,

Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea

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*Corresponding author contact information:

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E-mail: [email protected]

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Tel: +82-54-279-2281, Fax: +82-54-279-8299

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Keywords : Nano zero-valent iron (nZVI), Arabidopsis thaliana, Plasma membrane H+-

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ATPase, stomatal opening, CO2 uptake

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ACS Paragon Plus Environment


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TOC

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ABSTRACT

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Engineered nanomaterials (ENMs) enable the control and exploration of intermolecular

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interactions inside microscopic systems, but the potential environmental impacts of their

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inevitable release remain largely unknown. Plants exposed to ENMs display effects, such as

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increase in biomass and chlorophyll, distinct from those induced by exposure to their bulk

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counterparts, but few studies have addressed the mechanisms underlying such physiological

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results. The current investigation found that exposure of Arabidopsis thaliana to nano zero-

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valent iron (nZVI) triggered high plasma membrane H+-ATPase activity. The increase in

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activity caused a decrease in apoplastic pH, an increase in leaf area and also wider stomatal

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aperture. Analysis of gene expression indicated that the levels of the H+-ATPase isoform

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responsible for stomatal opening, AHA2, were 5-fold higher in plants exposed to nZVI than in

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unexposed control plants. This is the first study to show that nZVI enhances stomatal opening

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by inducing the activation of plasma membrane H+-ATPase, leading to the possibility of

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increased CO2 uptake.


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INTRODUCTION

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Engineered nanomaterials (ENMs) currently used in a variety of industrial fields can be

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generally divided into four categories, as follows: i) carbon-based materials, ii) metal-based

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materials, iii) dendrimers, and iv) micron-sized composites with nanoparticles.1 A number of

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products containing ENMs are readily available in the marketplace, including catalysts,

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electronics, personal care products, and medical devices.2-4 Such applications of ENMs in

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commercial products are expected to gradually advance and directly or indirectly influence

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our way of life; however, as it is unknown whether they have a toxic effect on components of

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ecosystems, the increasing production and extensive use of commercially available ENMs

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has raised environmental concerns. ENMs such as Ag nanoparticles reduce the ATP content

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of human lung cells and Mn3O4 nanoparticles increase reactive oxygen species production in

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the same cell type.5-6

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The toxicity of ENMs to living organisms remains largely uncertain, however. In study of

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metal-based ENMs, Fe3O4 nanoparticles had pH-dependent catalase- or peroxidase-like

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effects on human glioma cells and detoxified hydrogen peroxide into water and oxygen at

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neutral pH.7 Studies of the effects of metal-based ENMs on plants have been species-specific

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and ambiguous, and this needs to be resolved. Ag nanoparticles inhibited germination in

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barley (Hordeum vulgare)8 whereas those of Fe3O4 retarded root elongation of Arabidopsis

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(Arabidopsis thaliana), respectively.9 In contrast, Fe3O4 nanoparticles increased the

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chlorophyll content of soybean leaves,10 and low doses (0.01–0.05 mg/L) of Ag nanoparticles

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promoted soybean growth.11

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The unique properties of nanoparticles, which include their reactivity and size, have

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produced outstanding improvements in the field of environmental remediation.2,12-15 Nano

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zero-valent iron (nZVI) is a highly commercialized groundwater remediation reagent.

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Recently, investigation of the physiological changes of plants exposed to nZVI has been an



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important topic in environmental research. We previously demonstrated that it enhances root

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elongation in Arabidopsis by generating OH radicals and leading to the degradation of

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polysaccharides in the cell walls.16 This was the first study to describe the mechanism

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underlying nZVI-mediated increase in root length in plants; however, because the rising scale

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of remediation has increased nZVI production, more information on the subsequent impacts

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of nZVI on plants is required.17-20

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Plants respond to various external stimuli by extruding active agents into their

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environment; therefore, nZVI released into a plant’s rhizosphere can be considered an

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external stimulus.9,21 In particular, the strong oxidizing capacity of nZVI alters pH by

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producing OH- and this consequently leads to lower solubility of Fe ions in the rhizosphere.22-

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difficult for plants to use the particles as an iron source directly in the absence of Fe ion-

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reductase or any acidifiers.25-27 Plants activate plasma membrane (PM) H+-ATPase to extrude

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protons and acidify their rhizosphere, and thus increased activity of PM H+-ATPase is

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frequently observed in plants growing in conditions of low Fe-availability.25-27 Santi and

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Schmidt found that of the different PM H+-ATPase isoforms, Arabidopsis H+-ATPase 2

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(AHA2) is the major Fe-responsive gene for rhizosphere acidification.25

Moreover, because the surface of nZVI consists of several iron oxide-hydroxides,24 it is

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It is well known that stomatal opening on leaves can be promoted by activation of PM

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H+-ATPase.28-31 Recent research into the effects of well-known components (including blue

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light receptors, plasma membrane H+-ATPase and K+in channel) regulating light-induced

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stomatal opening showed AHA2 to be the major gene related to the stomatal opening

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process.28 It is notable that AHA2, the PM H+-ATPase isoform gene responsible for

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rhizosphere acidification, is identified as an essential component of stomatal opening. We

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therefore hypothesized that the presence of nZVI in rhizosphere would increase PM H+-

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ATPase activity in plants and thus enhance stomatal opening. To the best of our knowledge,


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no study has addressed both these phenomena together or considered the effects of ENMs on

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PM H+-ATPase activation.

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We measured sequentially the apoplastic pH in roots and the activity of PM H+-ATPase in

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shoot. In addition, as the stoma is the key organ for CO2 uptake for photosynthesis in plants,

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understanding how stomatal opening is mediated by the activation of PM H+-ATPase by

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nZVI may be useful in developing green technology to remove atmospheric CO2.



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MATERIAL AND METHODS

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Experimental engineered nanomaterial

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Commercial RNIP-10DS (Toda, Japan) was used as representative of nZVI in this study.

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XRD pattern was recorded on a PANalytical X’Pert diffractometer (Cu Kα radiation) with an

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X’Celerator detector, and TEM image was obtained by using JEOL-2100 high-resolution

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transmission electron microscope (HR-TEM) with Cs correction at 200 kV. Surface area was

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measured by N2 adsorption method using a Mirae SI nanoPorosity-XG analyzer. The average

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particle size and content of Fe0 (α-Fe) in nZVI were calculated using Scherrer’s formula with

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full width at half maximum (FWHM)32 (FWHM=0.28°, 2θ = 44.6°) and using GC-TCD

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(Gas chromatography thermal conductivity detector), respectively.

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Plant growth conditions To prepare nZVI slurry, the particles were washed and rinsed with 99% ethanol and degassed deionized water.

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i) Hydroponic culture: A half strength of standard Murashige and Skoog medium (1/2 MS

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medium; Duchefa Biochemie) was used. The concentration of additives such as sucrose

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(Duchefa Bichemie), MES (aMResco), and agar (Junsei) were 20, 0.05, and 0.9%,

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respectively. 70% ethanol and 20% Clorox was prepared to sterilize Arabidopsis thaliana

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(Colombia ecotype) seeds and the rest procedure were described in more detail in previous

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work,16 and the concentration of nZVI used was 0.1 g/L. Growth chamber condition was 16

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h/8 h light/dark and 22 ℃ with 30% relative humidity. Measurement of leaf area and


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apoplastic pH of plants was conducted using Arabidopsis grown on the 1/2 MS medium.

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Using plants grown in medium, the measurement of apoplastic pH, leaf area, and expression

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of AHA2 were conducted.

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ii) Soil culture: Autoclaved soil and nZVI slurry were mixed together to prepare nZVI

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mixed soil. The final concentration of nZVI to soil was 0.5 g/kg. Arabidopsis was cultivated

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on the soil and grown in the greenhouse with 16 h/8 h light/dark and 20-22 ℃. The initial

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water content of the nZVI mixed soil was 60-70%. Using plants grown in soil, the rest of

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experiments as follows were conducted; PM H+-ATPase activity, stomatal aperture, and water

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loss.

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Measurement of apoplastic pH in roots

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10-day-old seedling were harvested and immersed in 0.005% bromocresol purple

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solutions (Sigma-aldrich) pH indicator containing 1/2 MS medium solution without agar for 2

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days. The initial pH of the indicator solution for control and nZVI exposed seedlings were

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different, which were pH 5.8 and pH 6.0, respectively. The solution for nZVI exposed

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seedlings contained the same concentration of nZVI (0.1 g/L) to reflect the same condition of

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the rhizosphere in actual plates.

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Stomatal aperture measurement

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Stomatal apertures of 3-week-old seedlings grown in soil were measured as described by

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Hwang et al. with some modifications.33 For this assay, any buffers such as K+-MES that was

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able to stimulate stomatal opening were not used at all since the purpose of this assay was the

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comparison of the present state of stomatal apertures sizes when exposed to nZVI. Epidermal



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tissues were placed on a microscope slide and covered, and stomatal apertures were measured

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microscopically (Zeiss Axioplan). Statistical analysis was carried out using Excel program

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(Microsoft Corp.).

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Preparation of plasma membranes 3-week-old plants were used to isolate protoplasts following previous study

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. The

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protoplasts were pelleted by centrifugation at 500 rpm for 4 min and then, resuspended in 5

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ml lysis buffer (0.25 M sucrose, 1 mM EDTA, 20 mM Tris-HCl, pH 7.8). The protoplasts

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were homogenized by filter gently three times with two layered 11 µm pore nylon filters

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(Millipore). The extracts were centrifuged at 1000 g for 10 min to remove to chloroplasts and

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nuclear membranes. Supernatants were layered on 6 ml 30% percoll in lysis buffer and

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performed the centrifugation at 28,000 rpm for 30 min in a Beckman ultracentrifuge with

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SW41Ti rotor. Plasma membrane fraction was visible as a ring shape in ultracentrifuge tube

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and collected. The removal of percoll was carried out by dilution with reaction buffer A

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(30mM imidazole-HCl pH 7.4, 130mM NaCl, 20mM KCl, 4mM MgCl2). Next, 2 hr

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ultracentrifugation at 30,000 rpm was performed. Finally, 150 µl supernatant was obtained by

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a 10 kDa cut-off centricon (Millipore).

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Measurement of PM H+-ATPase activity

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Plasma membrane (PM) H+-ATPase activity was measured in 23 ℃ for 10 min with 10-

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20 µg plasma membrane proteins. To reveal pumping out activity of PM H+-ATPase, 0.05%

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Brij 58 (Sigma) was employed to select to inside-out vesicles. Reaction buffer B (30 mM

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imidazole-HCl pH 7.4, 4 mM MgCl2, 2 mM Sodium orthovanadate, 0.05% Brij 58) was used


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to eliminate the background signal of the ATPase activity. The 1 ml mixture of plasma

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membrane fraction with reaction buffer A, B was incubated on ice at dark for 1hr. The

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addition of 4 mM Tris-ATP was the start cue of this assay. After 23 ℃ incubation for 10 min,

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the reaction was terminated by 100 µl 20% SDS. To develop color as activity reporter, 500 µl

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reaction mixture added to 500 µl reagent A which consist with 3% ascorbic acid, 0.5N HCl,

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and 0.5% ammonium molybdate and incubated on ice for 10 min. 37 ℃ incubation for 10

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min was performed, after the insertion of 500 µl reagent B (2% Sodium meta-arsenite, 2%

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trisodium citrate, 2% acetic acid) to 500 µl reaction mixture and activity was detected by 850

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nm wavelength.

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Confocal microscopy for analysis of endocytosis

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We modified little the previous Kim et al.’s method.16 From both two groups, 2-week-old

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seedlings were harvested and whole leaves, not only cuticle, were immersed in 1 µM of FM

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4-64 solutions (Invitrogen) for 10 min. The emission and excitation wavelength employed for

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the fluorescence images were 543 nm and 560-615 nm, respectively (Zeiss Axioplan).

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Quantitative real-time PCR (qRT-PCR)

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Total RNA was separately isolated from 2-week-old plants roots and leaves using a

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RNeasy plant mini kit (Qiagen). cDNA conversion was performed with cDNA reverse

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transcription kit (Biosystems). Quantitative real-time PCR (qRT-PCR) was carried out with a

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SYBR green kit to detect to amplified probes. Actin 2 was empolyed as an endogeneous

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control. 10ng cDNA was used to PCR in a 20 µl reactio volume with 0.5 µM primers, 1 unit

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Taq polymerase. Primer sequences are F 5' TGACTGATCTTCGATCCTCTC 3', R



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5'GAGAATGTGCATGTGCCAAAA 3' for AHA2, F 5' TATGAATTACCCGATGGGCAAG

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3', R 5' TGGAACAAGACTTCTGGGCAT 3' for Actin 2. Amplification conditions were as

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follows: denaturation at 94 °C for 4 min, followed by 30 cycles of 94 °C for 15 s, 55 °C for

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30 s, and 72 °C for 60 s.


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RESULTS AND DISCUSSION

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Characterization of nano zero-valent iron

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The chemicophysical properties of nZVI particles used, including transmission electron

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microscopy (TEM) image, X-ray diffraction (XRD) pattern, surface area, particle size and

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weight percentage (wt%) of Fe0, are shown in Figure 1. Three peaks were present in the

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crystal at 44.6, 66.0, and 82.3°, indicating a typical Fe0 XRD pattern (Figure 1A). The TEM

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image of nZVI was shown in Figure 1B. Single particles appeared to be less than 100 nm in

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size, but agglomeration of particles occurred due to chemical characteristics such as magnetic

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interaction and Van der Walls attractions.35 The surface area was 30 ± 2 m2/g and the mean

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particle size (calculated using Sherrer’s formula32) was 54 ± 1 nm (Figure 1C). The weight

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percent of Fe0 in nZVI had slight higher value compared with an earlier analysis.24

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nZVI increases plasma membrane H+-ATPase activity

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Two groups of 10-day-old seedlings, one treated with nZVI and the other untreated

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control groups, were incubated in bromoceresol purple solutions (0.005%) for 2 days to

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measure changes in the apoplastic pH. The densities of the solutions were quantitatively

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determined using UV spectrophotometry (Figure 2A). The negative value seen when

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Arabidopsis seedlings were exposed to nZVI indicated their apoplastic pH was more acidic

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than pH 5.8. By contrast, the apoplastic pH of control seedlings remained almost constant

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throughout the experiment; thus the apoplastic pH decreased following exposure to nZVI.

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This acidification of the root apoplast was correlated with an increase in PM H+-ATPase

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activity. PM H+-ATPase plays an important role in transporting protons29,36-37 and, by



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pumping protons out of the roots, plants are able to acidify the rhizosphere when sparingly

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soluble metal ions such as iron are present.25-26

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Nano zero-valent iron has unique redox reactivity. On exposure to aerobic conditions,

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insoluble iron oxide-hydroxides, such as FeOOH and Fe(OH)3, form rapidly on the surface of

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each particle because of their strong oxidizing capacity.24,38-40 Additionally, nZVI can

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thermodynamically lead to lower Fe solubility by increasing pH via water decomposition

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following electrochemical/corrosion reactions.40 Together, these effects of nZVI reduce Fe

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availability in the rhizosphere, which enables the operation of the proton pump in plants.

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Fe0(s) + 2H2O(aq)

Fe2+/3+(aq) + H2(g)

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Fe3+(aq) + 3OH-

Fe(OH)3(s)

(2)

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Fe(OH)3(s)

FeOOH(s) + H2O

(3)

+

2OH-(aq)

(1)

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Therefore, the acidic apoplastic pH observed in Arabidopsis exposed to nZVI indicates that

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its presence may produce rhizosphere conditions requiring acidification, thereby triggering

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the activation of PM H+-ATPase.

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To determine whether PM H+-ATPase increased in the guard cells of Arabidopsis exposed

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to nZVI, the activity of PM H+-ATPase was directly measured in 3-week-old seedlings.

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Figure 2B shows PM H+-ATPase activity of the experimental and control plants. Plants

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exposed to nZVI exhibited much higher levels (an 80-90% increase) of PM H+-ATPase

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activity in their aerial part than the control plants, which implies that nZVI triggered

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activation of PM H+-ATPase across the whole plant, possibly due to cross-talk between PM

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H+-ATPase and auxin.36,41-42

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Root and shoot exchange signals to perform communication with each other.43 Activation


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of PM H+-ATPase can be mediated by auxin, which is the most important signal transmitted

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from root to shoot; PM H+-ATPase activation is likewise required for auxin transport.41 A

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chemiosmotic model44 suggest that intercellular transport of auxin is accelerated while the

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PM H+-ATPase channel, through which protons are pumped out, is open (“activated”). This

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provides a possible mechanism by which PM H+-ATPase activity could increase in the shoots

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of Arabidopsis exposed to nZVI, and suggests that such exposure promotes the transport of

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auxin from root to shoot. Therefore, the activation of PM H+-ATPase in root by nZVI

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promotes auxin transport, in accordance with the chemiosmotic model of polar auxin

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transport, which may lead to activation of PM H+-ATPase in shoot.

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

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AHA2 (the major H+-ATPase isoform involved in rhizosphere acidification)25 and thus

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specifically evaluate PM H+-ATPase expression at the transcriptional level. The increased

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expression of AHA2 in root of plants exposed to nZVI (Figure 2C) clearly supports the

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hypothesis that nZVI induces the need to acidify the rhizosphere.

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Increased stomatal aperture and tolerance to drought

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These observations led us to hypothesize that any increase in stomatal aperture observed

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following exposure of plants to nZVI would result from the activation of PM H+-ATPase in

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leaves. Guard cells swell due to a K+ or Na+ influx through inward-rectifying channels and

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the stomata open when PM H+-ATPase in guard cells is activated.45 Recently, AHA2 was

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found to have another role in leaves as a key component in stomatal opening,28 in addition to

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being the major Fe-responsive PM H+-ATPase isoform in roots.25 AHA2 levels increased

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almost 2-fold not only in roots but also in leaves of Arabidopsis exposed to nZVI (Figure

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2C).



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Wang et al. evaluated the function of AHA2, as well as other factors associated with light-

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induced stomatal opening, such as blue light receptor phototropin 2 (PHOT2) and PM

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inward-rectifying K+in channels (KAT1, AKT1,).28 The stomatal aperture was increased only

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by activation of PH H+-ATPase in transgenic Arabidopsis overexpressing AHA228: the

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stomatal aperture of transgenic plants were approximately 25% wider under light condition

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and expression of AHA2 was 3.4 to 5.2-fold higher; thus we predicted that stomatal apertures

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would also increase in Arabidopsis exposed to nZVI due to their higher levels of AHA2.

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To confirm this, the sizes of stomatal apertures in the two groups of plants were measured.

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Figure 3 shows images of Arabidopsis guard cells. The average size of the stomatal aperture

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of plants exposed to nZVI (3.7 ± 0.6 µm) was larger than that of the control (2.6 ± 0.7 µm).

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These results corresponded with higher activation of PM H+-ATPase in plants exposed to

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nZVI (Figure 2B) and thus supported our hypothesis. Therefore, we concluded that enhanced

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stomatal opening was induced by the activation of PM H+-ATPase by nZVI. Together with

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our earlier study, the finding of strong induction in AHA2 expression (Figure 2C) may

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answer the fundamental question of how exposure to nZVI increases the stomatal aperture of

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Arabidopsis. This is the first study not only to consider the effects of ENMs on PM H+-

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ATPase activation but also aspects of dual roles of AHA2 in roots and leaves.

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Although AHA2-transgenic plants have wider stomatal apertures than wild-type plants,

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they show a normal drought response in darkness and in response to the hormone abscisic

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acid.28 AHA2-overexpressing Arabidopsis exposed to nZVI are expected to have similar

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responses. To determine whether Arabidopsis exposed to nZVI exhibit a normal drought

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response, a water-loss experiment was performed using detached rosette leaves.

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The kinetics of weight decrease in detached rosette leaves from the treated and control

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plants were very similar (Figure 4), consistent with the behavior of AHA2-transgenic plants


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reported previously.28 Despite the wider opening of stomata following exposure to nZVI, the

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rates of water loss were nearly identical under conditions of both normal humidity and

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dehydration, indicating that the plants exposed to nZVI retained normal sensitivity and the

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ability to close stomata upon dehydration. The observation that Arabidopsis with nZVI-

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mediated activation of PM H+-ATPase showed not only increased stomatal opening but also

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normal drought sensitivity raises the possibility that these plants may have an increased CO2

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assimilation rate and respond normally to diverse environmental changes. Enhancement of

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the CO2 assimilation rate by plants through manipulation of their stomatal movements has

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been proposed as an eco-friendly alternative approach to reduce CO2 and thus combat climate

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change.28,46

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PM H+-ATPase and enlarged leaf area

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Arabidopsis seedlings were grown for 14days in 1/2 MS medium with control condition

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or nZVI mixed condition (Figure 5A-D). The surface areas of the leaves were quantified

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after 2 and 3 weeks growth. The leaf area of Arabidopsis seedlings exposed to nZVI was

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approximately 50% larger than that of the control plants (Figure 5E). Some ENMs, such as

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Ag nanoparticles, are known to increase biomass.11 While evaluating the toxic effects of

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various concentrations of silver nanoparticles on Arabidopsis, Wang et al. found that a

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relatively low dose (0.05 mg/L) of silver nanoparticles increased root elongation and leaf area.

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They speculated that the enhancement of root elongation was caused by Ag ions released

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from Ag nanoparticles, which might cause changes to levels of phytohormones or induce

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hormesis; however, they did not provide any clear evidence or mechanism to explain how

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low-dose silver nanoparticle treatment increased the leaf area.11

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Activation of PM H+-ATPase is involved in cell-wall loosening,31,36,45 which is necessary



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for various aspects of plant development including elongation, ripening, and expansion.30 An

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increased level of PM H+-ATPase causes more protons to be pumped across the PM, resulting

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in acidification of the apoplast. The acidic conditions activate pH-sensitive proteins and

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enzymes such as expansins,47 xyloglucan hydrolase, xyloglucan endotransglycosylase,48 and

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yieldins,49 which cause the acid-induced extension of cell walls. Expansins are known to

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disrupt the hydrogen bonding that connects glycans and cellulose microfibril in the cell-wall

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matrix without degrading the cellulose, therefore allowing the slippage of cellulose

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microfibrils during expansion. Their activity of increases with pH between pH 4.5 and 6.30

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Hydrolysis of molecules between adjacent cellulose filaments and other polysaccharides in

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cell walls by these proteins and enzymes eventually leads to turgor-driven cell expansion.30

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To determine if leaf expansion was mediated by turgor-driven cell elongation, we

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compared the endocytosis rates of leaves from the two groups of plants, because turgor-

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driven cell-wall loosening is inversely correlated with the endocytosis rate. Figure 6 shows

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leaf cells stained with FM 4-64 dye, an endocytosis marker. The rate of endocytosis clearly

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differed between the two groups of leaves, being lower in nZVI-treated leaves. The

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endocytosis rate is regulated according to the degree of tension on cell walls. Cell expansion

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via turgor-driven cell-wall loosening increases the tension on cell walls and consequently

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inhibits endocytosis and facilitates exocytosis.50-51 The reduction in endocytosis seen in

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leaves of nZVI-treated seedlings (Figure 6) supports our hypothesis that leaf expansion

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occurred via turgor-driven cell-wall loosening. This would mean the enlarged leaf area of

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nZVI-treated plants resulted from an increased extrusion of protons into the apoplast due to

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the increased levels of PM H+-ATPase; thus the pH of the apoplast is lowered, producing the

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acidic conditions that allow turgor-driven cell-wall loosening.

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Environmental implications

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In the global ecosystem, one of the most important roles of plants is providing O2 while

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absorbing CO2. In higher plants, O2 and CO2 are exchanged in the stomata and, therefore,

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much attention has been paid to the development and application of advanced technologies

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that modulate stomatal aperture. In this context, the regulation of PM H+-ATPase activity is

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of particular interest.29,36-37,52-53 An earlier study reported the response of stomata to ENMs,

337

showing that Ag nanoparticles caused stomatal closure by decreasing hydraulic conductance

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of stems after penetrating into vessel; however, that study focused only on post-harvest

339

technology.54, and thus it is not directly relevant to the release of nanoparticles into the

340

environment. Here, it was shown that Arabidopsis plants exposed to nZVI exhibited higher

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levels of AHA2 expression as well as higher PM H+-ATPase activity, and showed behavior

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similar to that of transgenic plants overexpressing AHA2,28-29,45 at least in terms of the water

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loss and stomatal aperture. These results, therefore, raise the possibility of an eco-friendly

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alternative approach to CO2 reduction. For this to be applied, however, it will be necessary to

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characterize in detail the plant’s responses to nZVI exposure, including its effects on the rate

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of CO2 assimilation, biomass production and photosynthesis.

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ACKNOWLEDGMENTS

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This work was supported by the National Research Foundation of Korea (NRF) grant funded

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by the Korea government (MEST) (No. 2011-0028723) and “The GAIA Project” by the

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Korea Ministry of Environment.



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353

Figures

5000 4500

A

B

Intensity (a.u.)

4000 3500 3000 2500 2000 1500 1000

20

30

40

50

60

70

80

90

2θ (degree)

C 354

Surface area (m2/g)

Particle size (nm)a

wt% of Fe0 (%)b

30 ± 1

54 ± 1

40 ± 3

355

356

Figure 1. Chemicophysical properties of nano zero-valent iron particles used in the study. (A)

357

X-ray diffraction (XRD) pattern. (B) Transmission electron microscopy (TEM) image. (C)

358

Particle surface area, size, and weight percentage of Fe0(α-Fe). a: particle size was calculated

359

using Scherrer’s formula (2θ = 44.6°). b: measurement of amount of H2 evolution in

360

concentrated HCl using gas chromatography with thermal conductivity detector (GC-TCD).

361

Scale bar in Figure 1B: 50 nm.

362


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363

364

A

* B

C

*

* 365 366

Figure 2. Effects of nano zero-valent iron (nZVI) on apoplastic pH, H+-ATPase activity, and

367

AHA2 expression in Arabidopsis thaliana. (A) In vivo determination of the extrusion of

368

protons. 10-day-old Arabidopsis seedling were incubated in bromocresol purple solutions for

369

2 days (n = 10, triplicate). (B) PM H+-ATPase activity in 3-week-old Arabidopsis seedling.

370

(C) Expression of AHA2 in 2-week-old Arabidopsis seedlings. Data are mean and SE of three

371

replicate experiments; n=10 in each experiment. Untreated seedlings were used as controls in



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each experiment. * indicates statistically significant differences at P < 0.01 compared with the

373

control (Student’s t-test)

nZVI

Control A

B

C

D

*

E

374 375 376

Figure 3. Effect of nano zero-valent iron (nZVI) on stomatal aperture in Arabidopsis thaliana.

377

(A to D) Confocal microscopy images, taken at low and high magnification, of guard cells in

378

3-week-old seedling. (A and C) control seedlings. (B and D) nZVI-exposed seedlings. White

379

scale bars: 20 µm. Black scale bars: 5 µm. (E) The average stomatal apertures of control and

380

nZVI-exposed seedling. Data are means and SE of three replicate experiment; n=20 in each


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381

experiment. * indicates statistically significant differences at P < 0.01 compared with the

382

control (Student’s t-test).

383 384 385

Figure 4. Water loss from detached rosette leaves of 4-week-old Arabidopsis thaliana. The

386

Y-axis relative weight is presented as a percentage of the initial fresh weight. The fresh

387

weight of the detached leaves was measured at the indicated time points. a: N, normal

388

humidity; b: D, relatively low humidity.



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nZVI

Control B

A

2 cm

C

2 cm

D

1 cm

1 cm

**

E

**

390 391

Figure 5. Effects of nano zero-valent iron (nZVI) on leaf are. Arabidopsis thaliana seedlings

392

were grown for two weeks and photographed under low and high magnification. (A and C)

393

control seedlings. (B and D) nZVI-exposed seedlings. (E) Leaf areas of control and nZVI-

394

exposed seedlings measured after 2 and 3 weeks. Data are means and SE of three replicate

395

experiments; n = 40 in each experiment. ** indicates statistically significant differences at P


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< 0.001, compared with the control (Student’s t-test).

Bright field

Endocytosis marker B

C

D

nZVI

Control

A

397 398

Figure 6. Effect of nano zero-valent iron (nZVI) on endocytosis in Arabidopsis thaliana.

399

Images of leaf cells from control and nZVI-treated plants. Bright field (leaf column) and red

400

channel (right column) images of leaf cells strained with the endocytosis marker FM 4-64

401

from control (top) and nZVI-exposed (bottom) seedlings. Two-week-old seedlings were

402

incubated in 1 µM FM 4-64 for 10 min. Scale bars: 20 µm.

403



404 405 406 407

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Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth.

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Plasma membrane lipid-protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana.

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bHLH transcription factor bHLH115 regulates iron homeostasis in Arabidopsis thaliana.

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