on Dimorphandra wilsonii seed germination

Environmental Pollution xxx (2016) 1e8

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Effects of glyphosate acid and the glyphosate-commercial formulation (Roundup) on Dimorphandra wilsonii seed germination: Interference of seed respiratory metabolism* Marcelo Pedrosa Gomes a, *, Fernanda Vieira da Silva Cruz a, Elisa Monteze Bicalho a, gas Borges a, Marcia Bacelar Fonseca b, Philippe Juneau c, Queila Souza Garcia a Felipe Vie ^nica, Avenida Anto gicas, Departamento de Bota ^nio Carlos, 6627, Pampulha, Caixa Postal Universidade Federal de Minas Gerais, Instituto de Ci^ encias Biolo 486, 31270-970, Belo Horizonte, Minas Gerais, Brazil ~o Zoo-Bota ^nica de Belo Horizonte, Departamento de Jardim Bota ^nico, Avenida Otacílio Negra ~o de Lima, 8000, Pampulha, 31365-450, Belo Fundaça Horizonte, Minas Gerais, Brazil c Montr Universit e du Qu ebec a eal, Department of Biological Sciences, GRIL-TOXEN, Ecotoxicology of Aquatic Microorganisms Laboratory, Succ. Centre-Ville, H3C 3P8, Montr eal, Qu ebec, Canada a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 June 2016 Received in revised form 26 September 2016 Accepted 27 September 2016 Available online xxx

Glyphosate-formulations are widely used in the Brazilian Cerrado (neotropical savanna) with little or no control, threatening population of the endangered species Dimorphandra wilsonii. We investigated the toxicity of different concentrations (0, 5, 25 and 50 mg l1) of glyphosate acid and one of its formulations (Roundup®) on seed germination in D. wilsonii. Glyphosate acid and Roundup drastically decreased seed germination by decreasing seed respiration rates. The activation of antioxidant enzymes, ascorbate peroxidase and catalase assure no hydrogen peroxide accumulation in exposed seeds. Glyphosate acid and the Roundup-formulation negatively affected the activities of enzymes associated with the mitochondrial electron transport chain (ETC), with Complex III as its precise target. The toxicity of Roundupformulation was greater than that of glyphosate acid due to its greater effects on respiration. The herbicide glyphosate must impair D. wilsonii seed germination by disrupting the mitochondrial ETC, resulting in decreased energy (ATP) production. Our results therefore indicate the importance of avoiding (or closely regulating) the use of glyphosate-based herbicides in natural Cerrado habitats of D. wilsonni as they are toxic to seed germination and therefore threaten conservation efforts. It will likewise be important to investigate the effects of glyphosate on the seeds of other species and to investigate the impacts of these pesticides elsewhere in the world. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Agriculture Antioxidant Cerrado Herbicides Mitochondria Toxicity

1. Introduction Although the Brazilian neotropical savanna (also known as “Cerrado”) has been ranked among the 25 most important terrestrial biodiversity hotspots (Myers et al., 2000), it continues to be impacted by anthropogenic activities, principally agriculture (Fernandes and Rego, 2014a; Ratter et al., 1997). Agricultural frontiers have been expanding during recent years into the Cerrado biome due to its favorable topography, its grassy vegetation with few shrubs that facilitates mechanization, and the regional tropical

*

This paper has been recommended for acceptance by Dr. Chen Da. * Corresponding author. E-mail address: [email protected] (M.P. Gomes).

climate that favors high productivity (Zilli et al., 2007). Agriculture is therefore considered the major factor threatening conservation efforts in the Brazilian Cerrado. Dimorphandra wilsonii is a threatened plant species found in the Cerrado, and is included in the Red List of Threatened Species (IUCN, 2016). Investigations concerning the ability of this species to cope with the impacts of agriculture practices are therefore urgently needed. As commercial planting coincides with the rainy season in the Cerrado region, there is a great risk of soil and water contamination by agricultural wastes (including pesticides and fertilizers) that can negatively impact non-target organisms (Zilli et al., 2007). The widespread and uncontrolled use of glyphosate-based herbicides threatens conservation efforts directed towards endangered Cerrado species such as the legume D. wilsonni. In addition to

http://dx.doi.org/10.1016/j.envpol.2016.09.087 0269-7491/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Gomes, M.P., et al., Effects of glyphosate acid and the glyphosate-commercial formulation (Roundup) on Dimorphandra wilsonii seed germination: Interference of seed respiratory metabolism, Environmental Pollution (2016), http://dx.doi.org/ 10.1016/j.envpol.2016.09.087

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M.P. Gomes et al. / Environmental Pollution xxx (2016) 1e8

habitat losses caused by agricultural development, the small population of that species (fewer than 250 total individuals) (Fernandes and Rego, 2014a) is threatened by herbicide exposure, and glyphosate formulations (the most widely used herbicide globally) may prove to be harmful to D. wilsonni even before its precise effects on those plants have been determined. The herbicidal effects of glyphosate are due to its inhibition of 5enolpyruvylshikimate-3-phosphate synthase (EPSPS e an enzyme of the shikimate pathway), which, in turn, inhibits the synthesis of aromatic amino acids (thus interfering with protein synthesis) and various other important plant secondary compounds (i.e., auxins and polyphenols) that require aromatic amino acid precursors (Siehl, 1997). Several other secondary effects of this herbicide on plant physiology have also been identified (as reviewed by Gomes et al., 2014). Glyphosate has been shown to induce oxidative stress in plants through the accumulation of reactive oxygen species (ROS) (Gomes et al., 2016a, 2016b). ROS have important natural roles in plants, participating in seed endosperm weakening, seed reserve mobilization, protection against pathogens, programmed cell death, and as signalling molecules for environmental cues (Gomes and Garcia, 2013). Germination and internal ROS contents appear to be linked in seeds, with the activity of ROS-scavenging systems having central roles in the germination process (ElMaarouf-Bouteau and Bailly, 2008). If these systems fail to control ROS contents, however, oxidative bursts can disrupt germination. The deleterious effects of ROS on seeds are due to their high reactivity that can damage cell components such as proteins, lipids, and DNA (Gill and Tuteja, 2010). Recently we have shown the glyphosate's ability to interfere with mitochondrial electron transport chain in Lemna minor leaves, resulting in ROS accumulation (Gomes and Juneau, 2016). If the herbicide could have similar toxic effects on respiration metabolism of seeds, glyphosate could therefore affect seed germination by inducing oxidative stress. Very little, however, is actually known about its impact on that process. Research on the effects of glyphosate and glyphosate-based herbicides on seed germination has been scarce (Blackburn and Boutin, 2003), and mostly contradictory, with deleterious (Morash and Freedman, 1989; Shuma et al., 1995) or little or no effects being reported (Egley et al., 1978; Piotrowicz-Cieslak et al., 2010) (for more details, see Blackburn and Boutin, 2003). According to Blackburn and Boutin (2003), herbicide usage merits more attention as it can induce drastic ecological changes. In the case of the Brazilian Cerrado, it will be important to understand the effects of glyphosate in the environment to facilitate conservational efforts. As such, we investigated the effects of glyphosate acid and one of its formulations (Roundup) on the germination of the threatened Brazilian species D. wilsonii. We hypothesised that by interfering on respiratory metabolism glyphosate and Roundup-formulation can impair seed germination by inducing ROS accumulation. Therefore, we also investigated the physiological processes associated with seed sensitivity/tolerance to the herbicide, especially in terms of oxidative and respiratory metabolism. The differential effects glyphosate acid and Roundup-formulation on the germination of D. wilsonii seeds were studied to determine if their effects were associated with the glyphosate molecule or with other compounds present in its commercial formulation. 2. Materials and methods 2.1. Plant material and germination assays D. wilsonii seeds, collected in 2015 in Minas Gerais State, Brazil, in areas of its natural occurrence (Fernandes and Rego, 2014b), were acquired from the Botanical Garden of the Zoo-Botanical

Foundation (Belo Horizonte, MG, Brazil). The seeds were used immediately after harvesting, pre-treating them by mechanical scarification followed by surface sterilization in 5% sodium hypochlorite for 5 min and thorough rinsing with deionized water before sowing. Germination assays were carried out by placing four replicates of 20 seeds in germination boxes lined with filter paper (Whatman No. 1) moistened daily with 20 ml of deionized water or with treatment solutions. The germination boxes were arranged in a completely randomized order in a growth chamber at 30 C under a 12-h photoperiod (30 mmol m2 s1, Philips T2 40 W/3 lamps). Germination was evaluated daily. Seeds were considered to have germinated when approximately 2 mm of the primary root had emerged. The tests were terminated when there was no further germination after three consecutive days. Seed viability was evaluated by the tetrazolium test, following Gomes et al. (2016c). The initial viability of the seed lot was 90%. Analytical-grade glyphosate (Pestanal grade) obtained from Sigma-Aldrich (Oakville, Canada) and the commercial glyphosate Roundup formulation (Monsanto, Brazil) were used in the experiments. Stock solutions (1000 mg l1) of glyphosate and Roundup were prepared in ultrapure water and used to obtain the solutions (in ultrapure water) with desired concentrations of the chemical which were used to moisten seed (20 ml/day/germination boxe). Seeds were submitted to different concentrations of glyphosate and Roundup (0, 5, 25 and 50 mg active ingredient l1) that were based on the recommended concentration of the herbicide for field applications (19 g l1 of active ingredient [Gomes et al., 2016b]). 2.2. Respiration rates The effects of glyphosate acid and Roundup-formulation on seed respiratory activity were investigated using a closed-system method. At each evaluation time, six seeds were placed into sealed glass tubes and the initial (ti) headspace O2 and CO2 levels were determined using a portable gas analyzer (PBI Dansensor CheckPoint II, Ringsted, Denmark), with four replicates. After approximately one hour of incubation at test temperatures, final (tf) headspace O2 and CO2 levels were recorded. The respiration rate (RCO2) was calculated according to the following equation, and was expressed in ml CO2 h1.

RCO2 ¼

DCO2 ðDtÞ

where: DCO2 is the CO2 difference (ml) measured in the system at a given time t, at Dt ¼ time (hours) elapsed between measurements. 2.3. Biochemical evaluations Biochemical analyses were performed on seeds treated in parallel experiments under the same conditions used in the germination tests. At each harvesting time (12, 24, 48, 72 and 96 h after treatment induction) the embryos of six seeds from each germination box were manually extracted and ground in liquid nitrogen, constituting one replicate (with a total of 4 replicates/treatment). Samples were stored at 80 C until analyzed. Hydrogen peroxide (H2O2) concentrations were measured following Velikova et al. (2000), using 0.1 g of embryos. H2O2 was extracted in 2 ml of 0.1% tricloroacetic acid (TCA) and after centrifugation at 12000g for 15 min, 300 ml of the centrifuged supernatant was reacted with 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and 1 ml of 1 M KI. Samples were read at 390 nm and H2O2 concentrations were determined using an extinction coefficient (ε) of 0.28 mM1 cm1. Catalase (CAT; E.C. 1.11.1.6) and ascorbate peroxidase (APX; E.C. 1.11.1.11) activities of the embryos



were determined spectrophotometrically. Antioxidant enzymes were extracted by macerating 0.1 g of embryos in 1 ml of an extraction buffer containing 100 mmol l1 potassium phosphate buffer (pH 7.8), 100 mmol l1 EDTA, 1 mmol l1 L-ascorbic acid, and 2% PVP (m/v). The protein contents of all of the samples were determined using the Bradford method. Catalase assay was determined following Aebi (1984) at 25 C in a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 250 mM hydrogen peroxide and distilled water. Catalase activity was determined following the decomposition of H2O2, for 1.5 min at 10s intervals, monitoring changes in absorbance at 240 nm, with a molar extinction coefficient of 0.0394 mM1 cm1. Ascorbate peroxidase activity was determined following Nakano and Asada (1981) at 28 C in a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 10 mM L-ascorbic acid, 2 mM hydrogen peroxide and distilled water. The APX activity was estimated my monitoring ascorbate oxidation rate (ε ¼ 2.8 mM1 cm1) at 290 nm for 3 min at 15-s intervals. The enzymatic activities of the mitochondrial ETC Complexes I to IV were determined spectrophotometrically on embryo homogenates (200 mM phosphate buffer pH 7.5) using 30 mg of protein in each assay from seeds exposed to 0 or 50 mg l1 of the test herbicides. Complex I (NADH:ubiquinone oxidoreductase) and Complex II (succinate dehydrogenase) assays were performed following Estornell et al. (1999), and their activities were calculated by monitoring the rates of NADH (ε ¼ 5.5 mM1 cm1) and ubiquinone (ε ¼ 9.6 mM1 cm1) decreases per mg of protein. Complex III (ubiquinol-cytochrome c reductase) activity was determined following Birch-Machin et al. (1993a), monitoring cytochrome cIII reduction rates (ε ¼ 19 mM1 cm1). Complex IV (cytochrome c oxidase) activity was determined following (Birch-Machin et al., 1993b), calculating the rate of increase in absorbency caused by the oxidation of cytochrome cII to cytochrome cIII (ε ¼ 19 mM1 cm1). All activities were expressed per microgram of protein (which was determined by the Bradford method). The activity of Complex V (ATP-synthase) was determined by monitoring pH increases associated with ATP synthesis, following (Madeira et al., 1974). 2.4. Statistical analyses The results represent the average of four replicates. The results were tested for normality (ShapiroeWilk) and homogeneity (Brown-Forsythe) and then statistically evaluated using JMP software 10.0 (SAS Institute Inc). MANOVA univariate repeated measures with Time as the within-subject factor and glyphosate and Roundup as the main effects were used to analyze differences in the variables studied during exposure to the treatments. Herbicides (glyphosate acid and Roundup-formulation), and the interaction between herbicides and doses were included within the model. The sphericity of the data was tested using Mauchly's criteria to determine whether the univariate F tests for the within-subject effects were valid. In cases of invalid F, the Greenhouse-Geisser test was used to estimate epsilon (ε). Contrast analysis was used when there were significant differences in the variables between treatments. 3. Results 3.1. Seed germination, viability and oxidative stress markers Both glyphosate acid and Roundup demonstrated negative effects on seed germination (Fig. 1). Regardless the concentration, germinability was reduced in seeds exposed to the herbicide, but it was more reduced in seeds treated with the Roundup formulation

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(P < 0.05, Table 1). Significant interactions were observed between herbicides, concentrations, and times of exposure (Table 1). When treated with 25 and 50 mg l1, final germination percentage was higher in seeds treated with glyphosate acid than to seeds exposed to Roundup (Fig. 1). In relation to the control (90% germination), viability was significantly (P < 0.05) reduced in seeds treated with 50 mg l1 glyphosate acid (50.85%) and with all of the Roundup concentrations tested (with 75.66, 51.85, and 46.29% viability after treatment with 5, 25 and 50 mg l1 Roundup respectively). Hydrogen peroxide (H2O2) concentrations were not significantly different between the treatments over time (P > 0.05, Fig. 2). Significant interactions were observed between treatment concentrations and time for catalase (CAT) and ascorbate peroxidase (APX) activities (Table 1). The activities of both enzymes increased in embryos extracted from seeds treated with glyphosate acid and Roundup-formulation after 24 h of exposure (Fig. 2, Table 1, P < 0.05). 3.2. Respiration rates and activity of mitochondrial electron transport chain enzymes The herbicide reduced respiration rates (RCO2) in seeds (Fig. 3, Table 1), but RCO2 was more greatly reduced in seeds exposed to the Roundup formulation (P < 0.0001). Significant interactions were observed between herbicides, concentrations, and times of exposure (Table 1). In glyphosate acid treated seeds, RCO2 was greater in seeds exposed to 5 mg l1 than to 25 and 50 mg l1 (P < 0.001). RCO2 did not significantly differ among seeds exposed to different concentrations of Roundup (P ¼ 0.4186). When treated with 5 mg l1, RCO2 was greater in seeds exposed to glyphosate acid than to seeds exposed to Roundup (P < 0.0001) after 48e96 h of evaluation. The activities of all of the mitochondrial electron transport chain enzymes evaluated (Complexes I to V) were reduced in the embryos of seeds treated with the herbicide (50 mg l1 of glyphosate acid or Roundup) (Fig. 4, Table 1). The activities of Complexes III, IV and V showed less recovery in embryos of seeds treated with herbicide. For instance, at the end of the exposure (96 h), the activity of Complex III, IV and V was decreased by 28/42%, 30/31% and 25/40% while the activity of Complex I and II was reduced by 18/29% and 19/29% in seeds treated with glyphosate acid and Roundup, respectively. Moreover, the activity of Complex III was more sensitive to Roundup than glyphosate acid and after 48 h of exposure; Complex III activity was more greatly reduced in seeds exposed to Roundup than to glyphosate acid (P < 0.05, Fig. 4). 4. Discussion Seed germination is widely used in biological testing as a simple and inexpensive analytical measure of the biological activities of environmental pollutants such as herbicides (Piotrowicz-Cieslak et al., 2010). In the present study, we observed negative effects of glyphosate acid and Roundup-formulation on the germination and viability of D. wilsonii seeds (Fig. 1). These results are critical within the context of the growing use of glyphosate-based herbicides in the habitats of threatened species. In addition to the decreased germination of fresh D. wilsonii seeds, the use of glyphosateformulations will drastically reduce the viability of its seed bank, thus disrupting the natural emergence of plants and constraining recovery programs. Decreased seed germination and viability in response to glyphosate exposure have been observed in other species (Morash and Freedman, 1989; Shuma et al., 1995). As a postemergence herbicide, however, glyphosate was not designed to disrupt seed germination e raising the question of how glyphosate interferes with germination.


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Fig. 1. Germination percentages of D. wilsonii seeds exposed to different glyphosate acid and Roundup concentrations (0, 5, 25 and 50 mg l1). Values expressed as means ± SE of four replicates. Values followed by *, within the same concentrations and times of exposure, are significantly different (P < 0.05) by the contrast test.

Table 1 MANOVA Repeated-measures for the effects of different concentrations (mg l1) of glyphosate acid or Roundup formulation (herbicides) and different times of exposure (hours) on germination percentages, respiration rates (RCO2), and hydrogen peroxide concentrations (H2O2), as well as catalase (CAT), ascorbate peroxidase (APX), and mitochondrial electron transport chain enzyme activities (Complex [C] I to V) in Dimorphandra wilsonii seeds. Source of Variation

D.F

Germination

RCO2

H2O2

CAT

APX

CI

CII

CIII

CIV

CV

Herbicides Herbicides Herbicides Time Herbicides Herbicides Herbicides

1 3 3 3 3 9 9