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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
5
nanoparticles and autophagy, a housekeeping mechanism that removes damaged cellular
6
constituents. We show that TiO2 nanoparticles induce autophagy in the plant model system
7
Arabidopsis thaliana and that autophagy is an important mechanism for managing TiO2
8
nanoparticle-induced oxidative stress. Additionally, we find that TiO2 nanoparticles induce
9
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
11
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
15
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
23
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
25
damage to the chloroplastic envelope in algae.18
26 27
In mammalian systems, both the mechanisms of nTiO2 toxicity and nTiO2-induced stress
28
responses have been analyzed in detail.19 One of the most studied among these stress
29
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
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degradation of cellular compartments even under favorable conditions.24,
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eukaryotes, autophagy in plants is important for growth and development as it regulates
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essential processes such as stress resistance, nutrient recycling and seedling establishment.
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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
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subsequently fuses with the central vacuole, depositing the cellular cargo surrounded with the
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inner membrane (i.e., autophagic body) that is then degraded by vacuolar proteases. The core
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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
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complex and two ubiquitin-like conjugation systems. The ubiquitin-like conjugation systems
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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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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
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Although ROS have been previously shown to be higher in photosynthetic organisms after
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exposure to nTiO2,7, 9 the link between nTiO2 treatments, autophagy and oxidative stress has not
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yet been investigated. The aim of this study was to determine if autophagy is a mechanism for
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managing nTiO2-induced stress in plants and assess the impact of nTiO2 on subcellular
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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
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Biotechnology) and bafilomycin A1 (Santa Cruz Biotechnology), 2',7'-dichlorodihydrofluorescein
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diacetate (DCFDA, Biotium). Phospholipase D isolated from Streptomyces chromofuscus was
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obtained from Enzo. All working stocks were prepared in DMSO.
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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
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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
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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
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were measured in 38 mM nTiO2 suspensions in either deionized water, 0.1% DMSO, 0.1%
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DMSO with 10 M AZD8055, 0.1% DMSO with 5 M wortmannin or liquid MS/2 media (for
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MS/2 media composition, see Table S2). The solutions were sonicated for 10 minutes to
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improve dispersity43 and measurements were done using a Malvern Zetasizer Nano ZS (HeNe
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laser, 663 nm; detector angle, 173) at nanoComposix (https://nanocomposix.com). The results
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of this analysis are presented in the Supporting Information file (Table S1 and Figure S1).
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Plant material and growth conditions
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Plant lines used were Arabidopsis wild type Col-0, autophagy (atg) mutants atg5-1,26 atg7-2
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(ABRC stock CS369834), atg9-3 (ABRC stock CS68820), atg10-1,44 and the autophagy marker
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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).
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Plants were grown under sterile conditions in a controlled environmental chamber in continuous
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light (80 μmol m−2 s−1) at 24°C on either solid half-strength Murashige and Skoog media45
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(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
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S2. For experiments involving growth on solid media containing nTiO2, an aqueous nanoparticle
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suspension was added to sterilized media cooled to 45C. Prior to sowing, seeds were surface
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sterilized with 70% ethanol (5 minutes), followed by 50% commercial bleach (20 minutes) and
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three rinses of sterile water. After media solidified, individual sterilized seeds were sown with a
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consistent density between individual experimental replicates. For experiments involving growth
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in liquid MS/2 media, plants were grown in 6-well microtiter plates with continuous shaking (25
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rpm). For sowing into microtiter wells, 40 mg of seeds were sterilized, suspended in 200 μl of
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0.1% agarose dissolved in sterile water and seeded at a rate of 10 μl of seed suspension per
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well. After seven days of growth, tissue was removed from media, rinsed and incubated in the
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concentrations of nTiO2 denoted in the Figure legends for 4 hours with continuous shaking (25
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rpm).
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Microscopic analyses
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For all analyses, four-day-old seedlings grown on solid media were treated with the
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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
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an Olympus Fluoview FV1200. For the confocal analyses of green fluorescent protein (GFP),
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tissues were visualized using a 488 nm excitation laser with emission window set at 510-530
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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
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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
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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
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ImageJ.
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For ROS level determination, 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA), which upon
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oxidation forms the fluorescent molecule 2’,7’-dichlorofluorescein (DCF). Microscopic analyses
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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
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detection limit. Identical acquisition parameters were used for all subsequent replicates. For
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confocal microscopy experiments, DCF was detected using a 488 nm excitation laser with
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emission window set at 510-530 nm with an Olympus Fluoview FV1200. DCF and chlorophyll
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channels were then overlaid and the chlorophyll fluorescence was outlined and the overlapping
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DCF fluorescence (measured a mean gray value) was calculated per chloroplast using ImageJ
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software. For fluorescent microscope analyses of DCF fluorescence, micrographs were
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captured on an Olympus SZX12 microscope equipped with an excitation/emission filter of 457-
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487 nm/502-538 nm. For quantification, bright-field and fluorescent images were stacked,
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cotyledon area was outlined and DCF fluorescence was measured as mean gray value using
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ImageJ software.
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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
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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
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phenylmethylsulfonyl fluoride). Protein concentration was measured with a BioPhotometer
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(Eppendorf) using Bradford Quick Start protein assay kit (Bio-Rad) with bovine serum albumin
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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
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for 5 minutes, loaded onto acrylamide gels, subjected to SDS-PAGE and transferred to
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nitrocellulose membranes. Except for ATG8 and ASC-POX analyses, for which 15% separating
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gels containing 6 M urea were used, proteins were separated on 12% gels. The primary
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antibodies used were monoclonal anti-GFP (Catalogue #: GTX113617, Genetex, dilution:
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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
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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-
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mercaptoethanol). Protein concentrations were normalized and 50 l of the normalized extracts
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was added to 1 V of 2X SDS-PAGE loading buffer for the control immunoblots. For
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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
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darkness for one hour. The derivatization reaction was stopped by the addition of 1 V of 2X
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SDS-PAGE loading buffer and 2/3 V of 1 M Tris base. Proteins were separated on a 4-20%
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acrylamide gel (Bio-Rad), blotted on a nitrocellulose membrane and exposed to anti-2,4-
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dinitrophenylhydrazone (DNP) antibodies (Sigma D9781, dilution: 1:10,000).
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Chlorophyll measurements
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Seedlings were grown on solid MS/2 media for 4 days and exposed to nTiO2 for 4 hours in a
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controlled environmental growth chamber. Seedlings were removed from solution and
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chlorophyll levels in the cotyledons were measured using a CCM-300 chlorophyll content meter
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(Opti-Sciences).
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Data analyses
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All statistical analyses were conducted on data obtained from three or more biologically
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independent experimental replicates (as specified in the Figure legends) using GraphPad Prism
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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
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hypocotyl region. Exposure to nTiO2 led to a significant increase in the number of puncta (Figure
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1), suggesting that nTiO2 elicits an autophagic response. The 35S::GFP-ATG8a seedlings were
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also treated with nTiO2 in the presence of the autophagy activator AZD8055. As expected, the
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number of GFP-ATG8a-labeled puncta increased after a 4-hour-long treatment with AZD8055
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alone (Figure 1). Co-treatments with nTiO2, however, induced a small but significant decrease in
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the number of puncta compared to the AZD8055 control, suggesting that when autophagic
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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
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machinery (Figure 1). Next, the 35S::GFP-ATG8a seedlings were co-treated with nTiO2 in the
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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
218
measured in cells treated with nanoparticles only. Measurements of the hydrodynamic diameter
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and zeta potential of nTiO2 in control and treatment solutions (Table S1) showed that the
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compounds used in the co-treatment experiments did not interfere with nTiO2 suspension
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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.
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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.
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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
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and did not lead to nanoparticle-induced formation of aggregates of the overexpressed GFP-
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ATG8a protein.
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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
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autophagic bodies are degraded by vacuolar proteases, the GFP moiety remains stable in
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comparison to the ATG8 portion of the fusion protein, resulting in the accumulation of
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“processed” GFP (pGFP). Thus, pGFP levels can be used as a marker for autophagic flux, i.e.,
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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
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concentrations in water with or without AZD8055 and used for immunoblotting analyses with
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anti-GFP antibodies (Figures 2A and 2B). In plants treated with only nTiO2, all doses except
245
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).
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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.
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The second method used to differentiate between increased autophagosome formation and
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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
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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
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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
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this high dose was less effective at inducing autophagic flux compared to other nTiO2 treatments
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(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
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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
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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
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defects in the autophagy pathway might be hypersensitive to nTiO2 treatments. To test this
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hypothesis, the wild type and a set of autophagy (atg) mutants were germinated and grown on
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solid MS/2 media supplemented with a range of nTiO2 doses. The addition of nTiO2 to high ionic
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strength growth media has been previously shown to alter the aggregation status of
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nanoparticles.
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confirmed that the polydispersity index is high (Table S1), indicating a significant aggregation of
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nTiO2 in the media. Moreover, particle size distribution analysis revealed major peaks at 4 nm
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(6% of intensity), 19 nm (14.6% of intensity) and 241 nm (79.4% of intensity) (Figure S1).
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Addition of gelling agents such as agar to the high ionic strength growth media is expected to
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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
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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
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atg mutants to nTiO2 toxicity was possible. We tested four atg mutant lines: atg9-3, which is
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deficient in the endoplasmic-reticulum derived membrane trafficking for autophagosome
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formation, 52 as well as atg7-2, atg10-1 and atg5-1, which all carry lesions in key genes involved
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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
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differed in sensitivity to nTiO2 compared to the wild type at lower doses (Figure 3A). It is
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important to note that, as expected from the changes in nanosuspension properties, the nTiO2
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concentrations needed to elicit a toxic response in solid media were higher than those in the
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aqueous nTiO2 suspension used for treatments in experiments described so far. In spite of that,
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at the maximal doses tested, the strongest autophagy-deficient mutant atg7-2 showed a ~31%
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(6 mM nTiO2) and ~59% (8 mM nTiO2) decrease in cotyledon emergence and greening in
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comparison to the wild-type (Figure 3B), indicating that atg7-2 is hypersensitive to nTiO2-
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induced stress. Since autophagy is known to be an important mechanism for recycling cellular
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components during early seedling development,53 our data suggest that autophagy is indeed
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involved in defense against nTiO2-induced stress, but it is not the only mechanism that mitigates
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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
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cotyledons of atg7-2 seedlings had significantly stronger DCF signals compared to Col-0 after
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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
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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
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plants bearing lesions in key autophagy genes are hypersensitive to oxidative stress.41,
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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.
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To test if the enhanced upregulation of FeSOD in atg7-2 seedlings is a compensation
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mechanism to mitigate increased ROS accumulation in atg7-2 chloroplasts, we measured the
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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.
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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
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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).
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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