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Environmental Processes

Anatase TiO2 nanoparticles induce autophagy and chloroplast degradation in thale cress (Arabidopsis thaliana) Timothy E. Shull, Jasmina Kurepa, and Jan A. Smalle Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b01648 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on August 3, 2019

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Environmental Science & Technology

Anatase TiO2 nanoparticles induce autophagy and chloroplast degradation in thale cress (Arabidopsis thaliana) Timothy E. Shull, Jasmina Kurepa and Jan A. Smalle*

Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY USA *Correspondence to: [email protected]

1

Abstract

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The extensive use of TiO2 nanoparticles and their subsequent release into the environment

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imposed an important question about the effects of this nanomaterial on ecosystems. Here we

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analyzed the link between the damaging effects of reactive oxygen species generated by TiO2

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nanoparticles and autophagy, a housekeeping mechanism that removes damaged cellular

6

constituents. We show that TiO2 nanoparticles induce autophagy in the plant model system

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Arabidopsis thaliana and that autophagy is an important mechanism for managing TiO2

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nanoparticle-induced oxidative stress. Additionally, we find that TiO2 nanoparticles induce

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oxidative stress predominantly in chloroplasts and that this chloroplastic stress is mitigated by

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autophagy. Collectively, our results suggest that photosynthetic organisms are particularly

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susceptible to TiO2 nanoparticle toxicity.

12 13

INTRODUCTION

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TiO2 nanoparticles (nTiO2) are one of the most abundantly produced nanomaterials and are

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widely used in industrial and commercial products.1 This surge in use has resulted in the release

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of various forms of nTiO2 into the environment2,3 resulting in an intensification of research

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focused on the effects of nTiO2 on photosynthetic organisms.4-6 To date, a consensus-effect of

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nTiO2 on photosynthetic organisms has not been reached; both severe toxicity,7-10 as well as

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growth-promoting effects11-13 have been reported. The mechanisms of nanoparticle entry into

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the plant cell also remain poorly understood.6, 14 It is, however, generally accepted that nTiO2

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can be taken up by plants both after foliar exposure and by root uptake, and that it can pass the

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cell wall and plasma membrane.6, 15 In Arabidopsis thaliana, nTiO2 has been shown to disrupt

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microtubule dynamics16 and accumulate in distinct subcellular compartments, including the

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vacuole.17 Moreover, nTiO2 can accumulate within the stroma of chloroplasts and cause severe

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damage to the chloroplastic envelope in algae.18

26 27

In mammalian systems, both the mechanisms of nTiO2 toxicity and nTiO2-induced stress

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responses have been analyzed in detail.19 One of the most studied among these stress

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responses is macroautophagy, hereafter referred to as autophagy.20-23 Autophagy is a

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eukaryotic cellular housekeeping mechanism that orchestrates both the bulk degradation of

31

cellular components under suboptimal environmental conditions and the highly selective

32

degradation of cellular compartments even under favorable conditions.24,

33

eukaryotes, autophagy in plants is important for growth and development as it regulates

34

essential processes such as stress resistance, nutrient recycling and seedling establishment.

35

The process of autophagy in plants involves the formation of a double-membrane structure

36

called an autophagosome around a cellular target. The outer membrane of the autophagosome

37

subsequently fuses with the central vacuole, depositing the cellular cargo surrounded with the

38

inner membrane (i.e., autophagic body) that is then degraded by vacuolar proteases. The core

39

mechanisms of autophagosome formation can be divided into four functional groups: the ATG1-

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ATG13 kinase complex, ATG9 and ATG9-associated proteins, a phosphatidylinositol 3-kinase

41

complex and two ubiquitin-like conjugation systems. The ubiquitin-like conjugation systems

42

facilitate the attachment of the membrane lipid phosphatidylethanolamine (PE) to ATG8. The

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ATG8-PE adduct subsequently becomes an integral part of the outer and inner autophagic


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Similar to other

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membrane and is necessary for the proper formation of autophagosomes. In plants, disruption

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of either conjugation system leads to accelerated senescence26 and hypersensitivity to adverse

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growth conditions.27-31 An emerging field of plant autophagy research is centered around the

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selective autophagy-mediated elimination of chloroplasts termed “chlorophagy”.25, 28

48 49

Both chlorophagy and non-selective autophagy are induced by a number of biotic and abiotic

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stresses such as starvation,29 pathogen infection,30 high-light and salt stress.31-33 The common

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denominator for many stresses that induce autophagy is the generation of reactive oxygen

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species (ROS) which cause protein oxidation and misfolding, lipid peroxidation and

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consequently organellar and cellular dysfunction.34 The stressor-induced generation of ROS

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also leads to activation of ROS-dependent signaling cascades that induce oxidative stress

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defense and repair mechanisms.35-37 A number of recent studies convincingly show that plant

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autophagy is one of the defense and repair mechanisms induced by oxidative stress.37-42 The

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importance of autophagy in oxidative stress defense is highlighted by findings indicating that

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autophagy mutants are hypersensitive to a number of stresses that increase the cellular ROS

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levels.37, 40-42

60 61

Although ROS have been previously shown to be higher in photosynthetic organisms after

62

exposure to nTiO2,7, 9 the link between nTiO2 treatments, autophagy and oxidative stress has not

63

yet been investigated. The aim of this study was to determine if autophagy is a mechanism for

64

managing nTiO2-induced stress in plants and assess the impact of nTiO2 on subcellular

65

compartments in Arabidopsis thaliana. A detailed understanding of the interactions between

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plants and nTiO2 will allow the formulation of more precise guidelines for acceptable

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environmental levels of these nanomaterials.

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MATERIALS AND METHODS


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Materials

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The following compounds were used: the autophagy activator AZD8055 (Enzo), the autophagy

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inhibitor wortmannin (Enzo), the vacuolar H+-ATPase inhibitors concanamycin A (Santa Cruz

73

Biotechnology) and bafilomycin A1 (Santa Cruz Biotechnology), 2',7'-dichlorodihydrofluorescein

74

diacetate (DCFDA, Biotium). Phospholipase D isolated from Streptomyces chromofuscus was

75

obtained from Enzo. All working stocks were prepared in DMSO.

76 77

Ultra-small anatase TiO2 nanoparticles were obtained from US Research Nanomaterials Inc. as

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a 15 wt% aqueous nanopowder dispersion (1.9 M). The transmission electron microscopy

79

analyses and sample purity measurements, conducted by the manufacturer (https://us-

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nano.com/inc/sdetail/630), show spherical or slightly elliptical particles with a size distribution

81

between 5 and 15 nm and a composition that is 99.9% TiO2. To investigate the stability of

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nanoparticles under our experimental conditions, the hydrodynamic diameter and zeta potential

83

were measured in 38 mM nTiO2 suspensions in either deionized water, 0.1% DMSO, 0.1%

84

DMSO with 10 M AZD8055, 0.1% DMSO with 5 M wortmannin or liquid MS/2 media (for

85

MS/2 media composition, see Table S2). The solutions were sonicated for 10 minutes to

86

improve dispersity43 and measurements were done using a Malvern Zetasizer Nano ZS (HeNe

87

laser, 663 nm; detector angle, 173) at nanoComposix (https://nanocomposix.com). The results

88

of this analysis are presented in the Supporting Information file (Table S1 and Figure S1).

89 90

Plant material and growth conditions

91

Plant lines used were Arabidopsis wild type Col-0, autophagy (atg) mutants atg5-1,26 atg7-2

92

(ABRC stock CS369834), atg9-3 (ABRC stock CS68820), atg10-1,44 and the autophagy marker

93

line 35S::GFP-ATG8a.26 The 35S::GFP-ATG8a transgene was introduced into the atg7-2

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background and the double homozygous transgenic line was selected on half-strength


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Murashige and Skoog media containing Basta (selection for 35S::GFP-ATG8a) and sulfonamide

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(selection for atg7-2 background).

97 98

Plants were grown under sterile conditions in a controlled environmental chamber in continuous

99

light (80 μmol m−2 s−1) at 24°C on either solid half-strength Murashige and Skoog media45

100

(MS/2, pH 5.7; Phytotechnology) containing 1% sucrose and 0.8% phytoagar or in liquid MS/2

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supplemented with 0.5% sucrose. The composition of the MS/2 media is summarized in Table

102

S2. For experiments involving growth on solid media containing nTiO2, an aqueous nanoparticle

103

suspension was added to sterilized media cooled to 45C. Prior to sowing, seeds were surface

104

sterilized with 70% ethanol (5 minutes), followed by 50% commercial bleach (20 minutes) and

105

three rinses of sterile water. After media solidified, individual sterilized seeds were sown with a

106

consistent density between individual experimental replicates. For experiments involving growth

107

in liquid MS/2 media, plants were grown in 6-well microtiter plates with continuous shaking (25

108

rpm). For sowing into microtiter wells, 40 mg of seeds were sterilized, suspended in 200 μl of

109

0.1% agarose dissolved in sterile water and seeded at a rate of 10 μl of seed suspension per

110

well. After seven days of growth, tissue was removed from media, rinsed and incubated in the

111

concentrations of nTiO2 denoted in the Figure legends for 4 hours with continuous shaking (25

112

rpm).

113 114

Microscopic analyses

115

For all analyses, four-day-old seedlings grown on solid media were treated with the

116

concentrations of the aqueous nTiO2 suspension denoted in the text for four hours in a

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controlled environmental growth chamber. Confocal microscopic analyses were performed with

118

an Olympus Fluoview FV1200. For the confocal analyses of green fluorescent protein (GFP),

119

tissues were visualized using a 488 nm excitation laser with emission window set at 510-530

120

nm. GFP-labeled puncta per unit area (1000 μm2) were measured using ImageJ software


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(https://imagej.nih.gov/ij/) from z-stack projections of three slices 10 μm apart. For the confocal

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microscopic detection of chlorophylls, a 559 nm excitation laser with emission window set at

123

650-750 nm was used. For chloroplast count experiments, seedlings were visualized on a Nikon

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Eclipse TE2000 fluorescence microscope equipped with a filter set with an excitation/emission

125

of 390 nm/575-750 nm that allowed visualization of chloroplasts via chlorophyll fluorescence.

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The number of chloroplasts per unit area (1000 μm2) in the upper hypocotyl was counted using

127

ImageJ.

128 129

For ROS level determination, 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA), which upon

130

oxidation forms the fluorescent molecule 2’,7’-dichlorofluorescein (DCF). Microscopic analyses

131

of the DCF fluorescence was performed essentially as previously described.46 The acquisition

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parameters for both confocal and fluorescence microscopy experiments were adjusted such that

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the fluorescent signal from the control (i.e., untreated wild-type seedlings) was slightly above the

134

detection limit. Identical acquisition parameters were used for all subsequent replicates. For

135

confocal microscopy experiments, DCF was detected using a 488 nm excitation laser with

136

emission window set at 510-530 nm with an Olympus Fluoview FV1200. DCF and chlorophyll

137

channels were then overlaid and the chlorophyll fluorescence was outlined and the overlapping

138

DCF fluorescence (measured a mean gray value) was calculated per chloroplast using ImageJ

139

software. For fluorescent microscope analyses of DCF fluorescence, micrographs were

140

captured on an Olympus SZX12 microscope equipped with an excitation/emission filter of 457-

141

487 nm/502-538 nm. For quantification, bright-field and fluorescent images were stacked,

142

cotyledon area was outlined and DCF fluorescence was measured as mean gray value using

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ImageJ software.

144 145

Immunoblotting analyses


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For immunoblotting experiments, seedlings were grown for 7 days in 2 ml of liquid MS/2 in 6-

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well microtiter plates, after which the media was aspirated and the seedling were rinsed with

148

distilled water. The nanoparticle suspension (2 ml) was then added to the wells and the plates

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were transferred to a controlled environmental chamber for treatments for the time intervals

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denoted in the text. Samples were then snap frozen in liquid nitrogen and disrupted with

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zirconium beads in a BeadBug bead beater (MidSci) in 2 volumes (V) of extraction buffer (200

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mM sucrose, 2.4 mM sodium deoxycholate, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1 mM

153

phenylmethylsulfonyl fluoride). Protein concentration was measured with a BioPhotometer

154

(Eppendorf) using Bradford Quick Start protein assay kit (Bio-Rad) with bovine serum albumin

155

as a standard. Protein concentration in all samples was equalized using extraction buffer.

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Protein extracts were then mixed with 1 V of 2X SDS-PAGE loading buffer, denatured at 95°C

157

for 5 minutes, loaded onto acrylamide gels, subjected to SDS-PAGE and transferred to

158

nitrocellulose membranes. Except for ATG8 and ASC-POX analyses, for which 15% separating

159

gels containing 6 M urea were used, proteins were separated on 12% gels. The primary

160

antibodies used were monoclonal anti-GFP (Catalogue #: GTX113617, Genetex, dilution:

161

1:10,000), anti-ATG8a (AS14 2769, Agrisera, 1:1000), anti-chloroplastic FeSOD (AS06 125,

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Agrisera, 1:2000), anti-HSP70 (AS08 371, Agrisera, 1:5000), anti-HSP21 (AS08 285, Agrisera,

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1:4000), anti-HSP90 (Santa Cruz Biotechnology, 1:4000), anti-COXII (AS04 053A, Agrisera,

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1:5000), anti-Ɣ-ECS (AS06 186, Agrisera, 1:5000), anti-GS (AS08 295, Agrisera, 1:4000) and

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anti-BIP (sc-33757, Santa Cruz Biotechnology; 1:1000 dilution). Secondary antibodies used

166

were goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, 1:1000 dilution) and goat anti-rabbit

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IgG-HRP (Santa Cruz Biotechnology, 1:1000). Immunoblots were developed using SuperSignal

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West Femto Maximum Sensitivity Substrate (Thermo Scientific). Signals were captured using a

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ChemiDoc XRS and quantified using ImageJ software.

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Immunoblotting analyses of carbonylated proteins were done as previously described.47 In brief,

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treated seedlings were blotted dry, frozen in liquid nitrogen and proteins were extracted in 2 V of

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extraction buffer (50 mM potassium phosphate buffer pH 7, 2 mM MgCl2, 5% glycerol, 5 mM 2-

174

mercaptoethanol). Protein concentrations were normalized and 50 l of the normalized extracts

175

was added to 1 V of 2X SDS-PAGE loading buffer for the control immunoblots. For

176

derivatization, 2 V of 20 mM dinitrophenylhydrazine (DNPH) dissolved in 20% trifluoroacetic

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acid was added to the normalized extracts which were then incubated at room temperature in

178

darkness for one hour. The derivatization reaction was stopped by the addition of 1 V of 2X

179

SDS-PAGE loading buffer and 2/3 V of 1 M Tris base. Proteins were separated on a 4-20%

180

acrylamide gel (Bio-Rad), blotted on a nitrocellulose membrane and exposed to anti-2,4-

181

dinitrophenylhydrazone (DNP) antibodies (Sigma D9781, dilution: 1:10,000).

182 183

Chlorophyll measurements

184

Seedlings were grown on solid MS/2 media for 4 days and exposed to nTiO2 for 4 hours in a

185

controlled environmental growth chamber. Seedlings were removed from solution and

186

chlorophyll levels in the cotyledons were measured using a CCM-300 chlorophyll content meter

187

(Opti-Sciences).

188 189

Data analyses

190

All statistical analyses were conducted on data obtained from three or more biologically

191

independent experimental replicates (as specified in the Figure legends) using GraphPad Prism

192

6. Statistical comparison of means within groups was performed using one-way analysis of

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variance (ANOVA) and comparisons between groups were performed by two-way ANOVA, in

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both cases using Tukey’s multiple comparisons post-hoc testing. P-values ≥0.05 were

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considered non-significant and are not marked on the graphs.

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RESULTS AND DISCUSSION

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Exposure to nTiO2 leads to an autophagic response

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To investigate if nTiO2 exposure leads to changes in autophagy in Arabidopsis, we employed an

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in vivo monitoring method based on the detection of GFP-labeled autophagosomes and

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autophagic bodies which appear as puncta on confocal micrographs.26 To that end, seedlings

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constitutively expressing a GFP-tagged version of ATG8a (35S::GFP-ATG8a) in the wild-type

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background were incubated in water or an aqueous solution of 0.1 mM nTiO2 for 4 hours and

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analyzed. The number of GFP-ATG8a-labeled puncta per unit area was counted in the

205

hypocotyl region. Exposure to nTiO2 led to a significant increase in the number of puncta (Figure

206

1), suggesting that nTiO2 elicits an autophagic response. The 35S::GFP-ATG8a seedlings were

207

also treated with nTiO2 in the presence of the autophagy activator AZD8055. As expected, the

208

number of GFP-ATG8a-labeled puncta increased after a 4-hour-long treatment with AZD8055

209

alone (Figure 1). Co-treatments with nTiO2, however, induced a small but significant decrease in

210

the number of puncta compared to the AZD8055 control, suggesting that when autophagic

211

activity is at its maximal capacity, the additional challenge imposed by nTiO2 leads to either

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accelerated autophagic body degradation or a decrease in the efficacy of the autophagic

213

machinery (Figure 1). Next, the 35S::GFP-ATG8a seedlings were co-treated with nTiO2 in the

214

presence of the autophagy inhibitor wortmannin (Figure 1). The number of GFP-ATG8a-labeled

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puncta decreased after a 4-hour-long treatment with wortmannin alone to ~21% of the untreated

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control, whereas the wortmannin/nTiO2 co-treatments led to an increase in the number of GFP-

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ATG8a-labeled puncta (Figure 1). However, this increase reached only ~57% of the puncta

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measured in cells treated with nanoparticles only. Measurements of the hydrodynamic diameter

219

and zeta potential of nTiO2 in control and treatment solutions (Table S1) showed that the

220

compounds used in the co-treatment experiments did not interfere with nTiO2 suspension

221

stability. Thus, the observed effects of autophagy modulators in the co-treatment assays were


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indeed due to the induced changes of autophagic activity and not to altered availability or

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uptake of nTiO2.

224 225

Finally, 35S::GFP-ATG8a was introgressed into the strong autophagy-defective mutant atg7-2

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background and the resulting double homozygous seedlings were treated with nTiO2 or water.

227

As expected, no puncta were observed in the control seedlings and the nTiO2 treatment did not

228

lead to any changes (Figure 1A), signifying that nanoparticles indeed affect autophagic activity

229

and did not lead to nanoparticle-induced formation of aggregates of the overexpressed GFP-

230

ATG8a protein.

231 232

Exposure to nTiO2 increases autophagic flux

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Since the increase in density of GFP-ATG8a-labeled puncta suggests an autophagic response

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(Figure 1), we next used two additional approaches to determine whether the accumulation of

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puncta was due to higher rates of autophagosome synthesis or lower rates of autophagic body

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degradation. The first approach is based on the finding that when GFP-ATG8-labeled

237

autophagic bodies are degraded by vacuolar proteases, the GFP moiety remains stable in

238

comparison to the ATG8 portion of the fusion protein, resulting in the accumulation of

239

“processed” GFP (pGFP). Thus, pGFP levels can be used as a marker for autophagic flux, i.e.,

240

the rate of the progression of autophagy from autophagosome biogenesis to the degradation of

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autophagic bodies.48 To test if nTiO2 exposure results in pGFP accumulation, seven-day-old

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35S::GFP-ATG8a seedlings were treated for four hours with a broad range of nTiO2

243

concentrations in water with or without AZD8055 and used for immunoblotting analyses with

244

anti-GFP antibodies (Figures 2A and 2B). In plants treated with only nTiO2, all doses except

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0.001 mM led to a statistically significant increase in pGFP levels compared to the control

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(Figures 2A and 2B). However, this accumulation curve peaked at the 2 mM dose and declined

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thereafter. The pGFP dose-response profile in plants co-treated with AZD8055 showed a much


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shallower increase of pGFP levels compared to the nTiO2 only-treated plants and also a

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significant decrease at 4 mM nTiO2 compared to the AZD8055 control (Figures 2A and 2B).

250

These data imply that a high dose of nTiO2 (4 mM) impedes autophagy either at the level of

251

autophagosome biosynthesis or at the level of autophagic body degradation.

252 253

The second method used to differentiate between increased autophagosome formation and

254

decreased autophagic body degradation is based on monitoring the accumulation of the ATG8-

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PE adduct in the presence of vacuolar protease inhibitors. To identify the ATG8-PE adduct,

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protein extracts from Col-0 and the ATG8-PE deficient atg7-2 mutant were treated with

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phospholipase D, which removes the PE moiety.48 A low molecular-weight band eliminated in

258

the wild type by phospholipase D treatment was also absent in the atg7-2 mutant (Figure S2A).

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This protein species also accumulated in wild-type plants treated with the autophagy activating

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compound AZD8055, further verifying that the species with an apparent molecular mass of 14

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kDa is the ATG8-PE adduct (Figure S2B).

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Next, wild-type seedlings were treated for 16 hours with the vacuolar H+-ATPase inhibitor

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concanamycin A dissolved in liquid media, which blocks vacuolar degradation of ATG8-PE by

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deactivating resident hydrolases.49 The treated seedlings were then washed, incubated in a

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range of concentrations of nTiO2 suspended in water and analyzed using immunoblotting. In the

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concanamycin A-only treated plants, the expected increase of ATG8-PE was observed (Figure

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2C). The nTiO2 co-treatments further increased the ATG8-PE adduct levels, confirming that

269

nTiO2 exposure indeed increases autophagic flux (Figure 2C). Similar results were obtained

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when seedlings were co-treated with bafilomycin A1, another inhibitor of vacuolar proteases

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(Figure S2C). The decrease in ATG8-PE levels in the 4 mM nTiO2-treated sample suggests that

272

this high dose was less effective at inducing autophagic flux compared to other nTiO2 treatments

273

(Figure 2C), which is in concurrence with the results of the pGFP accumulation assay. The


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results of the confocal microscopy analyses and both immunoblotting-based assays confirm that

275

nTiO2 induces autophagic flux. In addition, it seems that the high dose of nTiO2 induced

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autophagic dysfunction, a phenomenon which has also been described in other species

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exposed to high doses of nanoparticles.50

278 279

atg7-2 seedlings are hypersensitive to nTiO2-induced stress

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If autophagy is involved in mitigating the effects of nTiO2-induced stress, then mutants with

281

defects in the autophagy pathway might be hypersensitive to nTiO2 treatments. To test this

282

hypothesis, the wild type and a set of autophagy (atg) mutants were germinated and grown on

283

solid MS/2 media supplemented with a range of nTiO2 doses. The addition of nTiO2 to high ionic

284

strength growth media has been previously shown to alter the aggregation status of

285

nanoparticles.

286

confirmed that the polydispersity index is high (Table S1), indicating a significant aggregation of

287

nTiO2 in the media. Moreover, particle size distribution analysis revealed major peaks at 4 nm

288

(6% of intensity), 19 nm (14.6% of intensity) and 241 nm (79.4% of intensity) (Figure S1).

289

Addition of gelling agents such as agar to the high ionic strength growth media is expected to

290

increase the agglomeration and sedimentation of nanoparticles and thus, decrease nTiO2

291

bioavailability.

51

Dynamic light scattering measurements of nTiO2 added to liquid MS/2 media

292 293

Despite the altered characteristics of the nTiO2 suspension in the growth media and the

294

predicted decrease in bioavailability, a direct comparison of the sensitivity of the wild type and

295

atg mutants to nTiO2 toxicity was possible. We tested four atg mutant lines: atg9-3, which is

296

deficient in the endoplasmic-reticulum derived membrane trafficking for autophagosome

297

formation, 52 as well as atg7-2, atg10-1 and atg5-1, which all carry lesions in key genes involved

298

in the ubiquitin-like conjugation system responsible for the generation of the ATG8-PE adduct.25


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The shoot size of all lines was reduced in a dose-dependent manner, but none of the atg lines

300

differed in sensitivity to nTiO2 compared to the wild type at lower doses (Figure 3A). It is

301

important to note that, as expected from the changes in nanosuspension properties, the nTiO2

302

concentrations needed to elicit a toxic response in solid media were higher than those in the

303

aqueous nTiO2 suspension used for treatments in experiments described so far. In spite of that,

304

at the maximal doses tested, the strongest autophagy-deficient mutant atg7-2 showed a ~31%

305

(6 mM nTiO2) and ~59% (8 mM nTiO2) decrease in cotyledon emergence and greening in

306

comparison to the wild-type (Figure 3B), indicating that atg7-2 is hypersensitive to nTiO2-

307

induced stress. Since autophagy is known to be an important mechanism for recycling cellular

308

components during early seedling development,53 our data suggest that autophagy is indeed

309

involved in defense against nTiO2-induced stress, but it is not the only mechanism that mitigates

310

the detrimental effects of nanoparticle treatments.

311 312

Previous studies have shown that nTiO2 exposure leads to the formation of ROS and

313

consequently to oxidative damage of all cellular components.7,

314

between nTiO2-induced oxidative stress and autophagy, we incubated nTiO2-treated seedlings

315

in the ROS indicator 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) which upon oxidation

316

forms the fluorescent molecule 2’,7’-dichlorofluorescein (DCF).55 In agreement with the

317

hypothesis that autophagy mutants are hypersensitive to nTiO2-induced oxidative stress, the

318

cotyledons of atg7-2 seedlings had significantly stronger DCF signals compared to Col-0 after

319

0.1 mM and 2 mM nTiO2 treatments (Figure 4A, Figure S3). Additionally, we measured nTiO2-

320

induced protein oxidation in Col-0 and atg7-2 using a DNPH derivatization assay.56 This

321

experiment revealed that the autophagy mutant accumulated more carbonylated proteins both

322

in untreated and nTiO2-treated seedlings (Figure 4B). These findings align with evidence that

323

plants bearing lesions in key autophagy genes are hypersensitive to oxidative stress.41,


9, 54

To explore the interaction

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Moreover, these results suggest that autophagy is an important mechanism for mitigating

325

damage resulting from exposure to nTiO2-generated ROS.

326 327

Autophagy deficiency enhances nTiO2-induced chloroplastic oxidative stress

328

To test whether the hypersensitivity of autophagy mutants to nTiO2-induced ROS is associated

329

with a specific cellular process, we first analyzed the response of wild-type plants to 2 mM

330

nTiO2. To that end, we monitored the accumulation of a number of proteins which are either

331

canonical plant stress response proteins or homologs to proteins shown to have altered

332

accumulation in response to nTiO2 in other model systems.55,

333

GS1/2, BiP, HSP90, ASC-POX and -ECS did not change in response to the nTiO2 treatment

334

(Figure 4C). The only two proteins whose abundance changed in response to nTiO2 were the

335

chloroplastic chaperone HSP21 and chloroplast-localized FeSOD, which converts superoxide

336

radicals to hydrogen peroxide66 (Figure 4C). nTiO2 has been shown to cause changes in

337

chloroplast morphology and photosynthesis,18,

338

FeSOD was not unexpected. However, considering the broad range of nTiO2-induced cellular

339

stress responses documented in other model systems,54 it was surprising that the only proteins

340

which responded to treatment were chloroplastic. We concluded that, at least under our

341

experimental conditions, nTiO2-induced oxidative stress maximally impacts chloroplasts.

67

58-65

The abundance of COXII,

therefore, the accumulation of HSP21 and

342 343

We next analyzed FeSOD accumulation in wild-type and atg7-2 seedlings exposed to 0.1 mM or

344

2 mM nTiO2 (Figure 4D). As expected from the preliminary antibody screen (Figure 4C), FeSOD

345

levels were higher in the wild-type plants treated with 2 mM nTiO2 while 0.1 mM treatment did

346

not elicit a response. In comparison to the wild type, atg7-2 seedlings were hypersensitive to

347

nTiO2-induced chloroplastic stress, as illustrated by upregulation of FeSOD in response to both

348

nTiO2 concentrations.

349


14

Page 15 of 35


350

To test if the enhanced upregulation of FeSOD in atg7-2 seedlings is a compensation

351

mechanism to mitigate increased ROS accumulation in atg7-2 chloroplasts, we measured the

352

intensity of chloroplast-localized fluorescence of the ROS indicator dye DCF using confocal

353

microscopy. Four-day-old Col-0 and atg7-2 seedlings were treated with 0.1 mM nTiO2, water or

354

the chloroplastic oxidative stress inducer methyl viologen68 for four hours and analyzed. As

355

expected, the oxidative stress inducer methyl viologen caused a strong increase in chloroplastic

356

DCF fluorescence (Figure 5). The mean DCF fluorescence was ~30% higher in untreated atg7-

357

2 seedlings compared to the untreated wild type (Figure 5). In the nTiO2-treated seedlings, 0.1

358

mM nTiO2 increased DCF fluorescence in the wild type by ~80% and atg7-2 by ~150% (Figure

359

5). These results suggest that autophagy is an important pathway for mitigating nTiO2-induced

360

chloroplastic stress.

361 362

nTiO2-induced chloroplast degradation is autophagy-dependent

363

Previous studies have shown that photoinhibition induces chloroplastic oxidative stress and

364

causes chlorophagy,69 the autophagy-dependent degradation of damaged chloroplasts.70

365

Considering this, we next tested if nTiO2 treatments also cause chlorophagy. Four-day-old

366

seedlings were incubated in 0.1 mM nTiO2, 0.5 mM nTiO2 or water. After treatment, hypocotyls

367

were visualized on a fluorescence microscope and the number of chloroplasts per unit area was

368

counted. Both nTiO2 treatments caused a decrease in the number of chloroplasts in wild-type

369

seedlings whereas atg7-2 seedlings did not respond to the treatments (Figures 6A and 6B).

370

Consequently, the chlorophyll levels in Col-0 seedlings decreased to ~65% and ~61% in

371

samples treated with 0.1 mM and 0.5 mM nTiO2, respectively, whereas the chlorophyll content

372

of atg7-2 seedlings remained unchanged (Figure 6C). These results are similar to the previously

373

described delayed degradation of chlorophyll and stromal proteins in autophagy mutants

374

subjected to abiotic stress,71 and suggest that nTiO2-induced chlorophyll degradation is

375

autophagy-dependent.


15


Page 16 of 35

376 377

To further test if the autophagy pathway is involved in the degradation of chloroplasts damaged

378

by nTiO2, four-day-old 35S::GFP-ATG8a seedlings were treated with 0.5 mM nTiO2 and the

379

relative position of autophagosomes and chloroplasts was monitored. We observed that

380

autophagosomes in the hypocotyls of treated seedlings predominantly associated with

381

chloroplasts, which was confirmed by quantification of GFP and chlorophyll fluorescence

382

intensity at region of interest #1 (Figures 7A and 7B, Videos S1 and S2). We concluded that

383

nTiO2 exposure indeed leads to autophagy dependent-chloroplast degradation and that the

384

inability to degrade damaged chloroplasts in atg7-2 seedlings contributes to their nTiO2

385

hypersensitivity.

386

mechanisms have been described,72,32,73 but further research is needed to determine which is

387

responsible for degradation of chloroplasts in response to nTiO2 exposure.

Currently,

two

distinct

autophagy-dependent

chloroplast

degradation

388 389

Collectively, the results presented in this study reveal that anatase nTiO2 with a diameter

390

ranging from 5-15 nm are phytotoxic and induce autophagy to protect the plant cell from

391

nanoparticle-induced damage and in particular, oxidative damage to the chloroplasts.

392 393

ENVIRONMENTAL IMPLICATIONS

394

The low production cost and remarkable photocatalytic activity of anatase nTiO2 have led to its

395

increased use in dye-sensitized solar panels.74 Moreover, nTiO2 is often used as an additive in

396

various building materials, which ultimately leads to environmental contamination as a result of

397

weathering.75 Studies voicing concern regarding the impact of nTiO2 on the environment have

398

made a case for the need to understand interactions between nanomaterials and photosynthetic

399

organisms.76 The influence of anatase nTiO2 on chloroplast functionality described in this study

400

and by others18,

67

reveals that the interactions of this nanomaterial with photosynthetic


16

Page 17 of 35


401

organisms are largely detrimental and have the potential to disrupt the ecologically important

402

carbon-assimilation reactions carried out by photosynthetic eukaryotes.

403 404

ACKNOWLEDGEMENTS

405

This work was supported by the USDA National Institute of Food and Agriculture, AFRI funded

406

grant 1005253 and by the Kentucky Tobacco Research and Development Center. We would

407

like to thank Dr. Richard Vierstra for the 35S::GFP-ATG8a line and Dr. Seth Debolt for help with

408

the confocal microscope which was supported by the National Science Foundation under

409

Cooperative Agreement No. 1355438.

410 411

Conflicts of Interest

412

The authors declare no conflicts of interest

413 414

Authors’ contributions

415

T.E.S, J.K. conducted experiments. T.E.S, J.K and J.S. designed the experiments and wrote the

416

paper. All authors read and approved the final manuscript.

417 418

SUPPORTING INFORMATION

419

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

420

Characterization of anatase TiO2 nanoparticles (nTiO2) (Table S1), size distribution of anatase

421

nTiO2 in half-strength Murashige and Skoog (MS/2) media (Figure S1), half-strength Murashige

422

and Skoog Basal Medium (MS/2) (Table S2), verification of the ATG8-PE adduct (Figure S2),

423

representative 2',7'-dichlorodihydrofluorescein (DCF) micrographs (Figure S3), time-lapse

424

confocal microscopy of seedlings incubated with 0.5 mM nTiO2 or water for four-hours (Videos

425

S1 and S2).


17


Page 18 of 35

426 427

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Shandilya, N.; Le Bihan, O.; Bressot, C.; Morgeneyer, M., Emission of titanium dioxide

Du, W.; Tan, W.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L.; Ji, R.; Yin, Y.; Guo, H.,

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FIGURES 2 0 1 8 .6 .2 8 C o n fo c a l Q u a n tific a tio n fr o m

A DMSO

Col-0 AZD8055

wortmannin

atg7-2 DMSO

a n d 6 .2 7 s e ts

B

88

AZD8055

Puncta/unit area

DMSO

Control

66

****

* wortmannin

0.1 mM nTiO 2

44

****

22

0

0.1

0 0.1 0 nTiO2 (mM)

0.1

D

D

M M SO S O 0 A 0. A ZD 1 Z D 0 0 W .1 W M M 0 0 .1

00

Figure 1. Exposure to nTiO2 induces 35S::GFP-ATG8a labeled puncta A. nTiO2 induces the formation of GFP-labeled puncta. 35S::GFP-ATG8a plants in either wild-type (Col-0) or atg7-2 background were grown for 4 days on solid MS/2 media supplemented with 1% sucrose. Plants were then transferred to either water or aqueous 0.1 mM nTiO2 suspension each of which contained either DMSO (solvent control), AZD8055 (10 µM) or wortmannin (5 µM). After 4-hour-long treatment in the light, seedlings were rinsed, mounted in water and visualized on a laser scanning confocal microscope using an excitation wavelength of 488 nm and detected at 510-530 nm. Micrographs are z-stack projections of hypocotyl cells consisting of three slices 10 µm apart. Arrowhead: examples of GFP-ATG8a puncta quantified in B. Scale bar: 10 µm.

B. Quantification of z-stack projections. Micrographs of at least three biological replicates per treatment were overlaid with a 10002 µm grid (unit area) and puncta were counted per section. Data is presented as mean ± SD (n≥40). *, p

Arabidopsis thaliana - ACS Publications - American Chemical Society

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