Acta Physiol Plant (2008) 30:469–479 DOI 10.1007/s11738-008-0144-8

ORIGINAL PAPER

Biochemical responses of glyphosate resistant and susceptible soybean plants exposed to glyphosate Carlos Alberto Moldes Æ Leonardo Oliveira Medici Æ Othon Silva Abraha˜o Æ Siu Miu Tsai Æ Ricardo Antunes Azevedo

Received: 20 September 2007 / Revised: 10 January 2008 / Accepted: 22 January 2008 / Published online: 13 February 2008 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2008

Abstract Glyphosate is a wide spectrum, non-selective, post-emergence herbicide. It acts on the shikimic acid pathway inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), thus obstructing the synthesis of tryptophan, phenylalanine, tyrosine and other secondary products, leading to plant death. Transgenic glyphosateresistant (GR) soybean [Glycine max (L.)] expressing an glyphosate-insensitive EPSPS enzyme has provided new opportunities for weed control in soybean production. The effect of glyphosate application on chlorophyll level, lipid peroxidation, catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GOPX) and superoxide dismutase (SOD) activities, soluble amino acid levels and protein profile, in leaves and roots, was examined in two conventional (non-GR) and two transgenic (GR) soybean.

Communicated by A. Tukiendorf. C. A. Moldes Facultad de Ciencias Exactas y Naturales, Universidad Nacional de La Pampa, Santa Rosa 6300, Argentina L. O. Medici Departamento de Cieˆncias Fisiolo´gicas, Universidade Federal Rural do Rio de Janeiro, Serope´dica, RJ CEP 23890-000, Brazil O. S. Abraha˜o
S. M. Tsai Centro de Energia Nuclear na Agricultura, Universidade de Sa˜o Paulo, Piracicaba, SP 13400-970, Brazil R. A. Azevedo (&) Escola Superior de Agricultura ‘‘Luiz de Queiroz’’, Universidade de Sa˜o Paulo, Piracicaba, SP CEP 13418-900, Brazil e-mail: [email protected]

Glyphosate treatment had no significant impact on lipid peroxidation, whilst the chlorophyll content decreased in only one non-GR cultivar. However, there was a significant increase in the levels of soluble amino acid in roots and leaves, more so in non-GR than in GR soybean cultivars. Root CAT activity increased in non-GR cultivars and was not altered in GR cultivars. In leaves, CAT activity was inhibited in one non-GR and one GR cultivar. GOPX activity increased in one GR cultivar and in both non-GR cultivars. Root APX activity increased in one GR cultivar. The soluble protein profiles as assessed by 1-D gel electrophoresis of selected non-GR and GR soybean lines were unaffected by glyphosate treatment. Neither was formation of new isoenzymes of SOD and CAT observed when these lines were treated by glyphosate. The slight oxidative stress generated by glyphosate has no relevance to plant mortality. The potential antioxidant action of soluble amino acids may be responsible for the lack of lipid peroxidation observed. CAT activity in the roots and soluble amino acids in the leaves can be used as indicators of glyphosate resistance. Keywords Antioxidant enzymes
Glyphosate resistance
Oxidative stress
Soybean Abbreviations AGA After glyphosate application ALA d-Aminolevulinic acid APX Ascorbate peroxidase CAT Catalase CI Chlorophyll Index EPSPS 5-enolpyruvylshikimate-3-phosphate synthase GOPX Guaiacol peroxidase GR Glyphosate resistant GtR Glutathione reductase

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LP MDA PEP POX PVPP ROS SOD TBA TCA

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Lipid peroxidation Malondialdehyde Phosphoenolpyruvate Peroxidase Polyvinylpyrrolidone Reactive oxygen species Superoxide dismutase Thiobarbituric acid Trichloroacetic acid

Introduction The residual effects caused by agrochemicals such as herbicides, fungicides and insecticides can represent severe risks to the production of safe high quality food for human or animal consumption. Herbicides prevent the competition of weeds with crops, thus saving soil nutrients, water and arable land. However, the use of herbicides needs to be carefully managed, as they may damage the co-existing plants in areas adjacent to the herbicide-applied zones. The success of crop production relies on crop resistance and weed susceptibility to the target herbicide (Sunohara and Matsumoto 2004; Qi et al. 2008). Glyphosate is a wide spectrum, non-selective postemergence herbicide that affects the shikimic acid pathway by the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19), thus preventing the synthesis of the aromatic amino acids. As a consequence, plants die following a slow down in protein synthesis. In addition, there is a reduction in secondary products of the shikimate pathway and a diversion of carbon into an accumulated pool of shikimate (Tan et al. 2006). Transgenic glyphosate-resistant (GR) soybean [Glycine max (L.)] expressing an glyphosate-insensitive EPSPS enzyme has provided new insights for weed control in soybean production. The vast majority of the commercial glyphosate resistant varieties on the market today contain a gene derived from Agrobacterium sp. strain CP4, encoding a glyphosate-tolerant enzyme, the so-called CP4 EPSPS (Funke et al. 2006). Although GR soybean is resistant to glyphosate, application of glyphosate has resulted in significant injury under certain conditions and herbicide formulations (Zablotowicz and Reddy 2007). It has also been reported that low water availability may also contribute to glyphosate induced yield reduction (King et al. 2001), however, Elmore et al. (2001) has indicated that soybean yield and plant characteristics in most cases were not affected by glyphosate.

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The first visible symptom following the application of glyphosate, in susceptible plants, is normally the appearance of chlorosis in newly formed leaves. Subsequent known symptoms of glyphosate application include bleaching, chlorosis and stunted growth, which are mainly concentrated in metabolically active sink tissues such as immature leaves, shoot tips, buds and roots (Fuchs et al. 2002). A lower amount of leaf chlorophyll is a distinguishing characteristic in plants exposed to sublethal concentrations of glyphosate (Wong 2000; Tan et al. 2006). Studies have shown that glyphosate inhibited the enzymatic activity of cytochrome P450 expressed in yeast, suggesting a similar action in plants (Lamb et al. 1998; Xiang et al. 2005). Furthermore, glyphosate may also prevent chlorophyll synthesis by inhibiting the formation of the porphyrin precursor d-aminolevulinic acid (ALA) (Cole 1985; Zaidi et al. 2005). The photosynthetic electron transport system is the major source of reactive oxygen species (ROS) in plant tissues (Asada 1994) having the potential to generate singlet oxygen and superoxide (Jung et al. 2000). The superoxide ion can participate directly in oxidation and reduction reactions with cell components leading to toxic effects. It also reacts with H2O2 through the iron catalyzed Haber–Weiss reaction, giving rise to the more reactive hydroxyl radical (OH) (Grata˜o et al. 2005). The hydroxyl radical is an indiscriminate reactant capable of catalyzing the peroxidation of cell membranes (Halliwell and Gutteridge 1989), and protein and nucleic acid oxidation (Martinez-Cayuela 1998; Dro¨ge 2002). Antioxidant enzymes have been shown to respond to abiotic stresses (Vito´ria et al. 2001; Benavides et al. 2005; Grata˜o et al. 2005; Cao et al. 2006; Gulen et al. 2006; Lea and Azevedo 2006; Patykowski 2006; Tamas et al. 2006; Alla and Hassan 2007). Superoxide dismutase (SOD, EC 1.15.1.1) dismutates the superoxide radical into hydrogen peroxide and water. This enzyme can be present in distinct cell compartments such as the cytosol, mitochondria, chloroplasts and peroxisomes (Grata˜o et al. 2005). Other antioxidative enzymes, such as ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GtR, EC 1.6.4.2), which participate in detoxifying peroxide via the ascorbate–glutathione metabolic pathway, are also involved in the antioxidative stress system (Gomes-Junior et al. 2006a, b; 2007). There are few reports concerning the effects of glyphosate on the antioxidant system. The objective of this work was to study biochemical parameters that may be affected in roots and leaves of soybean plants exposed to glyphosate, focusing on the antioxidant response and soluble amino acid content, thus evaluating possible biochemical markers for differential characterization of glyphosate-resistant and conventional soybean lines.

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Materials and methods Plant materials, treatments and experimental design Four soybean cultivars were used, two glyphosate-resistant designated DM4800RG and Msoy7575RR and two glyphosate-susceptible DM48 and Msoy7501. For preliminary analyses, immunoassay tests with lateral flow strips (SDI TraitTM RUR Bulk Soybean Test, NY, USA) were conducted to verify the presence of the CP4 EPSPS protein in all plant material, with positive results for DM4800RG/ MSoy7575RR and negative for DM48/MSoy7501. A polymerase chain reactions (PCR) was carried out to verify the presence of the CP4 EPSPS gene, which indicated positive results for the transgenic materials and negative results for the conventional materials. Seeds of each soybean line were superficially sterilized by hypochlorite solution (2%), soaked in water and placed in sterile plates. Pre-germination was carried out in the dark at 30°C for 48 h. Pre-germinated seeds were sown in sterile sand:vermiculite (3:1) in 3 L pots. Three seedlings per pot were grown in a glasshouse at 15–30°C, 30–60% humidity under a natural light regime. Plants were supplied twice a week with 100 mL pot-1 of Murashige and Tucker (1969) nutrient solution without the addition of vitamins. Experiments were performed at ESALQ-USP, Piracicaba, Brazil, in 2005–2006. No insecticide and fungicide applications were necessary during the period of the experiment. Glyphosate (Agrisato 480 CS manufactured by ALKAGRO) was sprayed on 5-week-old plants, in an application chamber. The herbicide was diluted in water at 2:100 proportion and applied on the foliar surface using a compressor moved pressurized precision sprayer, equipped with continuous-deposition tips (XR110015), placed at 0.50 m from the pot upper surface. A 200 kPa work pressure allowed an intake corresponding to 200 L ha-1 of mix. Leaves and roots were harvested at 0, 24 and 72 h after glyphosate application, frozen in liquid nitrogen and stored at -80°C. Experiments were laid out in a complete randomized design with four replications.

Lipid peroxidation Lipid peroxidation was determined by estimating the malondialdehyde (MDA) content as describe previously (Gomes-Junior et al. 2007). Leaves and roots (250 mg fresh weight) were homogenized in a pestle and mortar with 20% (w/v) insoluble polyvinylpyrrolidone (PVPP) and 1.3 mL 0.1% trichloroacetic acid (TCA). The homogenate was centrifuged at 10,0009g for 5 min and 250 lL of the supernatant was added to 1 mL of 0.5% 2-thiobarbituric acid (TBA) and 20% TCA solution and incubated in a water

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bath at 95°C for 20 min. The concentration of MDA was calculated from the absorbance at 532 nm by using the absorbance coefficient 155 mM-1 cm-1, following a correction for unspecific turbidity determined by the absorbance at 600 nm.

Chlorophyll determination A Minolta SPAD-502 chlorophyll meter was used to determine a chlorophyll index (CI). The meter measures absorption at 650 and 940 nm wavelengths to estimate chlorophyll levels (Pinkard et al. 2006). SPAD readings were taken at the terminal leaflet of the second leaf from the apex of the shoot. The SPAD sensor was placed randomly on leaf mesophyll tissue only, with veins avoided. Six leaves were chosen per pot and measurements were immediately taken per leaf and averaged to provide a single CI per pot.

Amino acid extraction and quantification Frozen tissue (100 mg) was homogenized in 2 mL MCW extraction solution (methanol:chloroform:water, 12:5:3 v/v/ v) and centrifuged at 2,5009g for 20 min at 4°C. The supernatant was collected and added to 0.5 mL chloroform and 0.75 mL Milli-Q water. The water-soluble phase was used for further analysis. For amino acid quantification, 1 mL sample was mixed with 500 lL 0.2 M citrate buffer (pH 5.5), 200 lL 5% ninhydrin and 1 mL 0.2 mM potassium cyanide (KCN) in methyl glycol. The mixture was heated at 100°C for 20 min and cooled immediately. A measure of 1 mL of 60% ethanol was added and the quantification was carried out in a spectrophotometer at 570 nm using a leucine calibration curve as the standard (Azevedo et al. 2003).

Enzyme extraction and activity determination Soybean leaves and roots were homogenized in a pestle and mortar with 100 mM potassium phosphate buffer (pH 7.5) containing 1 mM ethylene diaminetetracetic acid (EDTA), 3 mM DL-dithiothreitol and 5% (w/v) insoluble PVPP. The homogenate was centrifuged at 10,0009g for 30 min and the supernatant was stored in separate aliquots at -80°C for all enzyme activity determinations (GomesJunior et al. 2007). CAT (EC 1.11.1.6) activity was assayed spectrophotometrically at 25°C in a 1 mL reaction mixture containing 100 mM potassium phosphate buffer (pH 7.5) and 0.0075% H2O2. The reaction was initiated by the addition of 20–40 lL plant extract and determined by monitoring

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H2O2 degradation at 240 nm over 1 min (Azevedo et al. 1998). CAT activity was calculated using an extinction coefficient for H2O2 of 39.4 mM-1 cm-1. Guaiacol peroxidase (GOPX, EC 1.11.1.7) activity was assayed in a 1 mL mixture reaction containing 0.2 M potassium phosphate–0.1 M sodium citrate buffer (pH 5.0), 0.01% guaiacol and 0.06% H2O2. The reaction was initiated by the addition of 5–10 lL plant extract and incubated at 30°C for 15 min. The reaction was stopped by rapid cooling in an ice water bath and the addition of 20 lL sodium metabisulphite. After vortexing, the reaction mixture was held for 10 min and the GOPX activity was determined by the absorbance at 450 nm. One enzyme activity unit corresponds to an increase of 0.001 in absorbance per min (Gomes-Junior et al. 2007). GOPX activity was expressed in units mg-1 protein. APX activity was determined by monitoring the rate of ascorbate oxidation at 290 nm at 30°C. The reaction was initiated by the addition of 40 lL plant extract to 1 mL of a medium containing 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM EDTA and 0.1 mM H2O2. APX activity, expressed as nmol ascorbate min-1 mg-1 protein was calculated using the extinction coefficient 2.8 mM-1 cm-1 for ascorbate (Nakano and Asada 1981). The protein concentration was determined by the method of Bradford (1976) using bovine serum albumin (BSA) as the standard.

Polyacrylamide gel electrophoresis (PAGE) Electrophoresis was carried out as described by Laemmli (1970). For non-denaturing gels, SDS was excluded. A 15 lg protein sample of each supernatant generated from the enzyme extraction procedure (see above) was used for SDSPAGE. Electrophoresis was carried out at a constant current set at 15 mA for *3 h. The gels were rinsed in distilleddeionized water and incubated overnight in 0.05% Coomassie blue R-250 in methanol:acetic acid:water 40:7:53 (v/v/v) solution and destained with successive washes of methanol:acetic acid:water 40:7:53 (v/v/v) solution.

Molecular mass calculation of SDS-PAGE stained bands was performed using Diversity-Database Fingerprinting Software (Bio-Rad) by a point-to-point semi-log method applied to Invitrogen Protein Ladder profile of molecular markers. Activity staining for SOD and CAT were carried out as described by Gomes-Junior et al. (2007) with minor modifications in the amount of protein loaded onto the gel, which was 60 lg.

Statistical analysis The data presented are means of one experiment with four independent replications. Variance analysis was performed on experimental data. Significant differences between the response of cultivars to glyphosate was determined by the Tukey multiple range test and linear regression analysis. Statistical analysis was performed using SAS statistical program (SAS Institute Inc.)

Results Lipid peroxidation No significant differences in lipid peroxidation were observed in leaves and roots for the four cultivars tested. However, in leaves a non-significant increase in lipid peroxidation at 24 h after glyphosate application (AGA) was observed in resistant cultivars DM4800RG and Msoy7575RR. Similarly, an increase in lipid peroxidation at 24 h AGA was observed in roots but there was no difference between susceptible and resistant cultivars (Table 1).

Chlorophyll contents At 72 h AGA some alteration in the treated plants was observed. Although chlorosis and necrosis were not very

Table 1 Lipid peroxidation estimated by TBARS products (MDA) for leaves and roots tissues at 0, 24 and 72 h after glyphosate application Cultivar

Lipid peroxidation (nmol MDA fresh weight-1) Leaf 0

Root 24

72

0

24

72

DM48

21.33 ± 3.35c

23.10 ± 2.81bc

20.31 ± 1.13c

4.76 ± 0.70b

6.22 ± 0.66ab

5.07 ± 0.97b

DM4800RG

19.86 ± 3.82c

25.84 ± 5.10abc

25.10 ± 3.28abc

4.86 ± 1.34b

4.63 ± 0.23b

4.90 ± 0.91b

MSOY7501

27.88 ± 3.85abc

30.96 ± 3.46ab

23.71 ± 5.09abc

6.42 ± 1.16ab

7.97 ± 1.13a

4.44 ± 0.45b

MSOY7575RR

23.49 ± 2.65ab

32.99 ± 6.20a

26.75 ± 2.60abc

5.28 ± 0.52b

6.24 ± 0.63ab

5.96 ± 0.90ab

Letters indicate significant differences by Tukey means test

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Table 2 Chlorophyll content at 0 and 72 h after glyphosate application Chlorophyll (SPAD units) 0h

72 h

DM48

35.63 ± 1.85a

30.20 ± 1.15b

DM4800RG

32.53 ± 1.14ab

32.63 ± 1.27ab

25.07 ± 1.30d 29.630 ± 1.42bc

25.43 ± 2.20cd 28.53 ± 2.15bcd

Msoy7501 Msoy7575RR

Letters indicate significant differences by Tukey means test

clear, in the susceptible cultivars (DM48 and Msoy7501), rolled leaves and slight pigment alterations were observed. However, the chlorophyll content decreased in only one susceptible cultivar DM48 (Table 2). Furthermore, the chlorophyll content of Msoy7501 was lower when compared to the other cultivars.

Total soluble amino acid content Changes were observed in the total soluble amino acid contents of leaves and roots. In leaves, the increase in soluble amino acids in susceptible cultivars was more pronounced than in resistant cultivars at 72 h AGA (Table 3). Regression analysis (Table 4) confirmed that the increase in amino acid contents of susceptible DM48 and Msoy7501 cultivars was significant and also higher than that of resistant soybean lines (Fig. 1). In roots, the total soluble amino acid content increased in all cultivars at 72 h AGA. However, the amino acid content of resistant soybean lines was constant during the first 24 h AGA, whilst in susceptible lines, the amino acid content increase was constant during the duration of the experiment (Tables 3, 4). The behavior observed for this parameter in leaves and roots suggests a different response to glyphosate application between glyphosate susceptible and resistant soybean cultivars. In leaves, the response was clearer than in roots.

Antioxidant enzymes Linear regression analysis revealed that leaf GOPX activity increased significantly AGA in DM48, Msoy7501 and Msoy7575RR (Table 4). Constitutive GOPX activity in DM4800RG was higher than in the others cultivars (Table 5). Therefore, at 72 h AGA all cultivars reached the same level of GOPX activity. The GOPX activity determined in leaves responded to glyphosate-stress mainly in non-GR cultivars, as indicated by the regression analysis (Table 4). In roots, GOPX activity did not exhibit any significant alteration in all four cultivars tested. Leaf CAT activity was not altered at 24 h AGA, but at 72 h AGA a tendency for a reduction in activity for all cultivars was observed (Table 5). This result was not confirmed by the regression analysis, nor Tukey tests, but non-denaturing PAGE of CAT activity revealed a slight decrease in the intensity of isoenzyme bands at 72 h for DM48 and DM4800RG cultivars (Fig. 2). In the roots, CAT activity was not altered significantly at 24 h AGA, but during the 24–72 h AGA period a significant increase in CAT activity was detected in susceptible cultivars Msoy7501 and DM48 (Table 5). Regression analysis of root CAT activity (Table 4) confirmed the different behavior between susceptible and resistant cultivars (Fig. 3). The APX activity in the leaves of the four cultivars did not exhibit any major alterations in glyphosate treated plants during the time length of the experiment (Table 5). However, an indication of increased APX activity at 24 h was observed in leaves of DM4800RG and in roots of Msoy7501 (Table 5). In roots, glyphosate treatment lead to a linear increase only for APX activity in Msoy7575RR cultivar (Table 4).

SDS-PAGE and non-denaturing PAGE for SOD and CAT SDS-PAGE analysis revealed an almost identical protein pattern and band intensity variation for the DM48-

Table 3 Total soluble amino acid content in roots and leaves at 0, 24 and 72 h after glyphosate application Cultivar

Amino acids content (nmol Leucine fresh weight-1) Leaf 0

Root 24

72 15.07 ± 2.30a

DM48

3.72 ± 1.04e

8.17 ± 0.76bcd

DM4800RG

5.07 ± 1.53de

6.25 ± 1.61bcde

MSOY7501

4.25 ± 1.04e

8.72 ± 0.70b

MSOY7575RR

5.27 ± 1.06cde

7.74 ± 0.69bcd

7.88 ± 1.80bcd 12.48 ± 1.37a 8.52 ± 1.30bc

0

24

72

0.58 ± 0.17e

0.96 ± 0.26bcde

1.48 ± 0.32a

0.89 ± 0.08bcde

0.69. ± 0.15cde

1.29 ± 0.28ab

0.50 ± 0.13e

0.70 ± 0.16cde

1.11 ± 0.23abc

0.74 ± 0.07cde

0.66 ± 0.13de

1.17 ± 0.25abcd

Letters indicate significant differences by Tukey means test

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Table 4 Results of linear regression analysis (y = A + Bx; R2 coefficient of determination; P calculate level of significance) Parameter LP-L LP-R

Cultivars

A

B

R2

P

Parameter

Cultivars

LP-L

Msoy7501

29.80

Msoy7575RR

27.10 7.36

DM48

22.24

-0.0205

0.0587

0.4482

DM4800RG

21.67

0.0602

0.1629

0.1932

DM48

5.34

0.0003

0.0001

0.9777

DM4800RG

4.76

0.0012

0.0019

0.8923

GOPX-L

DM48

0.56

0.0073

0.6793

0.0010

1.39

-0.0015

0.0220

0.6453

GOPX-R

DM48

29.43

0.0109

0.0024

0.8789

DM4800RG

29.96

0.0384

0.0279

0.6037

DM48

59.47

-0.2324

0.2017

0.1430

DM4800RG

CAT-L

A

R2

P

-0.0713

0.2070

0.1373

0.0203

0.0129

0.7255

-0.0340

0.3707

0.0356

B

LP-R

Msoy7501 Msoy7575RR

5.59

0.0073

0.0907

0.3415

GOPX-L

Msoy7501

0.55

0.0097

0.8778

0.0001

0.68

0.0061

0.5120

0.0089

GOPX-R

Msoy7501

19.95

0.0577

0.1125

0.2864

Msoy7575RR

18.76

0.0638

0.1648

0.1904

Msoy7501

75.67

-0.5234

0.6864

0.0009

Msoy7575RR

CAT-L

DM4800RG

88.03

-0.4553

0.5060

0.0095

Msoy7575RR

66.73

-0.0758

0.0438

0.5141

CAT-R

DM48 DM4800RG

71.52 98.37

3.0293 -0.0881

0.9109 0.0093

0.0001 0.7653

CAT-R

Msoy7501 Msoy7575RR

152.89 161.82

2.3642 0.1317

0.6451 0.0104

0.0017 0.7530

APX-L

DM48

190.83

0.7451

0.2859

0.0733

APX-L

Msoy7501

297.67

-0.3116

0.0605

0.4408

DM4800RG

273.56

-0.1235

0.0066

0.8011

Msoy7575RR

321.56

-0.1902

0.0061

0.8087

DM48

2602.49

-1.0133

0.0022

0.8857

DM4800RG

3076.29

-8.0927

0.1733

0.1783

APX-R AA-L AA-R

DM48

4.00

0.1556

0.9618

0.0001

DM4800RG

5.17

0.0383

0.3906

0.0298

DM48

0.61

0.0123

0.7307

0.0004

DM4800RG

0.75

0.0065

0.4240

0.0218

APX-R AA-L AA-R

Msoy7501

1663.15

8.8053

0.0830

0.3638

Msoy7575RR

1278.60

28.6156

0.8020

0.0001 0.0001

Msoy7501

4.99

0.1091

0.8762

Msoy7575RR

5.86

0.0410

0.5494

0.0058

Msoy7501

0.50

0.0085

0.739

0.0003

Msoy7575RR

0.64

0.0067

0.5503

0.0057

P values \0.05, in bold, indicate B value statistically different of 0. The analysis have been performed with data of Tables 1, 2, 3 and 5 LP lipid peroxidation, GOPX guaiacol peroxidase, CAT catalase, APX ascorbate peroxidase, AA soluble amino acid, L leaves, R roots

Fig. 1 Soluble amino acid contents of the leaves of DM48, DM4800RG, Msoy7501 and Msoy7575RR at 0, 24 and 72 h after glyphosate treatment. Regression equation and R2 are in the internal box

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Table 5 GOPX, CAT and APX activities in soybean leaves and roots at 0, 24 and 72 h after glyphosate application Guaiacol peroxidase activity (GOPX) (units mg-1 protein)

Cultivar

Leaf

Root

0

24

72

0

24

72

DM48

0.53 ± 0.12e

0.78 ± 0.22bcde

1.06 ± 0.14abcd

30.87 ± 6.96ab

27.56 ± 7.28ab

30.94 ± 7.97ab

DM4800RG

1.29 ± 0.32ab

1.52 ± 0.42a

1.23 ± 0.20abc

26.84 ± 7.65ab

35.55. ± 2.87a

31.16 ± 8.45ab

MSOY7501

0.61 ± 0.07de

0.70 ± 0.12cde

1.28 ± 0.12ab

17.69 ± 4.36b

24.72 ± 5.97ab

22.97 ± 3.95ab

MSOY7575RR

0.61 ± 0.12de

0.92 ± 0.12bcde

1.09 ± 0.24abcd

17.79 ± 4.41b

21.76 ± 5.66ab

22.87 ± 4.26ab

-1

Catalase activity (CAT) (nmol H2O2 min

-1

mg protein )

Leaf 0

Root 24

72

0

24

DM48

51.92 ± 12.30bc

65.22 ± 15.21abc

38.96 ± 10.84c

75.83 ± 17.47ef

DM4800RG

87.63 ± 23.07a

77.70 ± 7.72ab

55.05 ± 11.52bc

118.63 ± 22.83def

72

137.76 ± 25.00cdef 65.86 ± 10.25f

291.78 ± 54.11ab 102.15 ± 20.36def

MSOY7501

75.95 ± 12.80ab

62.68 ± 12.53abc

38.12 ± 11.29c

179.03 ± 42.67cd

170.42 ± 45.18cd

336.17 ± 63.01a

MSOY7575RR

59.46 ± 1.06abc

75.81 ± 7.55ab

57.63 ± 12.07abc

130.44 ± 21.49cdef

212.06 ± 24.46bc

155.61 ± 16.8cde

Ascorbate peroxidase (APX) (nmol ascorbate oxidized min-1 mg protein-1) Leaf 0

Root 24

72

0

24

72

DM48

175.49 ± 38.92c

231.72 ± 36.89bc

236.81 ± 31.58bc

2268 ± 452abcde

3080 ± 875abc

2362 ± 477abcd

DM4800RG

253.21 ± 41.10bc

301.13 ± 54.12ab

254.49 ± 40.30abc

2868 ± 390abc

3195 ± 618ab

2389 ± 618abcd

MSOY7501

289.04 ± 42.88abc

303.13 ± 49.02ab

270.92 ± 28.52abc

963 ± 289e

2925 ± 706abc

1947 ± 445bcde

MSOY7575RR

282.58 ± 58.65abc

375.47 ± 90.71a

288.37 ± 47.13abc

1357 ± 193de

1847 ± 336cde

3378 ± 738a

Letters indicate significant differences by Tukey means test

The leaf and root samples of the DM48 and DM4800RG cultivars were also analyzed for SOD and CAT activity staining by non-denaturing PAGE. SOD and CAT activity staining exhibited similar isoenzyme profiles and no new isoenzymes were specifically induced in response to glyphosate (Figs. 2, 5). CAT activity exhibited some variations in leaves with a reduction of band intensity (Fig. 2a) being detected during the time length of the experiment, whilst in the DM48 roots, an increase in CAT activity was observed at 72 h AGA (Fig. 2b), which confirmed the results obtained by the spectrophotometric CAT assay.

Fig. 2 CAT activity staining following PAGE of extracts of leaves (a) and roots (b). Lanes 1, 2 and 3 correspond to the DM48 cultivar at 0, 24 and 72 h AGA, respectively, whereas lanes 4, 5 and 6 correspond to the DM4800RR cultivar at 0, 24 and 72 h AGA, respectively. Lanes ‘‘S’’, bovine liver CAT standard

DM4800RG pair with the clear exception of the 45 kDa molecular mass protein band (indicate by an arrow in Fig. 4), which was strongly stimulated in the transgenic line, but was not dependent upon glyphosate treatment.

Discussion The oxidative response in plants can be exacerbated by stressful conditions (Grata˜o et al. 2005). At the molecular level, the extent and nature of this response can differ between species and even between closely related varieties of the same species. Yu et al. (2007) reported 24 differentially expressed genes in soybean leaves following glyphosate treatment of susceptible and tolerant lines.

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Fig. 3 CAT activity of the roots of DM48, DM4800RG, Msoy7501 and Msoy7575RR at 0, 24 and 72 h after glyphosate treatment. Regression equation and R2 are in the internal box

Fig. 4 SDS-PAGE protein profile of extracts of leaves. Lanes 1, 2 and 3 correspond to the DM48 cultivar at 0, 24 and 72 h AGA, respectively, whereas lanes 4, 5 and 6 correspond to the DM4800RR cultivar at 0, 24 and 72 h AGA, respectively. Lane ‘‘S’’, Molecular mass markers standards. The arrow indicates one particular protein band that is strongly induced in the DM4800RR cultivar

Among them, 16 genes were upregulated by glyphosate, while the other 8 genes were downregulated by glyphosate. Most of the glyphosate inducible genes identified were associated with plant stress responses (Yu et al. 2007). Screening techniques can be used for response-quantification to environmental stress allowing the possibility to rank

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species, strains or individuals according to their relative tolerance (Ekmekci and Terzioglu 2005). In the study described here, the responses of different soybean cultivars exposed to glyphosate treatment were assessed in terms of biochemical parameters related to oxidative stress, specifically the antioxidant enzyme response. The lack of a significant increase in lipid peroxidation (Table 1) in response to glyphosate indicates that membrane integrity appears not to have been affected and since lipid peroxidation is a strong indicator of oxidative stress, it could be argued that the glyphosate treatment did not induce oxidative stress to any great extent. In susceptible lines, the significant increase in the total soluble amino acid (Table 3) content may be due to the reduction in the rate of protein synthesis caused by glyphosate. Some amino acids such as asparagine and proline can play an important role in stress resistance (Lea et al. 2007; Lea and Azevedo 2007) and they may be responsible for the limited lipid peroxidation observed (Table 1). Although the antioxidant properties of amino acids are not as strong as other soluble antioxidants such as glutathione or ascorbate, they could become important in high quantities (Dro¨ge 2002). Although changes in glutathione, ascorbate, tocopherols and other non-enzymatic antioxidants, have not been measured in this study, they may well also have been involved in the prevention of lipid peroxidation.

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Fig. 5 SOD activity staining following PAGE of extracts of leaves (a) and roots (b). Lanes 1, 2 and 3 correspond to the DM48 cultivar at 0, 24 and 72 h AGA, respectively, whereas lanes 4, 5 and 6 correspond to the DM4800RR cultivar at 0, 24 and 72 h AGA, respectively. Lanes ‘‘S’’, bovine SOD standard

Molecules such as ascorbate, glutathione, tocopherol and amino acids are important antioxidants when increased production of ROS occurs. Antioxidant enzymes also have a major role in dismutating ROS (Grata˜o et al. 2005). An increase in the antioxidant enzyme system can also be advantageous for plants exposed to herbicides. A chillingtolerant cultivar of rice that was also less sensitive to paraquat, exhibited higher SOD and GtR activities than a sensitive cultivar (Guo et al. 2007). Donahue et al. (1997) found an interesting correlation between the paraquatresistance of Pisum sativum with an enhancement of GtR and APX activities in the leaves of resistant plants. In wheat, paraquat treatment resulted in differential responses between cultivated and wild lines, with the cultivated lines exhibiting higher enzyme activities of SOD, APX and peroxidases than the wild lines (Ekmekci and Terzioglu 2005). Increases in CAT, APX, GR and GST activities were also observed in the algae Scenedesmus obliquus when subjected to oxyfluorfen (Geoffroy et al. 2002) indicating that the induction of the antioxidant mechanisms was related to oxyfluorfen resistance. In the present study, an improved adaptive capacity of the antioxidant pathway for detoxification of oxidative stress appears to be generated during glyphosate action, since CAT activity increased in roots of non-GR soybean cultivars, GOPX activity increased in leaves of DM48, Msoy7501 and Msoy7575RR and APX activity increased in roots of Msoy7575RR. Transgenic rice plants expressing a Mixococcus xanthus protoporphirin oxidase gene non-sensitive to oxyfluorfen, a protoporphirin oxidase inhibitor, did not exhibit antioxidant activation of SOD, POX and CAT enzymes (Jung and Back 2005). However, the non-transgenic sensitive lines were shown to have induced antioxidant enzymes, in a similar manner to the pattern of CAT activity in roots observed in this study for resistant and susceptible soybean lines exposed to glyphosate. The slight antioxidant defense activation appears to be a characteristic of resistant transgenic rice exposed to oxyfluorfen (Jung and Back 2005) as

well as in transgenic soybean exposed to glyphosate in this study. Although susceptible and resistant soybean cultivars exhibited few changes in lipid peroxidation and antioxidant systems after glyphosate treatment, the oxidative stress generated seems to be an effect of secondary importance, with no relevance to the damage caused by glyphosate. The lack of variation in the protein profile as determined by SDS-PAGE could also be related to the slight effects of glyphosate on antioxidant enzyme activity. The potential oxidative damage in leaves of Pisum sativum exposed to imazethapyr, an acetolactate synthase (ALS) inhibitor, was indicated by non-significant lipid peroxidation or protein oxidation, and also in increased glutathione peroxidase activity (Zabalza et al. 2007). In this case, the authors suggested that the oxidative stress was not related to the mode of action of ALS inhibitor imazethapyr. So, perhaps a better predisposition of antioxidant defenses has little contribution to the resistance of soybean cultivars against glyphosate action. Glyphosate, fluazifop-buthyl and imazethapyr have a substantial difference in their mode of action when compared to herbicides that affect the photosynthetic apparatus such as paraquat, oxyfluorfen or norflurazon. Although the glyphosate primary target is the shikimate pathway, the first observable symptom of herbicide action is chlorosis in new leaves. Thus, the antioxidant response of susceptible soybean to glyphosate could be similar to those antioxidant responses of plants exposed to herbicides that affect the photosynthetic apparatus. Glyphosate treatment decreased the growth and pigment content of maize plants and increased lipid peroxidation, ion fluxes in leaf tissues and the activity of CAT, peroxidase and glutathione-S-transferase (Sergiev et al. 2006). Reports for maize, barley, tobacco and sunflower (Cole 1985; Eker et al. 2006) showed a high sensitivity of chlorophyll to glyphosate action. Nandula et al. (2007) verified that the chlorophyll content decreased at 7 days after glyphosate treatment in

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three GR soybean varieties. Nandula et al. (2007) also observed a differential reduction in chlorophyll by glyphosate between GR soybean varieties. The expected result in the present work was that susceptible cultivars DM48 and Msoy7501 would have a lower chlorophyll content AGA, however, only the DM48 soybean exhibited a reduction in chlorophyll content at 72 h AGA (Table 2). It is also important to note that the constitutive chlorophyll contents of Msoy7501 and Msoy7575RR were lower than that of DM48 and DM4800RG. Therefore, plants with low constitutive levels of chlorophyll might have slower reduction in chlorophyll synthesis rates induced by glyphosate. Moreover, these soybean lines have been tested and recommended for different ranges of climate conditions and latitudes. Msoy7501 and Msoy7575RR are cultivars recommended in Brazil while DM48 and DM4800RG are recommended in Argentina. Chlorophyll decreases can be caused by carotenoid loss induced by sub-lethal doses of glyphosate (Mun˜ozRueda et al. 1986). In sugar beet, decreases in pigments by photo destruction was caused by carotenoid losses (Servaites et al. 1987), but in Abutilon theophrasti, it was not clear whether the reduction in chlorophyll was due to chlorophyll biosynthesis interference, photo-destruction or both (Fuchs et al. 2002). In plants, ALA has a key role in porphyrin synthesis that is incorporated into cytochromes, CAT and POXs. Catalases, peroxidases and cytochromes contain heme-groups and therefore their activities can be affected by inhibition of ALA synthesis by glyphosate action. A reduction in CAT activity was reported for Cyperus rotundus (Abuirmaileh and Jordan 1978) and maize (Sergiev et al. 2006), 3 days after glyphosate treatment. In leaves, DM4800RG and Msoy7501 exhibited CAT inhibition, which may indicate independent effects that are not restricted to the primary target. In conclusion, some biochemical parameters responded differentially to glyphosate application in susceptible and resistant soybean cultivars, mainly when CAT activity in roots and total soluble amino acid contents in leaves are considered. In general, the total soluble amino acid content increased after glyphosate application, which might be responsible for reducing oxidative damage. Although glyphosate does not exert its main herbicide action by oxidative stress, evidence, such as increased activities of CAT, GOPX and APX indicates ROS generation and subsequent antioxidant responses after glyphosate treatment. The differential response of CAT activity in roots seemed to be dependent on the glyphosate susceptibility of cultivars. Furthermore, the study of antioxidant enzymes allowed the identification of other implicit mechanisms in mode of action of glyphosate.

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Acta Physiol Plant (2008) 30:469–479 Acknowledgments This work was funded by the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP, Grant no. 04/ 08444-6). The authors also thank the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq-Brazil) for the fellowship and scholarship, and the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES-Brazil) and Secretaria de Polı´ticas Universitarias (SPU-Argentina) for scholarship. We also would like to thank Dr. Diego Soldini (INTA-Marcos Juarez) and Dr. Valdemar L. Tornisielo (CENA, USP, Piracicaba) for the plant material used in present work and Prof. Peter J. Lea (University of Lancaster, UK) for the critical reading of the manuscript.

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