Oxidative stress response and proteomic analysis reveal the mechanisms of toxicity of imidazolium-based ionic liquids against Arabidopsis thaliana

doi:10.1016/j.envpol.2020.114013

Environmental Pollution

Volume 260, May 2020, 114013

https://doi.org/10.1016/j.envpol.2020.114013 Get rights and content

Highlights

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ROS and MDA increased with higher ILs concentration and longer carbon chain length.
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Antioxidase activity (SOD, CAT, POD and GPX) changed response to ILs stress.
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Differentially expressed proteins were identified in ILs treatment.
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Down-regulated protein enrichment levels were higher than up-regulated protein.
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The oxidative stress response and photosynthesis effects were proved by proteomic.

Abstract

Ionic liquids (ILs) are extensively used in various fields, posing a potential threat in the ecosystem because of their high stability, excellent solubility, and biological toxicity. In this study, the toxicity mechanism of three ILs, 1-octyl-3-methylimidazolium chloride ([C₈MIM]Cl), 1-decyl-3-methylimidazolium chloride ([C₁₀MIM]Cl), and 1-dodecyl-3-methylimidazolium chloride ([C₁₂MIM]Cl) on Arabidopsis thaliana were revealed. Reactive oxygen species (ROS) level increased with higher concentration and longer carbon chain length of ILs, which led to the increase of malondialdehyde (MDA) content and antioxidase activity, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and peroxidase (POD) activities. SOD, CAT, and GPX activities decreased in high ILs concentration due to the excessive ROS. Differentially expressed protein was analyzed based on Gene ontology (GO) and KEGG pathways analysis. 70, 45, 84 up-regulated proteins, and 72, 104, 79 down-regulated proteins were identified in [C₈MIM]Cl, [C₁₀MIM]Cl, and [C₁₂MIM]Cl treatment, respectively (fold change ≥ 1.5 with ≥95% confidence). Cellular aldehyde metabolic process, mitochondrial and mitochondrial respiratory chains, glutathione transferase and oxidoreductase activity were enriched as up-regulated proteins as the defense mechanism of A. thaliana to resist external stresses. Chloroplast, photosynthetic membrane and thylakoid, structural constituent of ribosome, and transmembrane transport were enriched as the down-regulated protein. Compared with the control, 8 and 14 KEGG pathways were identified forup-regulated and down-regulated proteins, respectively, in three IL treatments. Metabolic pathways, carbon metabolism, biosynthesis of amino acids, porphyrin and chlorophyll metabolism were significantly down-regulated. The GO terms annotation demonstrated the oxidative stress response and effects on photosynthesis of A. thaliana in ILs treatment from biological process, cellular component, and molecular function categories.

Graphical abstract

Introduction

Ionic liquids (ILs) are a new class of solvents that are extensively used in many areas owing to their superior chemical properties such as good solubility and low vapor pressure (Chatel et al., 2017; Salar-García et al., 2017). With expanding usage of ILs, there is a high potential for them to be introduced into the environment, which is a potential threat to the environment owing to their high stability, excellent water solubility, and biological toxicity (Amde et al., 2015; Pham et al., 2010). The biological toxicity of ILs against invertebrates, fish, algae, plant, bacteria, and fungi were revealed (Bubalo et al., 2014; Fan et al., 2019a). The toxicities of ILs were affected by their chemical structure, cation, anion, and length of an alkyl chain in the cation (Egorova and Ananikov, 2014). After exposed for 48 h, the LC₅₀ of Daphnia magna decreased 436-fold as the number of carbon atoms in the side chain of imidazolium ILs increased from 4 to 12 (Yu et al., 2009). The main toxic effect of [C₂MIM]R (R = Cl⁻, Br⁻, BF₄⁻) and [C_nMIM]Br (n = 2, 4, 8, 10, 12) on Danio rerio was related to alkyl-chain lengths (Zhang et al., 2018a). Growth and photosynthetic effects of imidazolium-based ionic liquids with varying carbon chain lengths ([C_nMIM]Cl, n = 8, 10, 12) on Arabidopsis thaliana (A. thaliana) were revealed (Liu et al., 2018b). The toxic effect of pyridinium- and pyrrolidinium-based ILs with various alkyl chain lengths and bromide anion on Pseudokirchneriella subcapitata was compared with log EC₅₀ values (Pham et al., 2016). The role of cations (imidazolium and pyridinium) and anions (NO₃⁻, Br⁻, and Cl⁻) on Scenedesmus obliquus (S. obliquus) were proved using chlorophyll fluorescence parameters and ultrastructure as indexes (Xia et al., 2018).

The mechanisms underlying the toxic effects of ILs on the plant have begun to receive considerable attention, and are the subject of many recent studies, elucidating their effects on various plants (Thamke et al., 2017). The reduction of photosynthetic efficiency was one of the important mechanisms identified in these studies (Reddy et al., 2017). ILs have adverse effects on photosystem II (PSII) of S. obliquus, they effect the primary photosynthetic reaction and diminish photosynthetic efficiency (Liu et al., 2018a). PSII activity was inhibited, and the genes involved in the photosynthetic Calvin cycle were down-regulated in maize seedlings by [C₄MIM]Cl exposure (Li et al., 2018b). Oxidative damage is considered to be another important mechanism through which ILs cause damage (Pawłowska et al., 2019). The growth of Phaeodactylum tricornutum was apparently inhibited under the stress of [C_nMIM]BF₄ (n = 4,8) exposure, superoxide dismutase (SOD) and peroxidase (POD) activity increased due to the excessive reactive oxygen species (ROS) (Deng et al., 2017). Oxidative stress in spring barley and common radishes was observed with H₂O₂ levels and malondialdehyde concentration increasing (Biczak et al., 2017). However, knowledge regarding the molecular mechanism of ILs toxicity against plants is still limited (Pawłowska et al., 2019).

Proteomics is a useful strategy for molecular mechanism study; it helps to identify the protein sequence in plants using a mass spectrometer, and compare with the existing sequences in the database to determine the protein change under stress (Heazlewood and Millar, 2003; Visconti et al., 2019). This method has been used in toxicity studies of heavy metals, nanoparticle, and pesticides (Bilal et al., 2019; Qian et al., 2016). However, the toxicity effect of ILs at protein expression level are scarce in the literature.

Because of its relatively short life cycle, complete genome and proteome database, A. thaliana has been used as a model plant in several studies to understanding the molecular biology (Ke et al., 2018; Wienkoop et al., 2010). This study aimed to reveal the stress mechanisms and compare the effect of toxicity of three methylimidazolium chloride ionic liquids with different alkyl-chain lengths on A. thaliana. The oxidative stress mechanism was revealed by detecting ROS level, malondialdehyde (MDA) content, and antioxidase activity, including SOD, catalase (CAT), POD, and glutathione peroxidase (GPX). Proteomic analysis was conducted to reflect the toxicity mechanism. The results will help to understand the possible toxic impact on environment caused by ILs exposure.

Section snippets

Chemicals

[C₈MIM]Cl, [C₁₀MIM]Cl, and [C₁₂MIM]Cl (Chengjie Chemical Co. LTD, Shanghai, China) were used for testing. The structures of three tested ILs are illustrated in Fig. S1.

Plant culture

A. thaliana (ecotype Colombia) seeds were disinfected for 15 min with a mixture of hydrogen peroxide (30%) and ethanol (75%) in a volume ratio of 1:1. After being rinsed with sterilized water, seeds were vernalized at 4 °C for 2 d away from light. The seeds were germinated using MS medium in dark and then cultured in a light

Reactive oxygen species level and MDA content change in ILs treatment

ROS, such as superoxide anion free radical (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl free radical (OH⁻) were highly active chemical species with toxic exposure (Gill and Tuteja, 2010; Pan et al., 2019). Under the stressors, excessive ROS leading to the production of MDA (i.e., the product of membrane lipid peroxidation) (Shao et al., 2018). High ROS levels, caused by environmental pollutants, indicate the loss of homeostasis in plants, leading to oxidative stress and even apoptosis (Fan et al.,

Conclusion

The toxicology mechanism of [C₈MIM]Cl, [C₁₀MIM]Cl, and [C₁₂MIM]Cl in A. thaliana were revealed in this study. After exposure to ILs, ROS and MDA content increased with a dose-effect relationship. SOD, CAT, and GPX activity increased first and then decreased with an increase in ILs concentration. POD activity increased with ILs concentration to eliminate H₂O₂ to maintain H₂O₂ homeostasis. High levels of down-regulated protein enrichment were detected in the [C₁₀MIM]Cl and [C₁₂MIM]Cl treatments.

CRediT authorship contribution statement

Mingkang Jin: Investigation, Data curation, Formal analysis, Writing - original draft. Huan Wang: Investigation, Data curation, Formal analysis. Huijun Liu: Conceptualization, Methodology, Writing - review & editing, Supervision. Yilu Xia: Investigation, Data curation. Songlin Ruan: Software, Formal analysis. Yuqing Huang: Software, Formal analysis. Jieren Qiu: Software, Formal analysis. Shaoting Du: Writing - review & editing, Project administration. Linglin Xu: Data curation.

Declaration of competing interest

We declare that we do not have any conflicts of interest in connection with the work submitted.

Acknowledgments

This work was financially supported by the Zhejiang Provincial Natural Science Foundation of China (No. Y5100376), and the National Natural Science Foundation of China (No. 21377115).

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Oxidative stress response and proteomic analysis reveal the mechanisms of toxicity of imidazolium-based ionic liquids against Arabidopsis thaliana☆

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals

Plant culture

Reactive oxygen species level and MDA content change in ILs treatment

Conclusion

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgments

Ecotoxicol. Environ. Saf.

J. Hazard Mater.

J. Hazard Mater.

Sci. Total Environ.

Environ. Pollut.

Sci. Total Environ.

Plant Physiol. Biochem.

Plant Physiol. Biochem.

J. Proteomics

Sci. Total Environ.

Plant Gene

Sci. Total Environ.

Ecotoxicol. Environ. Saf.

Ecotoxicol. Environ. Saf.

Sci. Total Environ.

J. Hazard Mater.

Environ. Pollut.

J. Hazard Mater.

J. Environ. Sci.

Ecotoxicol. Environ. Saf.

Chemosphere

Sci. Total Environ.

Colloids Surf., B

J. Hazard Mater.

Environ. Toxicol. Pharmacol.

Ecotoxicol. Environ. Saf.

Chemosphere

Water Res.

Plant Sci.

Sci. Total Environ.

BMC Plant Biol.

J. Proteomics

Chemosphere