Article pubs.acs.org/est
Exposure of Iron Nanoparticles to Arabidopsis thaliana Enhances Root Elongation by Triggering Cell Wall Loosening Jae-Hwan Kim,† Yongjik Lee,‡ Eun-Ju Kim,† Sungmin Gu,‡ Eun Ju Sohn,‡ Young Sook Seo,§ Hyun Joo An,§ and Yoon-Seok Chang†,* †
School of Environmental Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea ‡ Bioapplications Inc., Pohang 790-784, Republic of Korea § Cancer Research Institute and Graduate School of Analytical Science & Technology, Chungnam National University, Daejeon 305-764, Republic of Korea S Supporting Information *
ABSTRACT: In this study, we investigated the effect of nZVI on plant root elongation in Arabidopsis thaliana and showed, for the first time, that nZVI enhanced root elongation by inducing OH radical-induced cell wall loosening. Exposure of plants to 0.5 g/L nZVI enhanced root elongation by 150−200% over that in the control, and further mechanistic studies showed that this occurred via nZVI-mediated OH radical-induced cell wall loosening. The oxidation capacity of nZVI, leading to release of H2O2, allowed it to cause OH radical-induced cell wall loosening in roots. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometers (MALDI-TOFMS)-based analysis clearly revealed that pectin-polysaccharides in roots were degraded; they are one of the main matrix-polysaccharide-connecting and load-bearing polymers in cell walls. Rapid root elongation led to structural changes in root cell walls: reduction of cell wall thickness and a bias on the orientation of cellulose microfibrils. Additionally, the asymmetrical distribution of tensional strength resulted from the OH radical-induced cell wall loosening enhanced endocytosis. These findings emphasize that OH radical-induced cell wall loosening is important for mechanical regulation of the cell wall and provide new insights into the cellular responses of plants exposed to reactive metal nanoparticles.
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increasing the uptake of water.10 Furthermore, some metal MNPs (e.g., TiO2 and Al2O3) were found to enhance the elongation of roots in cucumber and A. thaliana.7,8 However, the mechanism(s) underlying this effect are not yet well understood. TiO2 was hypothesized to facilitate cucumber root elongation by stimulating nitrogen accumulation and increasing protein formation,7 but a simple increase in protein formation is insufficient to explain the observed elongation. One of iron MNPs, nano zerovalent iron (nZVI), is a prominent material in environmental industries11−14 due to its remarkable redox potential, which is not found in its micrometer-scale counterparts.15,16 During the past 20 years, nZVI slurries of 1.9 to 10 g/L have been used to remediate groundwater contaminations.17 Once nZVI is injected into the subsurface and dispersed in an unconfined aquifer with groundwater flow, the exposure of plants and animals (both
INTRODUCTION To date, a number of studies reporting the influences of manufactured nanoparticles (MNPs) on the environment have dealt with various ecological components, ranging from protozoa to human cells.1−6 The effects of MNPs have been widely investigated in plants, which form the base of the food chain for all higher-level consumers, including humans. However, few studies have intensively explored the cellular responses of plants to MNPs, limiting our ability to explain the influences of MNPs at a physiological level.7,8 The growth and development responses of plants have been shown to differ depending on the type of MNPs. For example, fullerene exposure triggered abnormal cell division in Arabidopsis thaliana roots via the disruption of auxin signaling; the adherence of ZnO to the roots of Lolium perenne led to root collapse; penetration of radish roots by CuO led to the formation of DNA lesions (FapyAde, FapyGua, and 8-OHGua).4,5,9 In contrast, other MNPs have positive effects on plant growth. For example, multiwalled carbon nanotubes (MWCNTs) were found to enhance tobacco cell growth by penetrating the seed coat and acting as water channels, thereby © 2014 American Chemical Society
Received: Revised: Accepted: Published: 3477
September 30, 2013 January 13, 2014 February 28, 2014 February 28, 2014 dx.doi.org/10.1021/es4043462 | Environ. Sci. Technol. 2014, 48, 3477−3485
Environmental Science & Technology
Article
Figure 1. Phenotype of Arabidopsis thaliana seedlings grown on medium with the indicated concentration of (A) RNIP and (B) Fe2+ ions for 21 and 14 days after sowing (DAS), respectively. (C) Root images of the control, RNIP-, and Fe2+ ion-treated seedlings on 21 DAS.
cell wall loosening in roots or hypocotyls by degrading pectin polysaccharides in cell walls; this was proposed to be mediated by apoplastic OH radicals, and was thus called “OH radicalinduced cell wall loosening”.26 Cell wall loosening is essential for plant growth and development, especially elongation, because cell wall functions as exoskeleton of cytoplasm. OH radical-induced cell wall loosening was demonstrated by Schopfer that plants treated by H2O2 and transition metal ions (e.g., Fe2+ or Cu2+) showed increased root length.26 Therefore, we hypothesized, based on the oxidation capacity of nZVI, exposure of nZVI to plant may give rise to enhanced root elongation due to OH radical-induced cell wall loosening. The OH radical-induced cell wall loosening is nonenzymatic reaction, and tensional stress toward the longitudinal axis in cell walls is released by degradation of pectin polysaccharides, resulting in elongation of organs.26−28 However, compared to the other cell wall loosening reactions mediated by enzymes including expansin and endo-(1,4)-β-D-glucanase24,26 little is yet known about OH radical-induced cell wall loosening, especially no study has performed with nano materials. In the present study, we first showed that nZVI caused OH radicalinduced cell wall loosening and enhanced root elongation in A. thaliana and confirmed how root elongation was induced by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometers (MALDI-TOFMS)-based analysis of pectin
aquatic and terrestrial) is likely. Recently, germination and shoot and root growth of crop, such as ryegrass, barely, flax, and Thypa, were reported to be inhibited by nZVI, and also the growth inhibition phenomenon for plant tissues were found to be accelerated with increasing nZVI concentration.18,19 However, despite the common use of nZVI in groundwater remediation, relatively few studies have determined the biocompatibility of plants and nZVI,8,20 compared to the bodies of literature investigating MWCNTs,10 fullerene,4 and other metal oxides.5,7,9,21 Nano zerovalent iron oxidizing capacity has been shown to be capable of generating H2O2 and OH radicals via the Fenton reaction,22,23 as follows: Fe 0 + O2 + 2H+ → Fe 2 + + H 2O2
(1)
Fe 0 + H 2O2 → Fe 2 + + 2OH−
(2)
Fe 2 + + H 2O2 → Fe3 + + OH • + OH−
(3)
The generation and actions of reactive oxygen species (ROS) should be considered when assessing the effect of metal MNPs on plants. The OH radical, which is a ROS, is known to negatively affect the growth of all living organisms, from plants and prokaryotes to human cell.9,24,25 However, in the case of plants, apoplastic OH radicals can play the role of a trigger for 3478
dx.doi.org/10.1021/es4043462 | Environ. Sci. Technol. 2014, 48, 3477−3485
Environmental Science & Technology
Article
SigmaPlot 12.0 software, and a value of P < 0.01 was considered as statistically significant. Confocal Microscopy Analysis. (i) For endocytosis scanning, we used the method of Paciolek et al. with minor modification.30 The control and RNIP-treated seedlings harvested at 14 DAS were incubated in 1 μM of FM 4−64 dye (Invitrogen) for 2 min. The fluorescence images of the roots were obtained using confocal laser scanning microscopy (Olympus FV-1000) with emission at 543 nm and excitation at 560−615 nm. (ii) For 2′,7′-dichlorofluorescin-diacetate (DCFH-DA) assays, same sized pieces (
