Comparative Physiological and Transcriptomic Analyses Reveal the

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Comparative physiological and transcriptomic analyses reveal the toxic effects of ZnO nanoparticles on plant growth jinpeng wan, ruting wang, ruling wang, qiong ju, yibo wang, and Jin Xu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06641 • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019

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Environmental Science & Technology

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Running title: ZnO nanoparticles repress plant growth

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Title: Comparative physiological and transcriptomic analyses

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reveal the toxic effects of ZnO nanoparticles on plant growth

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Authors: Jinpeng Wan†,‡, Ruting Wang†,§, Ruling Wang†, Qiong Ju†, Yibo Wang#,*,

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Jin Xu†,#,*

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† CAS

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Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, Mengla, Yunnan

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666303, China

9

‡

University of Chinese Academy of Sciences, Beijing 100049, China

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§

College of Agriculture and forestry, Puer University, Puer, Yunnan 665000,China

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#

GanSu Key Laboratory for Utilization of Agricultural Solid Waste Resources, College

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of Bioengineering and Biotechnology, TianShui Normal University, TianShui, GanSu

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741000, China

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Corresponding authors:

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Jin Xu, Ph D

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CAS Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna

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Tropical Botanical Garden, Chinese Academy of Sciences,

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Menglun, Mengla, Yunnan 666303, China

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Tel: 86 871 65140420

Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna

1

ACS Paragon Plus Environment


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Email: [email protected]

21

Yibo Wang, Ph D

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GanSu Key Laboratory for Utilization of Agricultural Solid Waste Resources, College of

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Bioengineering and Biotechnology, TianShui Normal University, TianShui, GanSu

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741000, China

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Email: [email protected]

26 27

TOC

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Comparative physiological and transcriptomic analyses reveal

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the toxic effects of ZnO nanoparticles on plant growth

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Jinpeng Wan†,‡, Ruting Wang†,§, Ruling Wang†, Qiong Ju†, Yibo Wang#,*, Jin

31

Xu†,#,*

32

† CAS

33

Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, Mengla, Yunnan

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666303, China

35

‡

University of Chinese Academy of Sciences, Beijing 100049, China

36

§

College of Agriculture and forestry, Puer University, Puer, Yunnan 665000,China

37

#

GanSu Key Laboratory for Utilization of Agricultural Solid Waste Resources, College

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of Bioengineering and Biotechnology, TianShui Normal University, TianShui, GanSu

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741000, China

Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna

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ABSTRACT: Zinc oxide (ZnO) nanoparticles (nZnO) are among the most commonly

41

used nanoparticles (NPs), and they have been shown to have harmful effects on plants.

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However, the molecular mechanisms underlying nZnO tolerance and root sensing of NP

43

stresses have not been elucidated. Here, we compared the differential toxic effects of

44

nZnO and Zn2+ toxicity on plants during exposure and recovery using a combination of

45

transcriptomic and physiological analyses. Although both nZnO and Zn2+ inhibited

46

primary root (PR) growth, nZnO had a stronger inhibitory effect on the growth of

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elongation zones, whereas Zn2+ toxicity had a stronger toxic effect on meristem cells.

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Timely recovery from stresses is critical for plant survival. Despite the stronger inhibitory

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effect of nZnO on PR growth, nZnO-exposed plants recovered from stress more rapidly

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than did Zn2+-exposed plants upon transfer to normal conditions, and transcriptome data

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supported these results. In contrast to Zn2+ toxicity, nZnO induced endocytosis and

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caused microfilaments rearrangement in the epidermal cells of elongation zones, thereby

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repressing PR growth. nZnO also repressed PR growth by disrupting cell wall

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organization and structure through both physical interactions and transcriptional

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regulation. The present study provides new insight into the comprehensive understanding

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and re-evaluation of NP toxicity in plants.

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INTRODUCTION With the rapid development of nanomaterials, the interactions of plants with

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nanoparticles (NPs) have attracted increasing attention. Macromolecules can enter plant

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cells by endocytosis or nonendocytic penetration,1 and several studies have demonstrated

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that NPs (up to 40 nm) can be taken up by plants and transported by the vascular system,

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ultimately entering plant cells.2-4 For example, NPs enter the protoplasts of sycamore

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maple (Acer pseudoplatanus) by endocytosis and are then deposited in central vacuoles.5

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Larger NPs adhere to cell walls and accumulate in apoplastic spaces.6 The physical

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interactions of NPs with cell walls affect cell wall organization and structure in plants,

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thereby modulating growth.

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NPs have recently been widely employed to enhance plant stress resistance to both

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biotic and abiotic stresses. 7-10 Copper (Cu)-containing NPs have wide applications as

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bactericides and fungicides in agriculture. 7 Treatment with cerium oxide (CeO2) NPs (50

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mg L-1) improves salt tolerance in Arabidopsis. 9 CeO2 NPs (250 mg L-1) also suppress

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fungal pathogens in tomato. 10 Moreover, a number of studies have demonstrated that

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CeO2 NPs possess antioxidant activity and catalytic reactive oxygen species (ROS)

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scavenging properties and can thereby improve stress tolerance in plants. 11-13

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Owing to their unique characteristics, such as broad host range applicability and the

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ability to traverse plant cell walls without external force, NPs have recently shown great

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potential in plant genetic engineering. 14 Thus, elucidating the toxicity of NPs in plants is

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critical for optimizing the efficiency of nanocarrier delivery.

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Numerous studies have revealed both positive and negative effects of NPs in plants.

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For example, several studies have demonstrated that NPs such as silver (Ag) NPs (0.5-3

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mg L-1) and zinc oxide (ZnO) NPs (nZnO, 0.01 mg L-1) induce oxidative damage and

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lipid membrane peroxidation in the aquatic plant Spirodela punctuta.15-16 Aluminum

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oxide (Al2O3) NPs, even at very high concentrations (4 g L−1), do not show toxicity with

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regard to root growth and development in Arabidopsis.17 Titanium dioxide (TiO2) NPs

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(0.025-0.4%) promote photosynthesis and nitrogen metabolism, thereby improving the

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growth of spinach.18-19 In contrast, nZnO (200 and 300 mg L-1) inhibits chlorophyll

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biosynthesis, reducing photosynthetic efficiency.20

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nZnO is among the most commonly used NP in paints, cosmetics, sunscreens, and

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ultraviolet (UV) light blockers.20 The worldwide production of ZnO is estimated to

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exceed 1.2 million tons per year.21 Many studies have shown the harmful effects of nZnO

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on plants, 6, 20, 22-27 indicating that nZnO represses plant growth by reducing

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photosynthesis and inducing ROS accumulation and subsequent oxidative damage.

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Previous studies have also suggested that nZnO-induced inhibition of plant growth may

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by mediated by either the particles themselves or by Zn2+ released from nZnO.6, 22

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However, Landa et al.28 found similar differential gene expression profiles between

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Arabidopsis seedlings treated with Zn2+ and those treated with different sizes of nZnO,

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indicating that the toxicity of nZnO was caused by the Zn2+ released. In contrast, Poynton

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et al.22 found different gene expression profiles in Daphnia magna treated with nZnO or

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with Zn2+ at sublethal concentrations (2.2 mg L-1 nZnO and 0.13 mg L-1 Zn2+). These

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divergent results may have resulted from the different plant materials and treatment

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periods used in these studies. However, NPs show strong toxicity even at concentrations 6


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lower than those necessary for ion toxicity, and this toxicity of NPs cannot be explained

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by ion release from the NPs.21 Furthermore, nZnO and Zn2+ induce different

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morphological changes and differential expression of metal homeostasis- and

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phytohormone regulation-related genes in Arabidopsis seedlings, indicating that the toxic

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mechanisms of nZnO and Zn2+ differ. 29 Nonetheless, the molecular mechanisms

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underlying NP tolerance, root NP sensing and responses to NP stresses have not been

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elucidated. Several studies have demonstrated that agar media can be a suitable culture

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system to study the toxicity of NPs in plants because it can prevent the aggregation of

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NPs.29-30 In this study, we compared the differential toxic effects of nZnO and Zn2+ on

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plants using a combination of transcriptomic and physiological analyses using agar

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media,. Our results reveal the differential responses of plants to nZnO and Zn2+ toxicity

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during exposure and recovery. Notably, nZnO-treated seedlings can more rapidly recover

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from stress than can Zn2+-treated seedlings. This finding may help us broaden the

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application of NPs as nanocarriers in plant genetic engineering.

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MATERIALS AND METHODS

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Plant Growth and ZnO Nanoparticles Exposure. Arabidopsis thaliana

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Columbia wild-type plants (Col-0) and the ROS-deficient mutants respiratory burst

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oxidase homolog D (rbohD) and rbohF were used in the experiments. The seeds were

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surface sterilized with 5% bleach for 5 min, washed 5 times with aseptic water, and

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stratified at 4°C for 2 d before being sown on vertically oriented agar medium containing

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one-half-strength Murashige and Skoog (MS) medium (Sigma-Aldrich, St. Louis, MO,

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USA), pH 5.7, 1% (w/v) agar and 1% (w/v) sucrose. Seedlings were grown vertically in

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growth chambers at 22°C under 16/8 h day/light periods. nZnO, 20-45 nm in size, were

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purchased from Sigma-Aldrich (St. Louis, MO, USA). Characterization of nZnO,

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including morphological analysis with transmission electron microscopy (TEM), size

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distribution analysis with dynamic light scattering (DLS) spectrometry, and dissolution

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analysis, has previously been performed.29 The nZnO was suspended in deionized water

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(ddH2O). After stirring for 2 h with a mechanical agitator, the stock solution was

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sonicated with an ultrasonicator (20 kHz, 30 min, Qsonica Q700, USA) until the nZnO

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was well distributed, as described by Nair and Chung.29 The percent dissolution of 20, 50,

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100, and 200 mg L-1 nZnO was 63.9 %, 35.7 %, 31,4 %, and 23.3 % (i.e. the Zn2+

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releases were 12.79, 17.83, 31.39, and 46.62 mg L-1 from 20, 50, 100, and 200 mg L-1

133

nZnO), respectively, after a 24-h period. 29 Five-day-old seedlings were transferred to

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plates with different chemicals, i.e., nZnO (0, 20, 50, 100, 200 mg L-1), zinc sulfate

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(ZnSO4; 0, 100, 200 mg L-1), and 0.2 mM catalase (CAT), and grown for an additional 1-

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5 d.

137 8


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Determination of Primary Root (PR) Length. Five-day-old seedlings were

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transferred to fresh 1/2 MS medium containing the chemicals for the indicated times.

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Images were obtained using an Epson Perfection V500 Photo Scanner (Japan), and the

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PR length was measured using ImageJ software (version 1.51j8). The experiments were

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repeated three times with at least 30 plants in each replicate.

143 144

Histochemical

Staining.

The

cell

cycle

markers pCYCLINB1;1

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(CYCB1;1):CYCB1;1-GUS, pCYCB3;1:CYCB3;1-GUS, and pCYCA3;1:CYCA3;1-GUS;

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the quiescent center (QC) markers QC25-GUS and pWUSCHEL-related homeobox5

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(WOX5):GUS; and the auxin-responsive DR5-GUS and domain II (DII)-VENUS

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transgenic lines were used for histochemical GUS staining analysis. The treated seedlings

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were incubated in GUS buffer containing 1 mM 5-bromo-4-chloro-3-indolyl-b-D-GlcA

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cyclohexyl-ammonium (Sigma-Aldrich). The samples were then washed and placed in 75%

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(w/v) ethanol, and photographs were taken using a Carl Zeiss imaging system.

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Fluorescence Microscopy. After the seedlings were exposed to the different

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chemicals for the indicated times, green fluorescent protein (GFP) or yellow fluorescent

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protein (YFP) fluorescence in the roots of actin-binding domain 2 (ABD2):GFP,

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PLETHORA2

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RESISTANT1 (AUX1)-YFP, PIN-FORMED1 (PIN1)-GFP, PIN2-GFP, PIN3-GFP, PIN4-

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GFP, and PIN7-GFP were detected. The fluorescence was examined using a confocal

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laser-scanning microscope (LSM710). The excitation wavelength for GFP was 488 nm,

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and the emission wavelength was 509 nm; the excitation wavelength for YFP was 514

(PLT2)-GFP,

SHORTROOT

(SHR)-GFP,

9


DII-VENUS,

AUXIN-


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nm, and the emission wavelength was 527 nm.

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In Situ Zn Localization. To visualize in vivo localization of Zn in Arabidopsis roots,

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Zinpyr-1 was used for staining as described previously.

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chemicals for the indicated time, the seedlings were washed alternately three times in

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ddH2O and in 10 mM ethylenediaminetetraacetic acid (EDTA) and were then incubated

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in Zinpyr-1 solution (20 μM) for 3 h at room temperature in the dark. After washing the

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seedlings five times with ddH2O, images were taken using an LSM710 confocal laser

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scanning microscope (excitation, 488 nm; emission, 509 nm).

21

After exposure to various

170 171

Measurement

of

ROS

Level.

The

specific

fluorescence

probe

2,7-

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dichlorofluorescein diacetate (DCFH-DA) (Beyotime) was used to visualize the in vivo

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localization of ROS in Arabidopsis roots. Treated seedlings were incubated in 10 μM

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staining solution at 37°C in the dark for 10 min. After washing five times with ddH2O,

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the fluorescence was examined using a confocal microscope LSM710 (excitation

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wavelength, 488 nm; emission wavelength, 525 nm).

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FM4-64 staining. To monitor nZnO-induced endocytosis, we used N-(3-

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triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium

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dibromide (FM4-64) staining. FM4-64 staining is a valuable technique to detect

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endocytosis. FM4-64 rapidly stain the plasma membrane and is then integrated into the

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intracellular vesicular network through endomembrane system-dependent internalization

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processes. 31 For FM4-64 staining analysis, 5-day-old Col-0 seedlings were treated with 10


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or without 100 mg L-1 nZnO (for 6 h or 3 d), 25 μM brefeldin A (BFA) (for 3 h), or 200

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mg L-1 Zn2+ (for 3 d) or were treated with 100 mg L-1 nZnO for 3 d and then transferred

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to normal medium for recovery for 3 d. The treated seedlings were incubated in 50 μM

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FM4-64 solution for 5 min at room temperature under darkness and were then washed

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five times with ddH2O. Images of the seedling roots were taken using an LSM710

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confocal microscope.

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Quantitative Reverse Transcription PCR (qRT-PCR) and Digital

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transcriptomics. Total RNA was extracted using RNAiso Plus (TaKaRa, Japan) from

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5-day-old Arabidopsis Col-0 seedlings. qRT-PCR was performed using Platinum SYBR

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Green qPCR SuperMix-UDG (Invitrogen, USA) as described previously. 32 High-

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throughput sequencing was performed using the BGISEQ-500 platform. Additional

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methods are available in the Supporting Information.

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Nutrient Content Analysis. The seedlings for ionomic analysis were grown on 1/2

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MS medium for 7 d and then transferred to fresh medium containing 100 mg L-1 nZnO or

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200 mg L-1 ZnSO4 for an additional 3 d. The treated seedlings were immersed in 10 mM

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EDTA for 30 min to remove metal ions adhering to the surface and then rinsed five times

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with ddH2O. According to the methods described by Liu et al.,32 the samples were fixed

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in an oven at 105°C for 1 h and then dried at 75°C until they reached a constant weight.

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The samples were digested in concentrated nitric acid for 3 d and then boiled for 2 h until

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thoroughly digested. The elements Zn, manganese (Mn), iron (Fe), and potassium (K)

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were measured using inductively coupled plasma atomic emission spectroscopy (ICP11



207

AES, IRIS Advantage, iCAP 6300, USA).

208 209

Statistical Analysis. More than 30 seedlings were analyzed in each treatment. Three

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repetitions with similar results were performed for each experiment. Student’s t-test or

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Tukey’s test were used to determine the significance of differences (P < 0.01).

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RESULTS AND DISCUSSION Differential toxic effects of nZnO and Zn2+ on the growth of Arabidopsis

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seedlings. Five-day-old Arabidopsis seedlings were transferred to fresh 1/2 MS

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medium containing 20-200 mg L-1 nZnO or 100 or 200 mg L-1 ZnSO4 (Zn2+) to estimate

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toxicity. PR growth was inhibited by 53 % after 2 d of treatment with 20 mg L-1 nZnO

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and nearly ceased to growth after 2 d of treatment with > 100 mg L-1 (Figure 1a). Zn2+

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toxicity also markedly inhibited PR growth. PR growth was inhibited by 31 % after 2 d of

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treatment with 100 mg L-1 Zn2+ and nearly ceased to growth after 2 d of treatment with

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200 mg L-1 Zn2+ (Figure 1a). Although 100 mg L-1 nZnO resulted in stronger inhibitory

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effects on PR growth than did 100 mg L-1 Zn2+, Zinpyr-1 fluorescence probe staining

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indicated that Zn accumulation was significantly lower in the roots of nZnO-treated

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seedlings than in those of Zn2+-treated seedlings (Figure 1b). Treatment with 200 mg L-1

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Zn2+ inhibited PR growth similarly to 100 mg L-1 nZnO (almost completely ceasing PR

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elongation) (Figure 1a). Thus, we chose 200 mg L-1 Zn2+ and 100 mg L-1 nZnO for our

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comparative study.

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To elucidate the underlying mechanisms of nZnO-mediated root growth inhibition, we

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measured the growth of elongation zones (EZ) and meristem zones (MZ). Although 200

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mg L-1 Zn2+ and 100 mg L-1 nZnO exerted similar inhibitory effects on PR elongation, the

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MZ length was shorter in Zn2+-treated roots than in nZnO-treated roots (Figure 1c). In

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contrast, the EZ length was shorter in nZnO-treated roots than in Zn2+-treated roots

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(Figure 1d). Although both nZnO and Zn2+ inhibited PR growth, nZnO had a stronger

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effect on EZ growth, and Zn2+ toxicity had a stronger inhibitory effect on meristem cell 13



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activity. We then tested the possibility that the nZnO-mediated cessation of root growth

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might result from PR death. To address this, seedlings were transferred to normal

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medium for further culture after 3 d of treatment with 100 mg L-1 nZnO or 200 mg L-1

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Zn2+. The PR growth of Zn2+-treated seedlings did not fully recover, whereas that of

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nZnO-treated seedlings did fully recover after 6 d of culture in normal medium (Figure

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1e). These data indicated that although nZnO almost completely stopped PR growth, it

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did not lead to root death.

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Our previous study found that Zn2+ toxicity inhibits root growth by inhibiting

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meristematic cell activity.33 To elucidate how nZnO inhibits PR growth, we used WOX5-

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GUS, QC25-GUS, pPLT2:PLT2-GFP, and pSHR:SHR-GFP reporters to monitor stem

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cell niche activity and pCYCB3;1:CYCB3;1-GUS, pCYCB1;1:CYCB1;1-GUS, and

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pCYCA3;1:CYCA3;1-GUS reporters to monitor the meristematic cell division potential.34-

246

35

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treatment. Zn2+ toxicity resulted in a greater reduction in the activities of CYCB3;1-GUS

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and CYCA3;1-GUS than those of nZnO-treated roots, and CYCB1;1-GUS activity was

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also markedly reduced in Zn2+-treated roots, whereas it was almost unaffected by nZnO

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treatment (Figure 2). These data support the above results and indicate that Zn2+ toxicity

251

had a stronger toxicity effect on meristem cell activity than did nZnO.

Similar to Zn2+ toxicity, the stem cell niche activity was almost unaffected by nZnO

252 253

Both nZnO and Zn2+ disrupt auxin accumulation and induce ROS

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accumulation in root tips. Our previous studies indicated that Zn2+ toxicity inhibits

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PR growth by disrupting auxin accumulation and inducing ROS accumulation in roots.33, 14


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36

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accumulation in root tips using the DII-VENUS and DR5:GUS transgenic lines. After 12

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h-1 d of treatment, both nZnO- and excess Zn2+-treated roots showed increased DR5:GUS

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and decreased DII-VENUS expression compared with that in control roots (Figure 3),

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indicating that nZnO and Zn2+ toxicity induce auxin accumulation in root tips during the

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early stages of stress. However, the auxin levels in the root tips of both nZnO- and Zn2+-

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treated seedlings were significantly reduced after 3 d of treatment.

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To elucidate whether nZnO produces similar results, we first investigated auxin

As auxin transport plays a role in modulating auxin distribution in roots, 32 we

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examined the expression of auxin carriers using pAUX1:AUX1-YFP and

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pPIN1/2/3/4/7:PIN1/2/3/4/7-GFP reporter lines. After 1 d of treatment, both PIN3-GFP

266

and PIN4-GFP showed reduced abundance in the root tips of nZnO-treated seedlings;

267

however, only PIN4-GFP showed reduced abundance in the root tips of Zn2+-treated

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seedlings. The abundance of AUX1, PIN1, PIN2, and PIN7 was almost unaffected in

269

both nZnO- and excess Zn2+-treated roots after 1 d of treatment but was markedly

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reduced after 3 d of treatment (Figure 4).

271

Analysis of auxin carrier expression indicated that nZnO inhibited the abundance of

272

PIN3 and PIN4 and that Zn2+ toxicity inhibited the abundance of PIN4 in root tips. Both

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PIN3 and PIN4 regulate auxin levels and gradients in the root meristem, and loss-of-

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function pin3 and pin4 mutant seedlings accumulate higher auxin levels in root tips than

275

wild-type seedlings,37 suggesting that PIN3 and PIN4 at least partly mediate nZnO-

276

induced auxin accumulation to inhibit root growth. During the late stages of stress (after 3

277

d of treatment), the abundance of PIN1, PIN2, and PIN7, in addition to PIN3 and PIN4, 15



278

was markedly reduced in both nZnO- and Zn2+-treated roots, thereby leading to auxin

279

reduction and PR growth cessation.

280

We next examined ROS levels in root tips. Treatment with nZnO markedly induced

281

higher ROS accumulation in the roots compared with the Zn2+-treated seedlings,

282

especially in EZs (Figure 5a and 5b). These data suggested that nZnO induced greater

283

oxidative damage than did Zn2+ toxicity in Arabidopsis seedlings.

284

Using the ROS scavenger CAT, We then investigated the physiological role of ROS

285

accumulation in Zn2+- or nZnO-mediated PR growth inhibition and found that exogenous

286

CAT alleviated Zn2+- or nZnO-mediated PR growth inhibition (Figure 5c). We also

287

examined root growth in the ROS-deficient mutants rbohD and rbohF and found that PR

288

growth was less inhibited in these mutants than in Zn2+- or nZnO-treated Col-0 seedlings.

289

These data indicated that increased ROS accumulation in Zn2+- or nZnO-treated roots was

290

responsible for the observed PR growth inhibition (Figure 5d).

291 292

nZnO induce endocytosis and rearrange microfilaments in Arabidopsis

293

roots. Many studies have demonstrated that NP can enter root cells.2-4 Endocytosis is a

294

major pathway for NPs to enter animal cells. NP-induced endocytosis has also been

295

found in the protoplasts of sycamore maple (Acer pseudoplatanus).5, 38 We thus examined

296

whether nZnO was able to induce endocytosis in Arabidopsis root cells by using FM4-64

297

staining. As shown in Figure 6, exposure to nZnO markedly increased FM4-64 uptake in

298

root cells, and the endocytosis gradually disappeared after 3 d of recovery growth in

299

normal medium. In contrast, treatment with excess Zn2+ did not induce endocytosis 16


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(Figure 6a). These data confirmed that nZnO can enter root cells by inducing endocytosis

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in plants.

302

Previous studies have indicated that TiO2 NP disrupt the cytoskeleton by disrupting the

303

microtubule network in Arabidopsis and that changes in actin microfilaments modulate

304

endocytosis.32, 39 We thus investigated the actin microfilament structure in nZnO-treated

305

roots using the actin-binding domain 2 (ABD2):GFP transgenic line. Treatment with

306

nZnO led to a obvious change in the organization and orientation of the actin

307

microfilaments in the epidermal cells of the transition zone in roots, but gradual recovery

308

was observed when the seedlings were transferred to normal medium (Figure 6b).

309

However, treatment with excess Zn2+ did not induce changes in actin microfilaments

310

(Figure 6b).

311

Changes in microfilaments disrupt normal cell communication and modulate

312

endocytosis.32, 40 Endocytosis promotes NP entry into root cells and disrupts the

313

localization and transport of plasma membrane proteins, thereby disrupting auxin

314

transport and subsequently repressing root growth and development.40 After the seedlings

315

were transferred to normal conditions, endocytosis gradually disappeared, the

316

microfilaments gradually recovered, and PR growth subsequently recovered to normal

317

levels. Taken together, these data indicated that nZnO inhibited transition zones

318

elongation by inducing endocytosis and rearranged microfilaments in the epidermal cells

319

of root transition zones.

320

17



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Transcriptomic analysis revealed differential responses to nZnO and Zn2+

322

toxicity during exposure and recovery. To identify the molecular mechanisms

323

responsible for the observed differences between nZnO- and Zn2+-treated seedlings, we

324

analyzed RNA-seq data for seedlings treated with 100 mg L-1 nZnO or 200 mg L-1 Zn2+

325

for 3 d and then following recovery for 3 d. We sequenced 15 samples using the

326

BGISEQ-500 platform, and a total of 25,146 genes were detected. The differentially

327

expressed genes (DEGs) were identified by comparison with the untreated control (log2-

328

fold change ≥ 1 and adjusted P value ≤ 0.001). All sequencing data were archived in the

329

Short Read Archive (SRA) of the National Center for Biotechnology Information (NCBI)

330

under accession no PRJNA505965.

331

A total of 1024 genes were upregulated and 447 genes were downregulated in nZnO-

332

treated seedlings. In addition, 2184 genes were upregulated and 927 genes were

333

downregulated in Zn2+-treated seedlings. After 3 d of recovery, more DEGs had returned

334

to normal levels in nZnO-treated seedlings (71%) than in Zn2+-treated seedlings (54%)

335

(Supplemental Table S1-S4).

336

Based on the DEGs results, we performed Gene Ontology (GO) enrichment analysis

337

(Supplemental Figure S2). GO analysis indicated that DEGs in nZnO-treated seedlings

338

were enriched in the response to ROS (Supplemental Figure S1a and S1b). Four

339

antioxidative enzyme genes, Cu-Zn superoxide dismutase 2 (CSD2), catalase 3 (CAT3),

340

salivary peroxidase (SAPX), and ascorbate peroxidase 1 (APX1), were significantly

341

downregulated in nZnO-treated seedlings, whereas their expression was unaffected in

342

Zn2+-treated seedlings (Supplemental Table S5). These data supported the above

343

physiological results that nZnO induced greater ROS accumulation than that Zn2+ toxicity. 18


Page 18 of 38

Page 19 of 38

344


During the recovery stage, the DEGs in nZnO-treated seedlings were also enriched in

345

cell wall organization and structure (Supplemental Figure S1a and S1b). The expressions

346

of 59 genes involved in cell wall organization and structure were downregulated in

347

nZnO-treated seedlings, whereas only 11 such genes were downregulated in Zn2+-treated

348

seedlings (Supplemental Table S6). The majority of NP adhere to cell walls and

349

accumulate in apoplastic spaces, thereby disrupting normal cell wall structure and cell

350

elongation. Our results supported the results of previous study, 6 suggesting that one of

351

the targets of NP toxicity in plants is the cell wall and that NPs affect cell wall

352

organization and structure through both physical interactions and transcriptional

353

regulation, thereby repressing PR growth.We also performed Kyoto Encyclopedia of

354

Genes and Genomes (KEGG) functional enrichment analysis (Supplemental Figure S2).

355

The DEGs were enriched in nitrogen metabolism and amino acid metabolism in nZnO-

356

treated seedlings (Supplemental Figure S2a). Among 16 DEGs (11 upregulated and 5

357

downregulated) involved in nitrogen metabolism and amino acid metabolism in nZnO-

358

treated seedlings, 12 genes (7 upregulated and 5 downregulated) recovered to normal

359

levels after 3 d of recovery; however, among 20 DEGs (11 upregulated and 9

360

downregulated) involved in nitrogen metabolism and amino acid metabolism in Zn2+-

361

treated seedlings, only 11 (3 upregulated and 8 downregulated) returned to normal levels

362

after 3 d of recovery (Supplemental Table S7).

363

The DEGs were also enriched in protein processing in the endoplasmic reticulum (ER)

364

(Supplemental Figure S2a and S2b). Among 44 DEGs (40 upregulated and 4

365

downregulated) involved in protein processing in ER in nZnO-treated seedlings, 41 (38

366

upregulated and 3 downregulated) recovered to normal levels after 3 d of recovery; 19



367

however, among 38 DEG (35 upregulated and 3 downregulated) involved in protein

368

processing in ER in Zn2+-treated seedlings, only 18 (15 upregulated and 3 downregulated)

369

recovered to normal levels after 3 d of recovery (Supplemental Table S8). Expression of

370

several ER stress-related genes, such as AT1G69325, AT2G27140, ATP/ADP transporter

371

protein 1 (AATP1), and WRKY DNA-binding protein 33 (WRKY33), 41 showed higher

372

expression in Zn2+-treated seedlings and could not recover to normal levels after 3 d of

373

recovery, whereas they could recover to normal levels in nZnO-treated seedlings after 3 d

374

of recovery. Expression of several Heat Shock Proteins (RESTRICTED TEV

375

MOVEMENT 2 (RTM2), HSP17.6II, HSP17.6A, At5g20970, At5g37670, HSP18.2,

376

HSP22.0, At1g07400, and At1g53540)42 recovered to normal levels after 3 d of recovery

377

in nZnO-treated seedlings, whereas they could not recover in Zn2+-treated seedlings

378

(Supplemental Figure S3, Supplemental Table S8). These data further supported the

379

above-observed phenotype that nZnO-treated seedlings were able to recover more rapidly

380

than were Zn2+-treated seedlings upon transfer to normal medium. Rapid recovery of

381

expression of genes involved in primary metabolism and protein processing is beneficial

382

in restoring plant growth to normal levels. Taken together, these data indicated that

383

although nZnO had a stronger inhibitory effect on PR growth than Zn2+, nZnO-treated

384

plants recovered more rapidly from stress than Zn2+-treated plants after transfer to normal

385

conditions.

386 387

Different effects of nZnO and Zn2+ toxicity on metal accumulation in

388

plants. Compared with excess Zn2+-treated seedlings, GO analysis also indicated that

389

nZnO toxicity resulted in significant changes in metal ion homeostasis, especially Fe 20


Page 20 of 38

Page 21 of 38


390

homeostasis (Supplemental Figure S1a and S1b). There were 30 upregulated metal

391

transporter genes in nZnO-treated seedlings, among which only 17 were also upregulated

392

in Zn2+-treated seedlings. Among the 12 downregulated metal transporter genes in nZnO-

393

treated seedlings, only 4 were also downregulated in Zn2+-treated seedlings (Figure 7a,

394

Supplemental Table S9). The transcriptome data were further verified by qRT-PCR

395

(Figure 7b). Expression of BASIC HELIX-LOOP-HELIX 38 (bHLH38), bHLH39,

396

bHLH100, ZINC TRANSPORTER 9 (ZIP9), IRON-REGULATED TRANSPORTER 1

397

(IRT1), IRT2, NICOTIANAMINE SYNTHASE 2 (NAS2), and NATURAL RESISTANCE-

398

ASSOCIATED MACROPHAGE PROTEIN 4 (NRAMP4) were markedly upregulated in

399

nZnO-treated seedlings, whereas NAS3 and NRAMP1 were downregulated. Expression of

400

bHLH38, bHLH39, bHLH100, ZIP9, and IRT1 was also upregulated in Zn2+-treated

401

seedlings.

402

The above results implied that nZnO had a greater effect than Zn2+ toxicity on metal

403

ion accumulation, especially Fe accumulation. We thus examined metal content in nZnO-

404

and Zn2+-treated seedlings. Zn2+ toxicity reduced the Fe content in seedlings, whereas the

405

Fe content was slightly but nonsignificantly increased in nZnO-treated seedlings (Figure

406

7c). Both nZnO and Zn2+ treatment decreased the K content but increased the Mn content

407

in seedlings; however, the K content was lower in nZnO-treated seedlings than in Zn2+-

408

treated seedlings (Figure 7c). Supporting these results, our transcriptome data indicated

409

that treatment with nZnO induced a higher expression of genes involved in Fe

410

homeostasis and transport, 43 such as FERRIC REDUCTION OXIDASE 1 (FRO1)/2/3/5,

411

bHLH38/39/101, IRT1/2, MYB DOMAIN PROTEIN 72 (MYB72), NRAMP4,

412

OLIGOPEPTIDE TRANSPORTER 3 (OPT3), POPEYE (PYE), HMA2, and NAS2/4, 21



413

whereas HIGH AFFINITY K+ TRANSPORTER 5 (HAK5), a K+ transporter gene, was

414

markedly downregulated in nZnO-treated seedlings compared with Zn2+-treated seedlings

415

(Figure 7a). The elevated expression of many Fe homeostasis-related genes maintained

416

Fe uptake in nZnO-treated seedlings. These data were consistent with Nair and Chung’s29

417

results and indicated the different toxicity of nZnO and Zn2+ to plants.

418 419

ASSOCIATED CONTENT

420

Supporting Information

421

The Supporting Information is available free of charge on the ACS Publications website

422

at DOI:

423

The DEGs in nZnO- or Zn2+-treated seedlings and the seedlings of recovery for 3 d are

424

listed in Table S1-S4 (.xlsx), DEGs of antioxidative enzymes are listed in Table S5

425

(.xlsx), DEGs of cell wall organization and structure-related genes are listed in Table S6

426

(.xlsx), DEGs of nitrogen metabolism and amino acid metabolism-related genes are listed

427

in Table S7 (.xlsx), DEGs involved in protein processing are listed in Table S8 (.xlsx),

428

DEGs of metal transporters are listed in Table S9 (.xlsx). qRT-PCR primers are listed in

429

Table S10 (.xlsx). GO enrichment analysis is shown in Figure S1 (.tif), KEGG

430

enrichment analysis is shown in Figure S2 (.tif), Heat maps of ER stress-related genes

431

and HSP genes is shown in Figure S3 (.tif), and the supplemental Methods (.docx).

432 433

AUTHOR INFORMATION

434

Corresponding Authors

22


Page 22 of 38

Page 23 of 38


435

*Phone: +86 871 65140420; e-mail: [email protected] (Jin Xu); [email protected]

436

(Yibo Wang).

437

Notes

438

The authors declare no competing financial interest.

439 440

ACKNOWLEDGMENTS

441

The authors gratefully acknowledge the Public Technology Service Center of the

442

Xishuangbanna Tropical Botanical Garden of CAS for providing research facilities. This

443

research was supported by the China National Natural Sciences Foundation (31772383,

444

31272239, 31560164), the National Key Research and Development Program of China

445

(2016YFC0501901), Basic Research Program of Qinghai Province (2019-ZJ-7033), and

446

the Yunnan Province Foundation for academic leader (2014HB043).

23



447 448

REFERENCES (1) Rosenbluh, J.; Singh, S. K.; Gafni, Y.; Graessmann, A.; Loyter, A. Non-endocytic

449

penetration of core histones into petunia protoplasts and culture cells: a novel mechanism

450

for the introduction of macromolecules into plant cells. Biochim. Biophys. Acta. 2004,

451

1664, 230-240.

452

(2) Kurepa, J.; Paunesku, T.; Vogt, S.; Arora, H.; Rabatic, B. M.; Lu, J. J.; Wanzer, M.

453

B.; Woloschak, M. B. Smalle, J. A. Uptake and distribution of ultrasmall anatase TiO2

454

Alizarin red S nanoconjugates in Arabidopsis thaliana. Nano letters. 2010, 10, 2296-2302.

455

(3) Zhao, L. J.; Peralta-Videa, J. R.; Ren, M.; Varela-Ramirez, A.; Li, C.; Hernandez-

456

Viezcas, J. A.; Aguilera, R. J.; Gardea-Torresdey, J. L. Transport of Zn in a sandy loam

457

soil treated with ZnO NPs and uptake by corn plants: electron microprobe and confocal

458

microscopy studies. Chem. Eng. J. 2012, 184, 1-8.

459

(4) Schwabe, F.; Tanner, S.; Schulin, R.; Rotzetter, A.; Stark, W.; Von Quadt, A.;

460

Nowack, B. Dissolved cerium contributes to uptake of Ce in the presence of differently

461

sized CeO2-nanoparticles by three crop plants. Metallomics 2015, 7, 466-477.

462

(5) Jozef, S. Endocytosis in plants. Springer, Berlin. 2012.

463

(6) Lin, D.; Xing, B. Root uptake and phytotoxicity of ZnO nanoparticles. Environ. Sci.

464 465

Technol. 2008, 42, 5580-5585. (7) Zhao, L.; Huang, Y.; Zhou, H.; Adeleye, A. S.; Wang, H.; Ortiz, C.; Mazere, S. J.;

466

Keller, A. A. GC-TOF-MS based metabolomics and ICP-MS based metallomics of

467

cucumber (Cucumis sativus) fruits reveal alteration of metabolites profile and biological

468

pathway disruption induced by nano copper. Environ. Sci.: Nano 2016, 3, 1114-1123.

469

(8) Jogaiah, S.; Kurjogi, M.; Abdelrahman, M.; Hanumanthappa, N.; Tran, L. S. P. 24


Page 24 of 38

Page 25 of 38


470

Ganoderma applanatum-mediated green synthesis of silver nanoparticles: Structural

471

characterization, and in vitro and in vivo biomedical and agrochemical properties. Arab.

472

J.Chem. 2017 Doi: 10.1016/j.arabjc.2017.12.002.

473

(9) Wu, H.; Shabala, L.; Shabala, S.; Giraldo, J. P. Hydroxyl radical scavenging by

474

cerium oxide nanoparticles improves Arabidopsis salinity tolerance by enhancing leaf

475

mesophyll potassium retention. Environ. Sci.: Nano 2018. 5, 1567-1583.

476

(10) Adisa, I. O.; Reddy Pullagurala, V. L.; Rawat, S.; Hernandez-Viezcas, J. A.;

477

Dimkpa, C. O.; Elmer, W. H.; White, J. C.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L.

478

Role of Cerium Compounds in Fusarium Wilt Suppression and Growth Enhancement in

479

Tomato (Solanum lycopersicum). J. Agri. Food Chem. 2018, 66, 5959-5970.

480

(11) Lee, S. S.; Song, W.; Cho, M.; Puppala, H. L.; Nguyen, P.; Zhu, H.; Segatori, L.;

481

Colvin, V. L. Antioxidant Properties of Cerium Oxide Nanocrystals as a Function of

482

Nanocrystal Diameter and Surface Coating. ACS Nano 2013, 7, 9693-9703.

483

(12) Xue, Y.; Luan, Q.; Yang, D.; Yao, X.; Zhou, K. Direct Evidence for Hydroxyl

484

Radical Scavenging Activity of Cerium Oxide Nanoparticles. J. Phys. Chem. C 2011, 115,

485

4433-4438.

486 487

(13) Celardo, I.; Pedersen, J. Z.; Traversa, E.; Ghibelli, L. Pharmacological potential of cerium oxide nanoparticles. Nanoscale 2011, 3, 1411-1420.

488

(14) Cunningham, F. J., Goh, N. S., Demirer, G. S., Matos, J. L., Landry, M. P.

489

Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends

490

Biotechnol. 2018, 36, 882-897.

491

(15) Thwala, M.; Musee, N.; Sikhwivhilu, L.; Wepener, V. The oxidative toxicity of

492

Ag and ZnO nanoparticles towards the aquatic plant Spirodela punctuta and the role of 25



493 494

testing media parameters. Environ. Sci. Proc. Impacts 2013, 15, 1830-1843. (16) Qian, H.; Peng, X.; Han, X.; Ren, J.; Sun, L.; Fu, Z. Comparison of the toxicity of

495

silver nanoparticles and silver ions on the growth of terrestrial plant model Arabidopsis

496

thaliana. J. Environ. Sci. 2013, 25, 1947-1956.

497

(17) Lee, C. W.; Mahendra, S.; Zodrow, K.; Li, D.; Tsai, Y. C.; Braam, J.; Alvarez, P.

498

J. Developmental phytotoxicity of metal oxide nanoparticles to Arabidopsis

499

thaliana. Environ. Toxicol. Chem. 2010, 29, 669-675.

500

(18) Hong, F. S.; Zhou, J.; Liu, C.; Yang, F.; Wu, C.; Zheng, L.; Yang, P. Effect of

501

nano-TiO2 on photochemical reaction of chloroplasts of spinach. Biol. Trace Elem. Res.

502

2005, 105, 269-279.

503

(19) Yang, F.; Hong, F. S.; You, W. J.; Liu, C.; Gao, F. Q.; Wu, C.; Yang, P.

504

Influences of nano-anatase TiO2 on the nitrogen metabolism of growing spinach. Biol.

505

Trace Elem. Res. 2006, 110, 179-190.

506

(20) Wang, X.; Yang, X.; Chen, S.; Li, Q.; Wang, W.; Hou, C. J.; Gao, X.; Wang, L.;

507

Wang, S. C. Zinc oxide nanoparticles affect biomass accumulation and photosynthesis in

508

Arabidopsis. Front. Plant Sci. 2016, 6, 1243.

509

(21) Kumari, M.; Khan, S. S.; Pakrashi, S.; Mukherjee, A.; Chandrasekaran, N.

510

Cytogenetic and genotoxic effects of zinc oxide nanoparticles on root cells of Allium

511

cepa. J. Hazard. Mater. 2011, 190, 613-621.

512

(22) Poynton, H.C.; Lazorchak, J. M.; Impellitteri, C. A.; Smith, M. E.; Rogers, K.;

513

Patra, M.; Hammer, K. A.; Allen, H. J.; Vulpe, C. D. Differential gene expression in

514

Daphnia magna suggests distinct modes of action and bioavailability for ZnO

515

nanoparticles and Zn ions. Environ. Sci. Technol. 2010, 45, 762-768. 26


Page 26 of 38

Page 27 of 38

516


(23) Hernandez-Viezcas, J. A.; Castillo-Michel, H.; Servin, A. D.; Peralta-Videa, J. R.;

517

Gardea-Torresdey, J. L. Spectroscopic verification of zinc absorption and distribution in

518

the desert plant Prosopis juliflora-velutina (velvet mesquite) treated with ZnO

519

nanoparticles. Chem. Eng. J. 2011, 170, 346-352.

520

(24) Du, W.; Sun, Y.; Ji, R.; Zhu, J.; Wu, J.; Guo, H. TiO2 and ZnO nanoparticles

521

negatively affect wheat growth and soil enzyme activities in agricultural soil. J. Environ.

522

Monit. 2011, 13, 822-828.

523 524

(25) Lee, S.; Kim, S.; Kim, S.; Lee, I. Assessment of phytotoxicity of ZnO NPs on a medicinal plant, Fagopyrum esculentum. Environ. Sci. Pollut. Res. 2013, 20, 848-854.

525

(26) Mousavi Kouhi, S. M.; Lahouti, M.; Ganjeali, A.; Entezari, M. H. Comparative

526

phytotoxicity of ZnO nanoparticles, ZnO microparticles, and Zn2+ on rapeseed (Brassica

527

napus L.): investigating a wide range of concentrations. Toxico. Environ. Chem. 2014, 96,

528

861-868.

529

(27) Bandyopadhyay, S.; Plascencia-Villa, G.; Mukherjee, A.; Rico, C. M.; José-

530

Yacamán, M.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Comparative phytotoxicity of

531

ZnO NPs, bulk ZnO, and ionic zinc onto the alfalfa plants symbiotically associated with

532

Sinorhizobium meliloti in soil. Sci. Total Environ. 2015, 515, 60-69.

533

(28) Landa, P.; Prerostova, S.; Petrova, S.; Knirsch, V.; Vankova, R.; Vanek, T. The

534

transcriptomic response of Arabidopsis thaliana to zinc oxide: a comparison of the

535

impact of nanoparticle, bulk, and ionic zinc. Environ. Sci. Technol. 2015, 49, 14537-

536

14545.

537 538

(29) Nair, P. M. G.; Chung, I. M. Regulation of morphological, molecular and nutrient status in Arabidopsis thaliana seedlings in response to ZnO nanoparticles and Zn ion 27



539

exposure. Sci. Total Environ. 2017, 575, 187-198.

540

(30) Lee, W. M.; An, Y. J.; Yoon, H.; Kweon, H. S. Toxicity and bioavailability of

541

copper nanoparticles to the terrestrial plants mung bean (Phaseolus radiatus) and wheat

542

(Triticum aestivum): plant agar test for water-insoluble nanoparticles. Environ. Toxicol.

543

Chem. 2008, 27, 1915-1921.

544

(31) Rigal, A., Doyle, S. M., Robert, S. Live cell imaging of FM4-64, a tool for tracing

545

the endocytic pathways in Arabidopsis root cells. Plant Cell Expansion 2015, 93-103.

546

(32) Liu, Y. Y.; Wang, R. L.; Zhang, P.; Sun, L. L.; Xu, J. Involvement of reactive

547

oxygen species in lanthanum-induced inhibition of primary root growth. J. Exp.

548

Bot. 2016, 67, 6149-6159.

549

(33) Zhang, P.; Sun, L.; Qin, J.; Wan, J.; Wang, R.; Li, S.; Xu, J. cGMP is involved in

550

Zn tolerance through the modulation of auxin redistribution in root tips. Environ. Exp.

551

Bot. 2018, 147, 22-30.

552

(34) Sabatini, S.; Heidstra, R.; Wildwater, M.; Scheres, B. SCARECROW is involved

553

in positioning the stem cell niche in the Arabidopsis root meristem. Genes Dev. 2003, 17,

554

354-358.

555

(35) Colón-Carmona, A.; You, R.; Haimovitch-Gal, T.; Doerner, P. Spatio-temporal

556

analysis of mitotic activity with a labile cyclin-GUS fusion protein. Plant J. 1999, 20,

557

503-508.

558 559 560 561

(36) Xu, J.; Yin, H. X.; Li, Y. L.; Liu, X. J. Nitric oxide is associated with long-term zinc tolerance in Solanum nigrum. Plant Physiol. 2010, 154, 1319-1334. (37) Friml, J.; Benková, E.; Blilou, I.; Wisniewska, J.; Hamann, T.; Ljung, K.; Woody, S.; Sandberg, G.; Scheres, B.; Jurgens, G.; Palme, K. AtPIN4 mediates sink-driven auxin 28


Page 28 of 38

Page 29 of 38

562 563 564 565 566


gradients and root patterning in Arabidopsis. Cell 2002, 108, 661-673. (38) Dev, A.; Srivastava, A. K.; Karmakar, S. Nanomaterial toxicity for plants. Environ. Chem. Lett. 2018, 1-16. (39) Wang, S.; Kurepa, J.; Smalle, J. A. Ultra-small TiO2 nanoparticles disrupt microtubular networks in Arabidopsis thaliana. Plant Cell Environ. 2011, 34, 811-820.

567

(40) Nagawa, S.; Xu, T.; Lin, D.; Dhonukshe, P.; Zhang, X.; Friml, J.; Scheres, B.; Fu,

568

Y.; Yang, Z. ROP GTPase-dependent actin microfilaments promote PIN1 polarization by

569

localized inhibition of clathrin-dependent endocytosis. PLoS Biol. 2012, 10, e1001299.

570

(41) Yang, Z. T.; Wang, M. J.; Sun, L.; Lu, S. J.; Bi, D. L.; Sun, L.; ... Liu, J. X. The

571

membrane-associated transcription factor NAC089 controls ER-stress-induced

572

programmed cell death in plants. PLoS Genet. 2014, 10, e1004243.

573

(42) Liu, J. X.; Howell, S. H. Endoplasmic reticulum protein quality control and its

574

relationship to environmental stress responses in plants. Plant Cell 2010, 22, 2930-2942.

575

(43) Zhang, J.; Liu, B.; Li, M.; Feng, D.; Jin, H.; Wang, P.; Liu, J.; Xiong, F.; Wang, J.

576

F.; Wang, H. B. The bHLH transcription factor bHLH104 interacts with IAA-LEUCINE

577

RESISTANT3 and modulates iron homeostasis in Arabidopsis. Plant Cell 2015, 27, 787-

578

805.

29



579

FIGURE LEGENDS

580 581

Figure 1. Both nZnO and excess Zn2+ inhibit PR growth. (a) Five-day-old seedlings were

582

transferred to fresh 1/2 MS medium containing 20-200 mg L-1 nZnO or 100-200 mg L-1

583

Zn2+ for 1-4 d, and PR growth was measured. (b) Zinpyr-1 fluorescence probe staining in

584

Arabidopsis roots from five-day-old seedlings that were transferred to 1/2 MS medium

585

supplemented with 100 μM nZnO or 100 μM Zn2+ for 1 d. (c, d) Five-day-old seedlings

586

were transferred to fresh 1/2 MS medium containing 100 mg L-1 nZnO or 200 mg L-1

587

Zn2+ for 1-4 d, and the length of meristem zones (c) and the length of elongation zones (d)

588

were measured. (e) Five-day-old seedlings were transferred to fresh 1/2 MS medium

589

containing 100 mg L-1 nZnO or 200 mg L-1 Zn2+ for 3 d, and the seedlings were then

590

transferred to normal 1/2 MS medium for recovery growth for 1 d, 3 d, or 6 d (RE 1 d,

591

RE 3 d, RE 6 d, respectively). PR growth was measured. The error bars represent the SD.

592

Different letters indicate significantly different values (P < 0.01 based on Tukey’s test). 30


Page 30 of 38

Page 31 of 38


593 594

Figure 2. Effects of nZnO and Zn2+ on root meristem development. (a-d) Stem cell niche

595

activity. Image of GUS staining and the relative GUS activity of 5-day-old WOX5-GUS

596

(a) and QC25-GUS (b) seedlings exposed to 100 μM nZnO or 200 μM Zn2+ for 0 h, 12 h,

597

and 24 h. The GUS activity in the untreated roots (0 h) was set to 100. GFP fluorescence

598

and quantification of the PLT2:GFP (c) and SHR-GFP (d) fluorescence intensities in the

599

roots of 5-day-old PLT2:GFP and SHR-GFP seedlings exposed to 100 μM nZnO or 200

600

μM Zn2+ for 0 h, 12 h, and 24 h. The fluorescence intensity of the untreated roots (0 h) 31



601

was set to 100. (e-g) Meristematic cell division potential. Image of GUS staining and the

602

relative GUS activity of 5-day-old pCYCLINA3;1:CYCLINA3;1-GUS (e),

603

pCYCLINB3;1:CYCLINB3;1-GUS (f), and pCYCLINB1;1:CYCLINB1;1-GUS (g)

604

seedlings exposed to 100 μM nZnO or 200 μM Zn2+ for 0 h, 12 h, and 24 h. The GUS

605

activity in the untreated roots (0 h) was set to 100. The error bars represent the SD.

606

Different letters indicate significantly different values (P < 0.01 based on Tukey’s test).

607

32


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Page 33 of 38


608 609

Figure 3. Both nZnO and excess Zn2+ induce auxin accumulation in root tips. (a) Images

610

of GUS staining of DR5:GUS and the relative GUS activity of five-day-old seedlings that

611

were transferred to 1/2 MS medium supplemented with 100 μM nZnO or 200 μM Zn2+

612

for 0 h, 12 h, 1 d, and 3 d. The GUS activity in the untreated roots (0 h) was set to 100. (b)

613

YFP fluorescence and quantification of the DII-VENUS fluorescence intensities in the

614

roots of 5-day-old DII-VENUS seedlings exposed to 100 μM nZnO or 200 μM Zn2+ for 0

615

h, 12 h, 1 d, and 3 d. The fluorescence intensity of the untreated roots (0 h) was set to 100.

616

The error bars represent the SD. Different letters indicate significantly different values (P

617

< 0.01 based on Tukey’s test).

33



618 619

Figure 4. nZnO and excess Zn2+ affect the abundances of auxin carriers in root tips. YFP

620

or GFP fluorescence and quantification of the AUX1-YFP (a), PIN1-GFP (b), PIN2-GFP

621

(c), PIN3-GFP (d), PIN4-GFP (e), and PIN7-GFP (f) fluorescence intensities in the roots

622

of 5-day-old AUX1-YFP, PIN1-GFP, PIN2-GFP, PIN3-GFP, PIN4-GFP, and PIN7-GFP

623

seedlings exposed to 100 μM nZnO or 200 μM Zn2+ for 0 h, 12 h, 1 d, and 3 d. The

624

fluorescence intensity of the untreated roots (0 h) was set to 100. The error bars represent

625

the SD. *P < 0.01 according to Tukey’s test.

626

34


Page 34 of 38

Page 35 of 38


627 628

Figure 5. Both nZnO and excess Zn2+ induce ROS accumulation in root tips. (a,b)

629

Detection of ROS production in the roots of 5-day-old Col-0 seedlings treated with 100

630

μM nZnO or 200 μM Zn2+ for 0 h, 12 h, 1 d, and 3 d using the ROS-specific fluorescent

631

probe DCFH-DA (a) and quantification of the ROS fluorescence intensities (b) in roots.

632

(c) PR growth of wild type seedlings treated without or with 100 μM nZnO or 200 μM

633

Zn2+ in the presence or absence of 0.2 mM CAT for 3 d. (d) Relative root growth of Col-

634

0, rbohD, and rbohF seedlings treated with 100 μM nZnO or 200 μM Zn2+ for 3 d

635

compared with untreated seedlings. The error bars represent the SD. Different letters

636

indicate significantly different values (P < 0.01 based on Tukey’s test). 35



637 638

Figure 6. (a) nZnO-induced endocytosis in the root cells. Images of FM4-64 staining in

639

the root cells of 5-day-old Col-0 seedlings treated with or without 25 μM BFA (for 3 h),

640

200 μM Zn2+ (for 3 d), 100 μM nZnO (for 6 h or 3 d), or treated with 100 μM nZnO for 3

641

d and then transferred to normal medium for recovery for 3 d. (b) nZnO rearranges

642

microfilaments in the epidermal cells of roots. GFP fluorescence in the roots of 5-day-old

643

ABD2::ABD2-GFP seedlings treated with or without 200 μM Zn2+ or 100 μM nZnO for 3

644

d and then transferred to normal medium for recovery for 1 d or 3 d.

36


Page 36 of 38

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645 646

Figure 7. (a) Heat maps indicate the log2-fold change in the expression of genes involved

647

in metal ion homeostasis and transport relative to the untreated control. (b) qRT-PCR

648

analysis of the genes involved in Fe homeostasis and transport. Five-day-old wild-type

649

seedlings grown on 1/2 MS medium were treated with 100 μM nZnO (nZnO 3 d) or 200

650

μM Zn2+ (Zn ion 3 d) for 3 d, and then following recovery for 3 d (nZnO 3 d+RE 3 d and

651

Zn ion 3 d+RE 3 d, respectively). The expression levels of the indicated genes in

652

untreated seedlings were set to 1. Error bars represent the SD. Asterisks indicate a

653

significant difference from the control (Student’s t test, P < 0.01). (c) Metal contents in

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Arabidopsis seedlings. Five-day-old wild-type seedlings grown on 1/2 MS medium were 37



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treated with 100 μM nZnO or 200 μM Zn2+ for 3 d. n=6. The error bars represent the SD.

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Different letters indicate significantly different values (P < 0.01 based on Tukey’s test).

38


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Comparative Physiological and Transcriptomic Analyses Reveal the

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