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Mitochondrial complex II-derived superoxide is the primary source of mercury toxicity in barley root tip
Accepted Manuscript Title: Mitochondrial complex II-derived superoxide is the primary source of mercury toxicity in barley root tip Author: Ladislav Tam´as Veronika Zelinov´a PII: DOI: Reference:
S0176-1617(16)30266-8 http://dx.doi.org/doi:10.1016/j.jplph.2016.10.014 JPLPH 52482
To appear in: Received date: Revised date: Accepted date:
9-8-2016 18-10-2016 19-10-2016
Please cite this article as: Tam´as Ladislav, Zelinov´a Veronika.Mitochondrial complex II-derived superoxide is the primary source of mercury toxicity in barley root tip.Journal of Plant Physiology http://dx.doi.org/10.1016/j.jplph.2016.10.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mitochondrial complex II-derived superoxide is the primary source of mercury toxicity in barley root tip
Ladislav Tamás* and Veronika Zelinová
Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84523 Bratislava, Slovak Republic.
*Corresponding author E-mail address: [email protected]
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Abstract Enhanced superoxide generation and significant inhibition of succinate dehydrogenase (SDH) activity followed by a strong reduction of root growth were detected in barley seedlings exposed to a 5 µM Hg concentration for 30 min, which increased further in an Hg dosedependent manner. While at a 25 µM Hg concentration no cell death was detectable, a 50 µM Hg treatment triggered cell death in the root meristematic zone, which was markedly intensified after the treatment of roots with 100 µM Hg and was detectable in the whole root tips. Generation of superoxide and H2O2 was a very rapid response of root tips occurring even after 5 min of exposure to Hg. Application of an NADPH oxidase inhibitor or the inhibition of electron flow in mitochondria by the inhibition of complex I did not influence the Hg-induced H2O2 production. Treatment of roots with thenoyltrifluoroacetone, a non-competitive inhibitor of SDH, markedly reduced root growth and induced both superoxide and H2O2 production in a dose dependent manner. Similar to results obtained in intact roots, Hg strongly inhibited SDH activity in the crude mitochondrial fraction and caused a considerable increase of superoxide production, which was markedly reduced by the competitive inhibitors of SDH. These results indicate that the mitochondrial complex II-derived superoxide is the primary source of Hg toxicity in the barley root tip.
Abbreviations: DPI, diphenyleneiodonium; mROS, mitochondrial ROS; ROS, reactive oxygen species; SDH, succinate dehydrogenase; TIFA, thenoyltrifluoroacetone
Keywords: barley root; cell death; mercury; reactive oxygen species; succinate dehydrogenase; superoxide
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Introduction We must currently face the reality that the pollution of the environment as a negative effect of industrialization is a global problem. Heavy metals are the extremely dangerous substances between the pollutants due to their stability and toxicity even at very low concentrations. Mercury (Hg) is one of the most toxic environmental contaminants due to its low melting and boiling points, conversion between chemical forms and participation in several biological cycles. Moreover, the global atmospheric Hg deposition rate is currently approximately three times higher than during pre-industrial times (Hylander and Meili, 2003). However, a major sink for heavy metals, including Hg, is the pedosphere. Therefore, their concentrations in the soils may exceed several times the non-toxic level to most plant species (Han et al., 2002). In addition, many fertilizers, pesticides and various soil amendments contain a considerable amount of Hg, and thus directly contaminate arable soils. Hg is easily taken up by plant cells through the various transport systems for essential micronutrients (Esteban et al., 2008) and is mainly accumulated in the roots, interfering with several physiological and developmental processes. At the same time, a growing body of evidence indicates that the Hg-induced oxidative stress in cells plays a pivotal role in the development of Hg toxicity symptoms, including root growth inhibition (Chen and Yang, 2012). Numerous publications have described the elevated levels of lipid peroxides and oxidized proteins in both roots and shoots of various crop plants, leading to reduced biomass production (Cho and Park, 2000; Cargnelutti et al., 2006; Rellán-Álvarez et al., 2006; Zhou et al., 2007). The rapid activation of protective antioxidative defense mechanisms is a prerequisite for the successful detoxification of reactive oxygen species (ROS) in a toxic overdose. Therefore, the increased level of both enzymatic and non-enzymatic antioxidant systems are a common root response to elevated Hg level (Zhou et al., 2008; Sobrino-Plata et al., 2009; Lomonte et al., 2010). Because moderate ROS levels act as signals in the regulation of stress responses, Hg upregulated several genes encoding proteins involved in the defense responses of plants to both biotic and abiotic stresses (Didierjean et al., 1996; Sävenstrand and Strid, 2004). Due to the very high affinity of Hg to the sulfhydryl groups, the phytochelatins play a key role in the detoxification of Hg in the cytosol, forming phytochelatin-Hg multicomplexes (Iglesia-Turiño et al., 2006). Analysis of the soluble fraction of barley, maize and alfalfa roots exposed to Hg revealed that Hg was only associated with the phytochelatins (Carrasco-Gil et al., 2011). A transgenic Arabidopsis line with increased phytochelatin content showed enhanced tolerance to Hg in comparison to the wild type seedlings (Li et al., 2006). On the other hand, just due to this high affinity of Hg to the sulfhydryl groups, the various biomolecules with these moieties are the main targets of Hg toxicity (Patra et al., 2004). In photosynthetic cells, the chloroplast and peroxisome are the main sources of ROS production, while in roots or in the dark the mitochondria are the main sources of ROS production (Rhoads et al., 2006). Both chloroplastic and mitochondrial electron transport chains contain numerous proteins with high amounts of sulfhydryl groups, which, just after their modification or inhibition may generate a large amount of ROS. It has been well documented that Hg inhibits photosynthetic electron transport at multiple sites in a concentration dependent manner (Murthy and Mohanty, 1993; Bernier and Carpentier, 1995). Recently, numerous publications have reported the mitochondrial origin of increased ROS production in several plant species exposed to toxic concentrations of different metals (Keunen et al., 2011). However, the precise mechanism of Hg-induced ROS generation in roots remains unclear. In human monocytic cells, Hg markedly inhibited mitochondrial activity via oxidative stress (Messer et al. 2005). Furthermore, succinate dehydrogenase (SDH) as a possible target site of Hg toxicity has been suggested in fish liver and rat brain (Rao et al., 2010; Mieiro et al., 2015). SDH has been described as a source of ROS and as a regulator of development and
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stress, including defense responses in plants (Huang and Millar, 2013; Jardim-Messeder et al., 2015). An Arabidopsis mutant in the complex II subunit showed considerably reduced SDH activity and ROS production and exhibited increased susceptibility to pathogens, suggesting that ROS generated by mitochondrial SDH are involved in the activation of defense response to biotic stresses (Gleason et al., 2011). The aim of the present study was to analyze the possible involvement of mitochondrial SDH in ROS generation in the responses of the barley root tip to Hg stress. Materials and methods Plant material and growth conditions Barley seeds (Hordeum vulgare L.) cv. Slaven (Plant Breeding Station, Hordeum Ltd, Sládkovičovo-Nový Dvor, Slovakia) were imbibed in distilled water for 15 min followed by germination between two sheets of filter paper (density 110 g/m2) moistened with distilled water in Petri dishes at 25°C in darkness. The uniformly germinating seeds, 24 h after the onset of seed imbibition, were arranged into rows between two sheets of filter paper moistened with distilled water in rectangle trays. Trays were placed into a nearly vertical position to enable downward radical growth. Continuous moisture of filter papers was supplied from the reservoir with distilled water through the filter paper wick. Seedlings, with approximately 4 cm long primary roots, 60 h after the onset of seed imbibition, were used for treatments. Short-term treatments During the short-term treatments, roots were immersed into appropriate test solutions, such as distilled water (dw; control; pH 5.5), 1–100 µM HgCl2, pH 5.2 - 5.5 depending on Hg concentration, 100–500 µM TIFA, pH 5 - 5.5 depending on TIFA concentration (500 mM stock in DMSO, the final concentration of DMSO was 0.1%), 1 µM diphenyleneiodonium – DPI, pH 5.4 (10 mM stock in DMSO, the final concentration of DMSO was 0.1%), or 5 µM rotenone, pH 5.5 (4 mM stock in methanol, the final concentration of methanol was 0.25%), for 5 - 30 min. Following the rinse in dw for 5 min, the seedlings (3 mm long root tips) were immediately used for analysis or were incubated between two sheets of filter paper moistened with distilled water as described above for 6 or 9 h and used for root length measurement and cell death analysis. Root length measurement For the determination of root length changes, the position of root tips following the treatments was marked on the filter paper. After 6 h, the roots were excised at the position of marks, and the change in length was measured after recording with a stereomicroscope using an image analyzer. For localization of root swelling, the roots were stained with 0.05% Toluidine blue for 10 min and after washing with distilled water were photographed with stereomicroscope. Measurement of hydrogen peroxide production by root tips H2O2 production was monitored fluorimetrically using the Amplex Ultra Red Hydrogen Peroxide Assay Kit (Molecular Probes) according to manufacturer’s recommendations, with minor modifications. Segments (3 mm) from barley root tips (20 segments per reaction) were washed in 400 µl of 20 mM sodium phosphate buffer, pH 6.0, for 5 min. After this washing, the root tips were incubated in 400 µl of 20 mM sodium phosphate buffer, pH 6.0, containing 50 µM Amplex UltraRed reagent (from 10 mM DMSO stock solution) and 0.1 U of horseradish peroxidase for 15 min at 30°C. The fluorescence signal was recorded (300 µl of reaction mixture without root segments) with the microplate reader using excitation at 485 (filter 485/20) nm and fluorescence detection at 590 (filter 590/20) nm. H2O2 production was
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expressed as an increase in relative fluorescence unit (RFU) during 15 min incubation of root segments after the subtraction of background (incubation medium) fluorescence. Localization of superoxide production Intact roots were immersed in a solution of 1 mM NBT (nitro-blue tetrazolium chloride) and 10 mM sodium azide in 20 mM sodium phosphate buffer, pH 6.0, for 20 min in the dark at room temperature. After a brief period of washing with distilled water, the roots were photographed immediately with a stereomicroscope. Localization of cell death Intact roots were immersed into a solution of propidium iodide (PI; 3 µg/mL) for 60 min at room temperature. After washing with distilled water, fluorescence of whole roots was observed using fluorescence stereomicroscope (excitation 545 ± 25 nm; emission 606 ± 70 nm). Isolation of crude mitochondrial fraction Root tips (approximately 150, 3 mm in length) were homogenized in a pre-cooled mortar with 20 mM potassium phosphate buffer (pH 7.5) containing 1 mM EDTA and 300 mM mannitol. The homogenate was centrifuged first at 1000 x g for 5 min and then the supernatant was centrifuged at 12000 x g for 10 min. The resulting pellet was resuspended in the homogenization buffer and centrifuged again at 1000 x g and 12000 x g for 5 and 10 min respectively. The pellet was resuspended in 20 mM potassium phosphate buffer (pH 7.4) and was immediately used for the measurement of SDH activity and for the H2O2 production. The amount of proteins was quantified with bovine serum albumin as the calibration standard by the method of Bradford (1976). SDH activity assay SDH (EC 1.3.5.1) activity was measured by a spectrophotometric method according to Jardim-Messeder et al. (2015) with some modifications. The reaction mixture (250 μL) contained 5 mM sodium azide, 0.1 mM 2,6-dichlorophenolindophenol, 10 mM sodium succinate, 0.05 % Triton X-100 in 20 mM potassium phosphate buffer (pH 7.4), and 30 μg of proteins from crude mitochondrial fraction. Oxalacetate and Hg were added to the reaction mixture at concentrations of 10 or 100 µM and 5, 10 or 50 µM respectively. The rate of 2,6dichlorophenolindophenol reduction was measured at 600 nm over a time period of 30 min at 30°C. Specific SDH activities were expressed as Δ A600 mg-1 min-1. Hydrogen peroxide measurement in crude mitochondrial fraction Mitochondrial H2O2 production was monitored fluorimetrically using the Amplex Ultra Red Hydrogen Peroxide Assay Kit (Molecular Probes). The reaction mixture (250 μL) contained 50 µM Amplex UltraRed reagent (from 10 mM DMSO stock solution), 0.1 U of horseradish peroxidase, 5 U of superoxide dismutase and 30 μg of proteins from crude mitochondrial fraction in 20 mM potassium phosphate buffer, (pH 7.4), and different concentrations of Hg and 5 mM malonate or 5 mM malate or 1 mM oxalacetate or 1 mM succinate. The fluorescence signal was recorded with the microplate reader using excitation at 485 (filter 485/20) nm and fluorescence detection at 590 (filter 590/20) nm. H2O2 production was expressed as an increase in relative fluorescence unit (RFU) after the incubation for 30 min at 30°C and after the subtraction of background (incubation medium) fluorescence.
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Statistical analyses The experiments were carried out in five independent series with three replicates (20 root tips for root length and H2O2 measurement, 10 root tips for superoxide and cell death localization and 150 root tips for SDH activity analysis and for the isolation of crude mitochondrial fraction per replicate). The data were analyzed by one-way analysis of variance (ANOVA test), and the means were separated using Tukey’s test. Results Significant root growth inhibition was detected in the seedlings exposed to a low 5 µM concentration of Hg for 30 min, measured 6 h after this short-term treatment (Fig. 1A). The degree of root growth inhibition increased in an Hg concentration-dependent manner. No root growth was observed during the first 6 h after the short-term treatment of seedlings with 100 M Hg. Similar to the Hg-induced root growth inhibition, the inhibition of SDH activity was also observed in roots treated with a 5 µM concentration of Hg. In addition, this inhibition of SDH activity also increased in an Hg dose-dependent manner (Fig. 1B). However, the analysis of H2O2 production in the root tip segments (Fig. 2A) showed that a significant increase of H2O2 production occurred only at a 10 µM Hg concentration, which further increased with increasing Hg concentration. After the treatment of roots with a very high 100 µM Hg concentration, the production of H2O2 in the root tips was sevenfold greater than in the controls. In turn, similarly to the Hg-induced root growth and SDH activity inhibition, an enhanced superoxide generation was already detected after the treatment of roots with 5 µM Hg, and its production further increased with increasing Hg concentration (Fig. 2B). Apart from the Hg-induced root growth inhibition, a visible root swelling behind the root apex (representing transition and elongation zone during the treatment) was detected within 6 h after the short-term treatment of seedlings with 25 µM Hg (Fig. 3A). This radial expansion of root cells was also observed in the root tips exposed to 50 µM Hg, but it was much smaller than in roots treated with the 25 µM Hg concentration. By contrast, swollen root was not detected in the seedlings exposed to the 100 µM Hg concentration. While at the 25 µM Hg concentration root cell death was not detected, 50 µM Hg treatment caused cell death in the root meristematic zone, which was markedly intensified after the treatment of roots with 100 µM Hg and was detectable in the whole root tips (Fig. 3B). This intensive cell death in the 100 µM Hg-treated roots caused root growth arrest and even death (Fig. 3C). The root growth of the seedlings exposed to 50 µM Hg concentration was renewed between 6–9 h after the short-term treatment. Generation of H2O2 was a very rapid Hg-induced response of barley root tips. Even a 5 min exposure of roots to 25 µM Hg markedly increased its production measured in the root segments immediately after the treatment (Fig. 4A). This H2O2 production was higher after the exposure of roots to 50 µM Hg and in both cases further increased up to 15 min. These results were also confirmed by the localization of superoxide production in the root tips (Fig. 4B). In comparison to control roots, a considerable increase in NBT stain was detected even 5 min after the exposure of roots to the 25 µM Hg concentration. Similar to H2O2 production, the superoxide production intensified with increasing Hg concentration and with a longer exposure time. Application of NADPH oxidase inhibitor DPI or the inhibition of electron flow in mitochondria, due to the inhibition of complex I with rotenone, did not influence the Hg-induced H2O2 production in spite of the marked growth reduction of control roots after these treatments (Fig. 5). In addition, DPI had an additive effect on the Hg-induced root growth inhibition. Treatment of roots with TIFA, a non-competitive inhibitor of SDH, markedly reduced root growth and induced both H2O2 and superoxide production in the root tips in a dose dependent manner (Fig. 6).
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Similar to results obtained in the intact roots, Hg strongly inhibited the activity of SDH in the crude mitochondrial fraction. Statistically significant SDH inhibition began at 5 µM Hg concentration and intensified with increasing Hg concentration (Fig. 7A). Furthermore, Hg caused a considerable increase of superoxide production in this fraction even at the 2.5 µM concentration (Fig. 7B). This Hg-induced superoxide production in the crude mitochondrial fraction was not influenced by malate or succinate but was markedly reduced by malonate or oxalacetate, which are the competitive inhibitors of SDH (Fig. 8). Discussion The uptake of Hg by roots is a very rapid process showing a linear increase for the first 30 min (Esteban et al., 2008). In this study, we demonstrated that even a brief exposure of barley roots to a low Hg concentration caused a significant inhibition of SDH activity and an increase in superoxide production resulting in the inhibition of root growth. In addition, both superoxide production and SDH activity inhibition increased with increasing Hg concentration, leading to an extensive cell death in the root tips. Superoxide rapidly dismutates to H2O2, which is strongly accelerated by the antioxidant enzyme superoxide dismutase. In accordance with this fact, a marked Hg-induced H2O2 generation was detectable in the root tip segments. However, this Hg-induced H2O2 production was observed only at a higher Hg concentration in comparison to the Hg-induced superoxide generation. This difference is probably due the higher sensitivity of the NBT stain to superoxide in vivo in comparison to the sensitivity of Amplex Red reagent to H2O2 released from the root segments in vitro. In addition, a very high activity of cell wall peroxidases in roots may also decrease the amount of released H2O2 from roots into the reaction solution. Similar to our results, Hg treatment evoked, within a few minutes of exposure, a very rapid and robust generation of ROS in alfalfa seedlings (Ortega-Villasante et al., 2007). In addition, the peroxidation of membranes, as an indicator of oxidative stress, and loss of cell integrity were detected in the meristematic and elongation zone of the root tips of Hg-treated alfalfa seedlings (Zhou et al., 2007). The production of ROS and the accumulation of peroxidative products, as toxicity symptoms of Hg exposure were described also in green algae, where the increasing concentration of Hg was negatively correlated with the rate of cell growth (Elbaz et al., 2010). One of the most frequently characterized superoxide-generating enzymes in plants is the plasma membrane-localized NADPH oxidase, which is involved in various developmental processes and in responses to both biotic and abiotic stresses (Maksymiec, 2007; Kaur et al., 2014). In our experiments, the Hg-induced superoxide generation was not influenced by the co-treatment of roots with DPI, suggesting that it is not produced via the plasma membrane NADPH oxidase activity. Similarly, the Hg-induced ROS generation in alfalfa root segments was not affected by DPI pre-treatment, suggesting that an NADPH oxidase-independent mechanism is responsible for this Hg-induced ROS production (Ortega-Villasante et al., 2007). It is well known that, in illuminated photosynthetic tissues, the chloroplasts are the main sources of ROS production. It has been reported that Hg interacts with numerous proteins in the chloroplast of green algae (Xyländer et al., 1996), thus inhibiting the photosynthetic electron transport chain, leading to the generation of ROS and oxidative damages (Heidenreich et al., 2001). These results indicated that, in photosynthetic cells, the chloroplasts are the main target sites of Hg toxicity. In turn, in roots, the main sources of uncontrolled ROS release are mitochondria, which were also indicated as a common target site of several toxic metals (Keunen et al., 2011). The main sites of superoxide production in mitochondria are complex I and III in the electron transport chain (Noctor et al., 2007). Inhibition of complex I by rotenone produces a considerable amount of superoxide, but it is
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released into the matrix of mitochondria; therefore, it is not detectable in intact mitochondria and tissues due to the strong antioxidative systems of the mitochondrial matrix (Chen et al., 2003). The inhibition of complex III by antimycin A also caused a rapid and strong generation of superoxide into the intermembrane space (Muller et al., 2004). This superoxide can pass through the voltage-dependent anion channels of outer mitochondrial membrane into the cytosol (Han et al. 2003); therefore, it may be detected also in plant tissues. It has been demonstrated that superoxide generated at complex III can be inhibited by the inhibition of complex I due to the limitation of electron flow from complex I to complex III (Chen et al., 2003). However, the Hg-induced superoxide generation was not affected by rotenone cotreatment, suggesting that neither complex I nor complex III are the targets of Hg toxicity in barely roots. Recently, it was demonstrated that mitochondrial complex II can generate superoxide at high rates in both the forward and reverse reactions (Quinlan et al., 2012). We showed that TIFA, a non-competitive inhibitor of SDH, induced a marked superoxide generation in a dose-dependent manner in the barley root tips, which was accompanied by a strong reduction in root growth. Similar to TIFA, Hg treatment inhibited SDH activity in the root tips which was accompanied by a robust superoxide generation resulting in root growth inhibition. Furthermore, we showed that the inhibition of SDH activity and the generation of superoxide were also induced in vitro after the addition of Hg into the crude mitochondrial fraction. It has been reported that the inhibition of SDH activity by competitive inhibitors decreased the noncompetitive inhibitor-induced ROS production (Jardim-Messeder et al., 2015). According to this result, in our experiments, both malonate and oxalacetate, the competitive inhibitors of SDH, significantly reduced the Hg-induced superoxide generation in the crude mitochondrial fraction. In some previous studies, it has been shown that mitochondrial SDH is an important target of Hg toxicity. In marine fish, Hg caused an impairment of mitochondrial energy metabolism via the inhibition of several mitochondrial enzymes, including SDH (Mieiro et al., 2015). Hg-induced depletion of SDH activity was also reported in the rat brain (Rao et al., 2010). In pea roots, Hg treatment as early as after 1.5 h markedly increased the expression of gene encoding protein with a high amino acid sequence similarity to the mitochondrial rotenone-insensitive NADH dehydrogenase (Sävenstrand and Strid, 2004). It has been suggested that in Fe-deficient cucumber roots, a strong increase of these alternative dehydrogenases may bypass a marked loss of complex I and II activities (Vigani et al., 2009). Sweetlove et al. (2002) reported that oxidative stress in mitochondria induced the degradation of damaged proteins. Although the Hg affects directly mainly complex II, through the rapid generation of ROS it may damage numerous mitochondrial and also cytosolic components. On the other hand, at a lower Hg concentration the amount of SDH-generated ROS may function as a signal molecule activating different stress responses including a rapid reorientation of root growth. In conclusion, Hg along with the inhibition of SDH activity, similar to the noncompetitive SDH inhibitor TIFA, caused a rapid and marked superoxide generation in barley root tip. Hg evoked this inhibition of SDH activity and enhanced superoxide generation also in vitro, in the crude mitochondrial fraction. In vitro, the Hg-induced superoxide production was inhibited by the competitive inhibitors of SDH. These results indicated that the mitochondrial complex II-derived superoxide is the primary source of mercury toxicity in the barley root tip.
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Acknowledgements This work was supported by the Grant Agency VEGA, project No. 2/0039/16. The authors also thank the anonymous reviewers for their helpful criticisms, which improved the manuscript.
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Figure legends Fig. 1. Root length increments 6 h (A) and SDH activity (B) immediately after the short-term treatment of roots with 0–100 µM Hg for 30 min. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). Fig. 2. Hydrogen peroxide production by root tips (A), and localization of superoxide production (B) immediately after the short-term treatment of roots with 0–100 µM Hg for 30 min. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). The black arrows indicate the area of Hg-induced superoxide production in comparison to the control roots. Fig. 3. Root morphology 6 h (A), localization of cell death 6 h (B) and root morphology 9 h (C) after the short-term treatment of roots with 0–100 µM Hg for 30 min. The black triangles show the starting position of new root growth after the short-term treatments. S – swollen root part. Fig. 4. Hydrogen peroxide production by root tips (A) and localization of superoxide production (B) immediately after the short-term treatment of roots with 0, 25 or 50 µM Hg for 5, 10 or 15 min. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). The black arrows indicate the area of Hg-induced superoxide production in comparison to the control roots. Fig. 5. Root length increments 6 h (A) and hydrogen peroxide production by root tips (B) immediately after the short-term treatment of roots with 0, 25 or 50 µM Hg and co-treatment with 1 µM DPI or 5 µM rotenone for 30 min. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). Fig. 6. Root length increments 6 h (A) and hydrogen peroxide production by root tips (B) and localization of superoxide production (C) immediately after the short-term treatment of roots with 0–500 µM TIFA for 30 min. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). The black arrows indicate the area of Hg-induced superoxide production in comparison to the control roots. Fig. 7. SDH activity (A) and hydrogen peroxide production (B) in crude mitochondrial fraction in the presence of 0–50 µM Hg. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). Fig. 8. Hydrogen peroxide production in crude mitochondrial fraction in the presence of 0 or 10 µM Hg and 1 mM succinate or 5 mM malonate or 5 mM malate or 1 mM oxalacetate. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05).
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S0176-1617(16)30266-8 http://dx.doi.org/doi:10.1016/j.jplph.2016.10.014 JPLPH 52482
To appear in: Received date: Revised date: Accepted date:
9-8-2016 18-10-2016 19-10-2016
Please cite this article as: Tam´as Ladislav, Zelinov´a Veronika.Mitochondrial complex II-derived superoxide is the primary source of mercury toxicity in barley root tip.Journal of Plant Physiology http://dx.doi.org/10.1016/j.jplph.2016.10.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mitochondrial complex II-derived superoxide is the primary source of mercury toxicity in barley root tip
Ladislav Tamás* and Veronika Zelinová
Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84523 Bratislava, Slovak Republic.
*Corresponding author E-mail address: [email protected]
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Abstract Enhanced superoxide generation and significant inhibition of succinate dehydrogenase (SDH) activity followed by a strong reduction of root growth were detected in barley seedlings exposed to a 5 µM Hg concentration for 30 min, which increased further in an Hg dosedependent manner. While at a 25 µM Hg concentration no cell death was detectable, a 50 µM Hg treatment triggered cell death in the root meristematic zone, which was markedly intensified after the treatment of roots with 100 µM Hg and was detectable in the whole root tips. Generation of superoxide and H2O2 was a very rapid response of root tips occurring even after 5 min of exposure to Hg. Application of an NADPH oxidase inhibitor or the inhibition of electron flow in mitochondria by the inhibition of complex I did not influence the Hg-induced H2O2 production. Treatment of roots with thenoyltrifluoroacetone, a non-competitive inhibitor of SDH, markedly reduced root growth and induced both superoxide and H2O2 production in a dose dependent manner. Similar to results obtained in intact roots, Hg strongly inhibited SDH activity in the crude mitochondrial fraction and caused a considerable increase of superoxide production, which was markedly reduced by the competitive inhibitors of SDH. These results indicate that the mitochondrial complex II-derived superoxide is the primary source of Hg toxicity in the barley root tip.
Abbreviations: DPI, diphenyleneiodonium; mROS, mitochondrial ROS; ROS, reactive oxygen species; SDH, succinate dehydrogenase; TIFA, thenoyltrifluoroacetone
Keywords: barley root; cell death; mercury; reactive oxygen species; succinate dehydrogenase; superoxide
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Introduction We must currently face the reality that the pollution of the environment as a negative effect of industrialization is a global problem. Heavy metals are the extremely dangerous substances between the pollutants due to their stability and toxicity even at very low concentrations. Mercury (Hg) is one of the most toxic environmental contaminants due to its low melting and boiling points, conversion between chemical forms and participation in several biological cycles. Moreover, the global atmospheric Hg deposition rate is currently approximately three times higher than during pre-industrial times (Hylander and Meili, 2003). However, a major sink for heavy metals, including Hg, is the pedosphere. Therefore, their concentrations in the soils may exceed several times the non-toxic level to most plant species (Han et al., 2002). In addition, many fertilizers, pesticides and various soil amendments contain a considerable amount of Hg, and thus directly contaminate arable soils. Hg is easily taken up by plant cells through the various transport systems for essential micronutrients (Esteban et al., 2008) and is mainly accumulated in the roots, interfering with several physiological and developmental processes. At the same time, a growing body of evidence indicates that the Hg-induced oxidative stress in cells plays a pivotal role in the development of Hg toxicity symptoms, including root growth inhibition (Chen and Yang, 2012). Numerous publications have described the elevated levels of lipid peroxides and oxidized proteins in both roots and shoots of various crop plants, leading to reduced biomass production (Cho and Park, 2000; Cargnelutti et al., 2006; Rellán-Álvarez et al., 2006; Zhou et al., 2007). The rapid activation of protective antioxidative defense mechanisms is a prerequisite for the successful detoxification of reactive oxygen species (ROS) in a toxic overdose. Therefore, the increased level of both enzymatic and non-enzymatic antioxidant systems are a common root response to elevated Hg level (Zhou et al., 2008; Sobrino-Plata et al., 2009; Lomonte et al., 2010). Because moderate ROS levels act as signals in the regulation of stress responses, Hg upregulated several genes encoding proteins involved in the defense responses of plants to both biotic and abiotic stresses (Didierjean et al., 1996; Sävenstrand and Strid, 2004). Due to the very high affinity of Hg to the sulfhydryl groups, the phytochelatins play a key role in the detoxification of Hg in the cytosol, forming phytochelatin-Hg multicomplexes (Iglesia-Turiño et al., 2006). Analysis of the soluble fraction of barley, maize and alfalfa roots exposed to Hg revealed that Hg was only associated with the phytochelatins (Carrasco-Gil et al., 2011). A transgenic Arabidopsis line with increased phytochelatin content showed enhanced tolerance to Hg in comparison to the wild type seedlings (Li et al., 2006). On the other hand, just due to this high affinity of Hg to the sulfhydryl groups, the various biomolecules with these moieties are the main targets of Hg toxicity (Patra et al., 2004). In photosynthetic cells, the chloroplast and peroxisome are the main sources of ROS production, while in roots or in the dark the mitochondria are the main sources of ROS production (Rhoads et al., 2006). Both chloroplastic and mitochondrial electron transport chains contain numerous proteins with high amounts of sulfhydryl groups, which, just after their modification or inhibition may generate a large amount of ROS. It has been well documented that Hg inhibits photosynthetic electron transport at multiple sites in a concentration dependent manner (Murthy and Mohanty, 1993; Bernier and Carpentier, 1995). Recently, numerous publications have reported the mitochondrial origin of increased ROS production in several plant species exposed to toxic concentrations of different metals (Keunen et al., 2011). However, the precise mechanism of Hg-induced ROS generation in roots remains unclear. In human monocytic cells, Hg markedly inhibited mitochondrial activity via oxidative stress (Messer et al. 2005). Furthermore, succinate dehydrogenase (SDH) as a possible target site of Hg toxicity has been suggested in fish liver and rat brain (Rao et al., 2010; Mieiro et al., 2015). SDH has been described as a source of ROS and as a regulator of development and
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stress, including defense responses in plants (Huang and Millar, 2013; Jardim-Messeder et al., 2015). An Arabidopsis mutant in the complex II subunit showed considerably reduced SDH activity and ROS production and exhibited increased susceptibility to pathogens, suggesting that ROS generated by mitochondrial SDH are involved in the activation of defense response to biotic stresses (Gleason et al., 2011). The aim of the present study was to analyze the possible involvement of mitochondrial SDH in ROS generation in the responses of the barley root tip to Hg stress. Materials and methods Plant material and growth conditions Barley seeds (Hordeum vulgare L.) cv. Slaven (Plant Breeding Station, Hordeum Ltd, Sládkovičovo-Nový Dvor, Slovakia) were imbibed in distilled water for 15 min followed by germination between two sheets of filter paper (density 110 g/m2) moistened with distilled water in Petri dishes at 25°C in darkness. The uniformly germinating seeds, 24 h after the onset of seed imbibition, were arranged into rows between two sheets of filter paper moistened with distilled water in rectangle trays. Trays were placed into a nearly vertical position to enable downward radical growth. Continuous moisture of filter papers was supplied from the reservoir with distilled water through the filter paper wick. Seedlings, with approximately 4 cm long primary roots, 60 h after the onset of seed imbibition, were used for treatments. Short-term treatments During the short-term treatments, roots were immersed into appropriate test solutions, such as distilled water (dw; control; pH 5.5), 1–100 µM HgCl2, pH 5.2 - 5.5 depending on Hg concentration, 100–500 µM TIFA, pH 5 - 5.5 depending on TIFA concentration (500 mM stock in DMSO, the final concentration of DMSO was 0.1%), 1 µM diphenyleneiodonium – DPI, pH 5.4 (10 mM stock in DMSO, the final concentration of DMSO was 0.1%), or 5 µM rotenone, pH 5.5 (4 mM stock in methanol, the final concentration of methanol was 0.25%), for 5 - 30 min. Following the rinse in dw for 5 min, the seedlings (3 mm long root tips) were immediately used for analysis or were incubated between two sheets of filter paper moistened with distilled water as described above for 6 or 9 h and used for root length measurement and cell death analysis. Root length measurement For the determination of root length changes, the position of root tips following the treatments was marked on the filter paper. After 6 h, the roots were excised at the position of marks, and the change in length was measured after recording with a stereomicroscope using an image analyzer. For localization of root swelling, the roots were stained with 0.05% Toluidine blue for 10 min and after washing with distilled water were photographed with stereomicroscope. Measurement of hydrogen peroxide production by root tips H2O2 production was monitored fluorimetrically using the Amplex Ultra Red Hydrogen Peroxide Assay Kit (Molecular Probes) according to manufacturer’s recommendations, with minor modifications. Segments (3 mm) from barley root tips (20 segments per reaction) were washed in 400 µl of 20 mM sodium phosphate buffer, pH 6.0, for 5 min. After this washing, the root tips were incubated in 400 µl of 20 mM sodium phosphate buffer, pH 6.0, containing 50 µM Amplex UltraRed reagent (from 10 mM DMSO stock solution) and 0.1 U of horseradish peroxidase for 15 min at 30°C. The fluorescence signal was recorded (300 µl of reaction mixture without root segments) with the microplate reader using excitation at 485 (filter 485/20) nm and fluorescence detection at 590 (filter 590/20) nm. H2O2 production was
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expressed as an increase in relative fluorescence unit (RFU) during 15 min incubation of root segments after the subtraction of background (incubation medium) fluorescence. Localization of superoxide production Intact roots were immersed in a solution of 1 mM NBT (nitro-blue tetrazolium chloride) and 10 mM sodium azide in 20 mM sodium phosphate buffer, pH 6.0, for 20 min in the dark at room temperature. After a brief period of washing with distilled water, the roots were photographed immediately with a stereomicroscope. Localization of cell death Intact roots were immersed into a solution of propidium iodide (PI; 3 µg/mL) for 60 min at room temperature. After washing with distilled water, fluorescence of whole roots was observed using fluorescence stereomicroscope (excitation 545 ± 25 nm; emission 606 ± 70 nm). Isolation of crude mitochondrial fraction Root tips (approximately 150, 3 mm in length) were homogenized in a pre-cooled mortar with 20 mM potassium phosphate buffer (pH 7.5) containing 1 mM EDTA and 300 mM mannitol. The homogenate was centrifuged first at 1000 x g for 5 min and then the supernatant was centrifuged at 12000 x g for 10 min. The resulting pellet was resuspended in the homogenization buffer and centrifuged again at 1000 x g and 12000 x g for 5 and 10 min respectively. The pellet was resuspended in 20 mM potassium phosphate buffer (pH 7.4) and was immediately used for the measurement of SDH activity and for the H2O2 production. The amount of proteins was quantified with bovine serum albumin as the calibration standard by the method of Bradford (1976). SDH activity assay SDH (EC 1.3.5.1) activity was measured by a spectrophotometric method according to Jardim-Messeder et al. (2015) with some modifications. The reaction mixture (250 μL) contained 5 mM sodium azide, 0.1 mM 2,6-dichlorophenolindophenol, 10 mM sodium succinate, 0.05 % Triton X-100 in 20 mM potassium phosphate buffer (pH 7.4), and 30 μg of proteins from crude mitochondrial fraction. Oxalacetate and Hg were added to the reaction mixture at concentrations of 10 or 100 µM and 5, 10 or 50 µM respectively. The rate of 2,6dichlorophenolindophenol reduction was measured at 600 nm over a time period of 30 min at 30°C. Specific SDH activities were expressed as Δ A600 mg-1 min-1. Hydrogen peroxide measurement in crude mitochondrial fraction Mitochondrial H2O2 production was monitored fluorimetrically using the Amplex Ultra Red Hydrogen Peroxide Assay Kit (Molecular Probes). The reaction mixture (250 μL) contained 50 µM Amplex UltraRed reagent (from 10 mM DMSO stock solution), 0.1 U of horseradish peroxidase, 5 U of superoxide dismutase and 30 μg of proteins from crude mitochondrial fraction in 20 mM potassium phosphate buffer, (pH 7.4), and different concentrations of Hg and 5 mM malonate or 5 mM malate or 1 mM oxalacetate or 1 mM succinate. The fluorescence signal was recorded with the microplate reader using excitation at 485 (filter 485/20) nm and fluorescence detection at 590 (filter 590/20) nm. H2O2 production was expressed as an increase in relative fluorescence unit (RFU) after the incubation for 30 min at 30°C and after the subtraction of background (incubation medium) fluorescence.
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Statistical analyses The experiments were carried out in five independent series with three replicates (20 root tips for root length and H2O2 measurement, 10 root tips for superoxide and cell death localization and 150 root tips for SDH activity analysis and for the isolation of crude mitochondrial fraction per replicate). The data were analyzed by one-way analysis of variance (ANOVA test), and the means were separated using Tukey’s test. Results Significant root growth inhibition was detected in the seedlings exposed to a low 5 µM concentration of Hg for 30 min, measured 6 h after this short-term treatment (Fig. 1A). The degree of root growth inhibition increased in an Hg concentration-dependent manner. No root growth was observed during the first 6 h after the short-term treatment of seedlings with 100 M Hg. Similar to the Hg-induced root growth inhibition, the inhibition of SDH activity was also observed in roots treated with a 5 µM concentration of Hg. In addition, this inhibition of SDH activity also increased in an Hg dose-dependent manner (Fig. 1B). However, the analysis of H2O2 production in the root tip segments (Fig. 2A) showed that a significant increase of H2O2 production occurred only at a 10 µM Hg concentration, which further increased with increasing Hg concentration. After the treatment of roots with a very high 100 µM Hg concentration, the production of H2O2 in the root tips was sevenfold greater than in the controls. In turn, similarly to the Hg-induced root growth and SDH activity inhibition, an enhanced superoxide generation was already detected after the treatment of roots with 5 µM Hg, and its production further increased with increasing Hg concentration (Fig. 2B). Apart from the Hg-induced root growth inhibition, a visible root swelling behind the root apex (representing transition and elongation zone during the treatment) was detected within 6 h after the short-term treatment of seedlings with 25 µM Hg (Fig. 3A). This radial expansion of root cells was also observed in the root tips exposed to 50 µM Hg, but it was much smaller than in roots treated with the 25 µM Hg concentration. By contrast, swollen root was not detected in the seedlings exposed to the 100 µM Hg concentration. While at the 25 µM Hg concentration root cell death was not detected, 50 µM Hg treatment caused cell death in the root meristematic zone, which was markedly intensified after the treatment of roots with 100 µM Hg and was detectable in the whole root tips (Fig. 3B). This intensive cell death in the 100 µM Hg-treated roots caused root growth arrest and even death (Fig. 3C). The root growth of the seedlings exposed to 50 µM Hg concentration was renewed between 6–9 h after the short-term treatment. Generation of H2O2 was a very rapid Hg-induced response of barley root tips. Even a 5 min exposure of roots to 25 µM Hg markedly increased its production measured in the root segments immediately after the treatment (Fig. 4A). This H2O2 production was higher after the exposure of roots to 50 µM Hg and in both cases further increased up to 15 min. These results were also confirmed by the localization of superoxide production in the root tips (Fig. 4B). In comparison to control roots, a considerable increase in NBT stain was detected even 5 min after the exposure of roots to the 25 µM Hg concentration. Similar to H2O2 production, the superoxide production intensified with increasing Hg concentration and with a longer exposure time. Application of NADPH oxidase inhibitor DPI or the inhibition of electron flow in mitochondria, due to the inhibition of complex I with rotenone, did not influence the Hg-induced H2O2 production in spite of the marked growth reduction of control roots after these treatments (Fig. 5). In addition, DPI had an additive effect on the Hg-induced root growth inhibition. Treatment of roots with TIFA, a non-competitive inhibitor of SDH, markedly reduced root growth and induced both H2O2 and superoxide production in the root tips in a dose dependent manner (Fig. 6).
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Similar to results obtained in the intact roots, Hg strongly inhibited the activity of SDH in the crude mitochondrial fraction. Statistically significant SDH inhibition began at 5 µM Hg concentration and intensified with increasing Hg concentration (Fig. 7A). Furthermore, Hg caused a considerable increase of superoxide production in this fraction even at the 2.5 µM concentration (Fig. 7B). This Hg-induced superoxide production in the crude mitochondrial fraction was not influenced by malate or succinate but was markedly reduced by malonate or oxalacetate, which are the competitive inhibitors of SDH (Fig. 8). Discussion The uptake of Hg by roots is a very rapid process showing a linear increase for the first 30 min (Esteban et al., 2008). In this study, we demonstrated that even a brief exposure of barley roots to a low Hg concentration caused a significant inhibition of SDH activity and an increase in superoxide production resulting in the inhibition of root growth. In addition, both superoxide production and SDH activity inhibition increased with increasing Hg concentration, leading to an extensive cell death in the root tips. Superoxide rapidly dismutates to H2O2, which is strongly accelerated by the antioxidant enzyme superoxide dismutase. In accordance with this fact, a marked Hg-induced H2O2 generation was detectable in the root tip segments. However, this Hg-induced H2O2 production was observed only at a higher Hg concentration in comparison to the Hg-induced superoxide generation. This difference is probably due the higher sensitivity of the NBT stain to superoxide in vivo in comparison to the sensitivity of Amplex Red reagent to H2O2 released from the root segments in vitro. In addition, a very high activity of cell wall peroxidases in roots may also decrease the amount of released H2O2 from roots into the reaction solution. Similar to our results, Hg treatment evoked, within a few minutes of exposure, a very rapid and robust generation of ROS in alfalfa seedlings (Ortega-Villasante et al., 2007). In addition, the peroxidation of membranes, as an indicator of oxidative stress, and loss of cell integrity were detected in the meristematic and elongation zone of the root tips of Hg-treated alfalfa seedlings (Zhou et al., 2007). The production of ROS and the accumulation of peroxidative products, as toxicity symptoms of Hg exposure were described also in green algae, where the increasing concentration of Hg was negatively correlated with the rate of cell growth (Elbaz et al., 2010). One of the most frequently characterized superoxide-generating enzymes in plants is the plasma membrane-localized NADPH oxidase, which is involved in various developmental processes and in responses to both biotic and abiotic stresses (Maksymiec, 2007; Kaur et al., 2014). In our experiments, the Hg-induced superoxide generation was not influenced by the co-treatment of roots with DPI, suggesting that it is not produced via the plasma membrane NADPH oxidase activity. Similarly, the Hg-induced ROS generation in alfalfa root segments was not affected by DPI pre-treatment, suggesting that an NADPH oxidase-independent mechanism is responsible for this Hg-induced ROS production (Ortega-Villasante et al., 2007). It is well known that, in illuminated photosynthetic tissues, the chloroplasts are the main sources of ROS production. It has been reported that Hg interacts with numerous proteins in the chloroplast of green algae (Xyländer et al., 1996), thus inhibiting the photosynthetic electron transport chain, leading to the generation of ROS and oxidative damages (Heidenreich et al., 2001). These results indicated that, in photosynthetic cells, the chloroplasts are the main target sites of Hg toxicity. In turn, in roots, the main sources of uncontrolled ROS release are mitochondria, which were also indicated as a common target site of several toxic metals (Keunen et al., 2011). The main sites of superoxide production in mitochondria are complex I and III in the electron transport chain (Noctor et al., 2007). Inhibition of complex I by rotenone produces a considerable amount of superoxide, but it is
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released into the matrix of mitochondria; therefore, it is not detectable in intact mitochondria and tissues due to the strong antioxidative systems of the mitochondrial matrix (Chen et al., 2003). The inhibition of complex III by antimycin A also caused a rapid and strong generation of superoxide into the intermembrane space (Muller et al., 2004). This superoxide can pass through the voltage-dependent anion channels of outer mitochondrial membrane into the cytosol (Han et al. 2003); therefore, it may be detected also in plant tissues. It has been demonstrated that superoxide generated at complex III can be inhibited by the inhibition of complex I due to the limitation of electron flow from complex I to complex III (Chen et al., 2003). However, the Hg-induced superoxide generation was not affected by rotenone cotreatment, suggesting that neither complex I nor complex III are the targets of Hg toxicity in barely roots. Recently, it was demonstrated that mitochondrial complex II can generate superoxide at high rates in both the forward and reverse reactions (Quinlan et al., 2012). We showed that TIFA, a non-competitive inhibitor of SDH, induced a marked superoxide generation in a dose-dependent manner in the barley root tips, which was accompanied by a strong reduction in root growth. Similar to TIFA, Hg treatment inhibited SDH activity in the root tips which was accompanied by a robust superoxide generation resulting in root growth inhibition. Furthermore, we showed that the inhibition of SDH activity and the generation of superoxide were also induced in vitro after the addition of Hg into the crude mitochondrial fraction. It has been reported that the inhibition of SDH activity by competitive inhibitors decreased the noncompetitive inhibitor-induced ROS production (Jardim-Messeder et al., 2015). According to this result, in our experiments, both malonate and oxalacetate, the competitive inhibitors of SDH, significantly reduced the Hg-induced superoxide generation in the crude mitochondrial fraction. In some previous studies, it has been shown that mitochondrial SDH is an important target of Hg toxicity. In marine fish, Hg caused an impairment of mitochondrial energy metabolism via the inhibition of several mitochondrial enzymes, including SDH (Mieiro et al., 2015). Hg-induced depletion of SDH activity was also reported in the rat brain (Rao et al., 2010). In pea roots, Hg treatment as early as after 1.5 h markedly increased the expression of gene encoding protein with a high amino acid sequence similarity to the mitochondrial rotenone-insensitive NADH dehydrogenase (Sävenstrand and Strid, 2004). It has been suggested that in Fe-deficient cucumber roots, a strong increase of these alternative dehydrogenases may bypass a marked loss of complex I and II activities (Vigani et al., 2009). Sweetlove et al. (2002) reported that oxidative stress in mitochondria induced the degradation of damaged proteins. Although the Hg affects directly mainly complex II, through the rapid generation of ROS it may damage numerous mitochondrial and also cytosolic components. On the other hand, at a lower Hg concentration the amount of SDH-generated ROS may function as a signal molecule activating different stress responses including a rapid reorientation of root growth. In conclusion, Hg along with the inhibition of SDH activity, similar to the noncompetitive SDH inhibitor TIFA, caused a rapid and marked superoxide generation in barley root tip. Hg evoked this inhibition of SDH activity and enhanced superoxide generation also in vitro, in the crude mitochondrial fraction. In vitro, the Hg-induced superoxide production was inhibited by the competitive inhibitors of SDH. These results indicated that the mitochondrial complex II-derived superoxide is the primary source of mercury toxicity in the barley root tip.
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Acknowledgements This work was supported by the Grant Agency VEGA, project No. 2/0039/16. The authors also thank the anonymous reviewers for their helpful criticisms, which improved the manuscript.
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Figure legends Fig. 1. Root length increments 6 h (A) and SDH activity (B) immediately after the short-term treatment of roots with 0–100 µM Hg for 30 min. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). Fig. 2. Hydrogen peroxide production by root tips (A), and localization of superoxide production (B) immediately after the short-term treatment of roots with 0–100 µM Hg for 30 min. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). The black arrows indicate the area of Hg-induced superoxide production in comparison to the control roots. Fig. 3. Root morphology 6 h (A), localization of cell death 6 h (B) and root morphology 9 h (C) after the short-term treatment of roots with 0–100 µM Hg for 30 min. The black triangles show the starting position of new root growth after the short-term treatments. S – swollen root part. Fig. 4. Hydrogen peroxide production by root tips (A) and localization of superoxide production (B) immediately after the short-term treatment of roots with 0, 25 or 50 µM Hg for 5, 10 or 15 min. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). The black arrows indicate the area of Hg-induced superoxide production in comparison to the control roots. Fig. 5. Root length increments 6 h (A) and hydrogen peroxide production by root tips (B) immediately after the short-term treatment of roots with 0, 25 or 50 µM Hg and co-treatment with 1 µM DPI or 5 µM rotenone for 30 min. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). Fig. 6. Root length increments 6 h (A) and hydrogen peroxide production by root tips (B) and localization of superoxide production (C) immediately after the short-term treatment of roots with 0–500 µM TIFA for 30 min. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). The black arrows indicate the area of Hg-induced superoxide production in comparison to the control roots. Fig. 7. SDH activity (A) and hydrogen peroxide production (B) in crude mitochondrial fraction in the presence of 0–50 µM Hg. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05). Fig. 8. Hydrogen peroxide production in crude mitochondrial fraction in the presence of 0 or 10 µM Hg and 1 mM succinate or 5 mM malonate or 5 mM malate or 1 mM oxalacetate. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P 0.05).
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