Oxidative stress in duckweed (Lemna minor L.) induced by glyphosate: Is the mitochondrial electron transport chain a target of this herbicide?

doi:10.1016/j.envpol.2016.07.019

Environmental Pollution

Volume 218, November 2016, Pages 402-409

https://doi.org/10.1016/j.envpol.2016.07.019 Get rights and content

Highlights

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We investigated physiological responses of Lemna minor plants exposed to glyphosate.
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We located the primary ROS production site in plants exposed to glyphosate.
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Glyphosate interferes with the mitochondrial electron transport chain.

Abstract

We investigated the physiological responses of Lemna minor plants exposed to glyphosate. The deleterious effects of this herbicide on photosynthesis, respiration, and pigment concentrations were related to glyphosate-induced oxidative stress through hydrogen peroxide (H₂O₂) accumulation. By using photosynthetic and respiratory electron transport chain (ETC) inhibitors we located the primary site of reactive oxygen species (ROS) production in plants exposed to 500 mg glyphosate l⁻¹. Inhibition of mitochondrial ETC Complex I by rotenone reduced H₂O₂ concentrations in glyphosate-treated plants. Complex III activity was very sensitive to glyphosate which appears to act much like antimycin A (an inhibitor of mitochondrial ETC Complex III) by shunting electrons from semiquinone to oxygen, with resulting ROS formation. Confocal evaluations for ROS localization showed that ROS are initially produced outside of the chloroplasts upon initial glyphosate exposure. Our results indicate that in addition to interfering with the shikimate pathway, glyphosate can induce oxidative stress in plants through H₂O₂ formation by targeting the mitochondrial ETC, which would explain its observed effects on non-target organisms.

Graphical abstract

Introduction

The global use of glyphosate-based herbicides in agriculture increased enormously after the introduction of glyphosate-resistant (GR) plants. By inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate alters aromatic amino acid production and interferes with the synthesis of proteins and various important plant secondary metabolic compounds (i.e., auxins and polyphenols) which require these amino acids as precursors (Siehl, 1997). Apart from its inhibitory effect on the shikimate pathway, glyphosate has been shown to alter several plant physiological mechanisms such as photosynthesis, respiration, and mineral nutrition (Kielak et al., 2011, Zablotowicz and Reddy, 2007, Zobiole et al., 2012). Additionally, reported detrimental effects of glyphosate on animals (Chłopecka et al., 2014) (which do not possess the shikimate pathway) indicated that it can interact with metabolic pathways other than its primary target (EPSPS). We recently demonstrated a strict relationship between glyphosate-induced oxidative stress and its deleterious effects on photosynthetic processes (Gomes et al., 2016b, Gomes et al., 2016c). The increase in reactive oxygen species (ROS) content upon glyphosate exposure was found to be related to decreased photosynthetic electron transport rates and chlorophyll degradation. It still is not clear, however, how glyphosate induces ROS accumulation.

Chloroplasts and mitochondria constitute the major intercellular sources of ROS (Gill and Tuteja, 2010, Rhoads et al., 2006). Oxygen will accept electrons passing through the photosynthetic electron transport chain (ETC) between photosystem (PS) I and PSII as well as from triplet chlorophyll, resulting in the formation of ROS (O₂⁻, ₁O² and H₂O₂) (Gill and Tuteja, 2010), and approximately 2–3% of respiration oxygen is diverted to ROS production (Puntarulo et al., 1988). The mitochondrial ETC produces electrons with sufficient free energy to directly reduce O₂ and represents an unavoidable primary source of ROS in mitochondria (Rhoads et al., 2006). Superoxide anions (O₂⁻) are continuously produced by complexes I, II and III of the mitochondrial ETCs, which can then undergo dismutation to H₂O₂ via superoxide dismutase activity. After formation, H₂O₂ can react with reduced metals (such as Fe²⁺ and Cu²⁺) to produce radical dot OH which can leave the mitochondrion and migrate to other cell compartments (Rhoads et al., 2006). In addition to chloroplast and mitochondrial activities, NADPH oxidase in the plasma membrane is known to be a ROS production site, especially contributing to O₂⁻ production in metabolically active cells (Grant and Loake, 2000).

Reactive oxygen species function as important signalling molecules in several plant biological processes, serving, for example, as secondary messengers in plant hormonal responses (Gomes et al., 2014a). When accumulated, however, ROS can have phytotoxic effects. Elevated ROS concentrations cause the modification and damage of cellular components such as proteins, lipids, and DNA (Gill and Tuteja, 2010) and can impair metabolism. H₂O₂, for example, can disrupt plant photochemistry by suppressing the de novo synthesis of PSII proteins (especially the D1 protein) required for PSII repair and assembly (Takahashi and Murata, 2008). By inducing lipid peroxidation and interacting with the mitochondrial membrane system, high ROS concentrations can lead to the disruption of mitochondria cristae, mitochondrial fragmentation (Deryabina et al., 2014), and the inactivation of essential enzymes of the respiration pathway through protein carbonylation (Sohal et al., 2002). Within this context, the secondary effects of glyphosate in plants (such as decreased photosynthesis, respiration, and nutrient assimilation) (see also Gomes et al., 2014b) could be intrinsically related to oxidative stress induced by this herbicide.

We investigated the oxidative responses to glyphosate exposure in Lemna minor L. (popularly known as duckweed), an aquatic plant recommended for use as a reference organism for pesticide phytotoxicity assessments (Boutin et al., 1993). We therefore attempted to identify sites of ROS generation caused by glyphosate exposure using specific inhibitors of the activity of ROS-production sites (chloroplast, mitochondria, and plasma membrane ETC inhibitors), and investigated glyphosate effects on respiratory pathways. This study also has implications for eco-toxicology as we examined the effects of glyphosate on the major energy pathways involved in plant metabolism (photosynthesis and respiration), where deleterious effects can result in decreased plant yields and potential damage to untargeted organisms, with obvious environmental impacts. Respiration is involved in generating most intermediary metabolic compounds, so that interactions of this herbicide with mitochondrial functions could result in severe impairment of their overall metabolism (Peixoto, 2005) – and could cause detrimental effects even in organisms without the shikimate pathway, such as animals.

Section snippets

Materials and methods

Lemna minor was cultivated in sterile Bold’s Basal medium (BBM) (Bischoff and Bold, 1963) under standardized growth conditions: temperature 22 ± 2 °C, and continuous light (85 μmol photons m⁻² s⁻¹) provided by cool white fluorescent lamps. Tests were performed in 250 ml Erlenmeyer flasks stoppered with cotton wool to avoid evaporation and contamination. Experiments used 25 fronds of L. minor that had previously been acclimated for 25 days to the above mentioned conditions of temperature and

Physiological responses to glyphosate

As shown in Fig. 1, both photosynthetic and respiration rates decreased in plants exposed to glyphosate concentrations ≥10 mg l⁻¹ for 45 min (P < 0.05) while total chlorophyll concentrations decreased and the pheophytin a/chlorophyll a ratio increased (P < 0.05; Fig. 1). The increased hydrogen peroxide (H₂O₂) concentrations in plants exposed to glyphosate concentrations ≥ 10 mg l⁻¹ were followed by increases in lipid peroxidation (MDA) (P < 0.05; Fig. 1). Glyphosate-exposed plants demonstrated

Discussion

Exposure to glyphosate produced deleterious effects on the physiological processes of Lemna minor (Fig. 1). The observed decreases in photosynthesis, respiration, total chlorophyll concentration, as well as increased pheophytin/chlorophyll ratios in plants exposed to glyphosate concentrations ≥10 mg l⁻¹ can all be related to oxidative stress induced by H₂O₂ accumulation. The decrease in total chlorophyll concentration, concomitant with the increase in the pheophytin/chlorophyll ratio, indicated

Conclusion

Although it has been widely reported that glyphosate can induce oxidative stress in plants, to our knowledge this is the first investigation of the specific site of ROS induction by this herbicide. We have shown that, apart from its specific site of action (the inhibition of the shikimate pathway), glyphosate can interfere in mitochondrial activity by impairing normal electron flow in the respiratory ETC. Glyphosate apparently acts much like antimycin A, inhibiting Complex III activity, which

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgements

This research was supported by the Natural Science and Engineering Research Council of Canada (NSERC- #262210-2011). M.P.G received a Ph.D. scholarship from Fond de Recherche du Quebec–Nature et Technologies (FRQNT). The authors want to thank Marie-Claude Perron for her technical assistance.

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Citation Excerpt :
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^☆: This paper has been recommended for acceptance by Chen Da.

¹: Present address: Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Botânica, Avenida Antônio Carlos, 6627, Pampulha, Caixa Postal 486, 31270-970, Belo Horizonte, Minas Gerais, Brazil.

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Oxidative stress in duckweed (Lemna minor L.) induced by glyphosate: Is the mitochondrial electron transport chain a target of this herbicide?☆

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials and methods

Physiological responses to glyphosate

Discussion

Conclusion

Conflict of interest

Acknowledgements

Methods Enzymol.

Plant Physiol. Biochem.

Pestic. Biochem. Physiol.

Plant Physiol. Biochem.

Environ. Exp. Bot.

Pestic. Biochem. Physiol.

J. Hazard. Mater

Pestic. Biochem. Physiol.

Free Radic. Biol. Med.

J. Biol. Chem.

Chemosphere

Mutat. Res.

Pestic. Biochem. Physiol.

Free Radic. Biol. Med.

Biochem. Biophys. Res. Commun.

Trends Plant Sci.

Plant Sci.

Crop Prot.

Pestic. Biochem. Physiol.

Quantifying colocalization by correlation: the pearson correlation coefficient is superior to the Mander’s overlap coefficient

Cytom. Part A