Elevated antioxidant response and induction of tau-class glutathione S-transferase after glyphosate treatment in Vigna radiata (L.) Wilczek

Pesticide Biochemistry and Physiology 99 (2011) 111–117

Contents lists available at ScienceDirect

Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest

Elevated antioxidant response and induction of tau-class glutathione S-transferase after glyphosate treatment in Vigna radiata (L.) Wilczek Mahesh Basantani a, Alka Srivastava a,⇑, Somdutta Sen b a b

In Vitro Culture and Plant Genetics Unit, Department of Botany, University of Lucknow, Lucknow, India The Centre for Genomics Application, New Delhi, India

a r t i c l e

i n f o

Article history: Received 9 July 2010 Accepted 9 November 2010 Available online 24 November 2010 Keywords: Affinity chromatography Herbicide Glyphosate Vigna radiata Tau GST Antioxidant response

a b s t r a c t Glyphosate (N-(phosphonomethyl) glycine) is a broad-spectrum herbicide, acting on the shikimic acid pathway inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), thus obstructing the synthesis of tryptophan, phenylalanine, tyrosine and other secondary products. It has also been reported to generate oxidative stress which influences the antioxidant response of target plants. The effect of glyphosate application on total protein, CAT, POD and GST activities was investigated and elevated expression of the oxidative stress enzymes was obtained after glyphosate treatment. Tau-class GSTs are plant-specific, and are chiefly involved in xenobiotics and oxidative stress metabolisms. Many herbicides and safeners have been known to selectively induce tau-class GSTs in different plant species. Here we also report the induction of tau-class GSTs after glyphosate treatment in the seedling roots of two Vigna radiata varieties (PDM11 and PDM54). GSH-agarose affinity chromatography and mass spectrometry revealed that the tau-class GSTs induced in the two varieties were different; the tauclass GSTs present in the untreated controls were also different in the two varieties. The present study highlights the elevated antioxidant response, the induction of tau-class GST and the genotypic variation in the type of tau-GST in control and glyphosate treated varieties of V. radiata. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Glyphosate is used to control several weeds, which include grasses, sedges and other broad-leaved weeds. It acts as a potent inhibitor of shikimic acid pathway for the biosynthesis of aromatic amino acids. It is a competitive inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) with respect to phosphoenolpyruvate (PEP) and noncompetitive with respect to shikimate-3-phosphate (S3P) [1]. The inhibition of EPSP synthase leads to killing of plants due to scarcity of the three aromatic amino acids. Glyphosate is considered to be less toxic to animals due to the absence of shikimic acid pathway in animals, but certain studies have shown the detrimental effects of this herbicide on animal systems: it leads to cell cycle dysfunction [2], inhibits global transcription [3] and has teratogenic potential [4]. Catalases (CAT, EC 1.11.1.6) involved in herbicide tolerance, or an increase in CAT activity during herbicide exposure, have been reported from several plant species [5–7]. A few earlier reports too have shown an increase in CAT activity in Vigna radiata plants after herbicide exposure. 2-Benzoxazolinone (BOA) was found to cause oxidative stress in mung bean plants, which responded by an increase in the activity of ROS scavenging enzymes like CAT ⇑ Corresponding author. E-mail address: [email protected] (A. Srivastava). 0048-3575/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2010.11.007

and superoxide dismutase (SOD, EC 1.15.1.1) in the root and leaf tissues [8]. Sergiev et al. [9] demonstrated that catalase activity was increased after 6 and 10 days of glyphosate application in maize plants. Cañal et al. [10] could demonstrate the activation of different peroxidase (POD, EC 1.11.1.7) isozymes at different glyphosate concentrations in Cyperus esculentus L. plants. Glutathione S-transferase (GST, E.C. 2.5.1.18) act as major phase II detoxification enzymes like glycosyl transferases. The GST gene family in plants is represented by eight distinct classes: seven of these (phi, tau, zeta, theta, lambda, DHAR, TCHQD) are soluble, and one is microsomal [11]. The phi and tau classes are plant specific and the most abundant. Several members from both the classes involved in diverse metabolic processes are very well characterized from different plant species [12]. GSTs from both the phi and tau classes are induced upon exposure to herbicides and protect plants from herbicide injury. Tau-class GSTs have been mainly studied in relation to their role in xenobiotics and oxidative stress metabolisms. Roxas et al. [13] emphasized upon the role of tau-class GSTs in chilling and oxidative stress. They showed that tobacco seedlings overexpressing a tau-class GST, having a high GPOX activity, were more tolerant to oxidative stress. This was one of the first reports demonstrating the importance of tau-class GSTs in oxidative stress metabolism. Kilili et al. [14] identified five tau-class GSTs playing major role in oxidative stress in tomato. Moons [15] showed that tau-class GSTs are induced upon exposure to heavy metals as well. Tau GSTs

112

M. Basantani et al. / Pesticide Biochemistry and Physiology 99 (2011) 111–117

involved in herbicide metabolism have been identified in Triticum tauschii [16], soybean [17], wheat [18] etc. The effect of glyphosate on plants, its toxicity and tolerance, has been studied mainly in relation to EPSPS. There is dearth of literature on the role of GSTs in glyphosate metabolism. However, GST activity is found to be induced by glyphosate, and there are only a few reports demonstrating this induction [19,20,9]. This increase in GST activity enhances the tolerance of crop plants towards glyphosate. Moreover, the studies carried out so far have not shed any light on the class of GST involved in glyphosate metabolism. The aim of the present study was to identify the effects of glyphosate herbicide on V. radiata, to understand the role of antioxidant enzymes in the detoxification of glyphosate, and, most importantly, to purify and recognize the class of GSTs induced after glyphosate treatment. The activity of antioxidant enzymes CAT and POD was also measured. Moreover, the glyphosate-induced GST has been purified in order to be assigned to a particular class by using mass spectrometry analysis of the purified protein. 2. Materials and methods 2.1. Herbicide treatment The seeds of the V. radiata varieties PDM11 and PDM54 were procured from Indian Institute for Pulse Research, Kanpur. The chemicals used were purchased from HiMedia, Mumbai, India. GSH-agarose and protein molecular weight marker (Medium Range) were supplied by Bangalore Genei, Bangalore, India. The seeds were soaked for 24 h in increasing isopropylamine glyphosate (Monsanto, India) concentrations prepared in water. The seeds of both the varieties were treated with glyphosate solution (20 mL) at concentrations 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM. Control seeds were treated with water only. After soaking, the seeds were placed on moist filter paper in petridishes for germination. The germination and survival percentages were observed and seedling root length was measured. The survival percentage was measured after 12 days of germination. The roots were harvested after 12 days for protein extraction, GST purification and enzyme assays. 2.2. Protein isolation and estimation After germination, the seedling roots were harvested for protein extraction and GST purification. The tissue was ground to a fine powder in liquid nitrogen and homogenized in the extraction buffer (0.2 M Tris–HCl pH 7.8, 1 mM EDTA, 20% glycerol and 2 mM PMSF). The protein samples were quantified by the Bradford method [21], using BSA as the standard. 2.3. GST enzyme assay The GST activity was measured spectrophotometrically according to Habig et al. [22]. The final assay mixture consisted of 50 mM phosphate buffer pH 6.5, 1 mM CDNB, 1 mM GSH, 0.5 mM EDTA and the seedling root extract containing 100 lg protein. The final reaction volume was made to 2.5 mL with water. The reaction was started by the addition of the root extract. The reaction was monitored spectrophotometrically at 340 nm. The GST activity was expressed as as lmol min 1 mg 1 protein. It was the measure of DNP–GS complex formed. 2.4. CAT enzyme assay CAT activity was measured according to Euler and Josephson [23]. A 2.5% protein extract (in the extraction buffer) was prepared.

2 mL citrate phosphate buffer (pH 7.0), 1 mL water and 1 mL enzyme extract were taken in two sets of test tubes. One of the sets was labeled blank and the other as sample. In the sample, 1 mL H2O2 was added and exactly after 10 min the reaction was stopped by adding 2 mL 4 N H2SO4. In the blank, 2 mL 4 N H2SO4 was added first and then 1 mL H2O2 was added. The reaction mixtures were titrated against 0.01 N KMnO4 till the end point was (light pink) reached. The catalase activity was expressed as mL H2O2 decomposed g 1 fresh weight of tissue. 2.5. POD enzyme assay POD activity was estimated by a modified method of Luck [24]. 2.5% protein extract was prepared. Two milliliters citrate phosphate buffer (pH 6.0), 1 mL H2O2 and 1 mL p-phenylenediamine were taken in two sets of test tubes; one was blank and the other was labeled sample. In the sample, 1 mL enzyme extract was added and allowed to stand for 10 min. After 10 min 2 mL 4 N H2SO4 was added to stop the reaction. In the blank, 2 mL 4 N H2SO4 was added and then 1 mL enzyme extract was added. All the reaction mixtures were allowed to stand for 1 h at room temperature in the dark. After 1 h A485 was measured with the spectrophotometer. The enzyme activity was expressed as the difference in OD between blank and sample per gram fresh weight (DOD g 1 fresh wt). 2.6. GSH-agarose affinity chromatography GST enzyme was purified according to DeRidder et al. [25]. The tissue was ground to a fine powder in liquid nitrogen and homogenized in the extraction buffer containing 20 mM Tris–HCl pH 7.8, 1 mM EDTA and 5 mM DTT (buffer A). The homogenate was filtered through two layers of cheesecloth and centrifuged at 14,000 rpm for 20–30 min at 4 °C. GST purification using GSH-agarose affinity chromatography was conducted according to DeRidder et al. [25] with slight modifications. The GSH-agarose affinity column was run using gravity flow. The affinity column was first equilibrated with five column volumes of buffer A. After equilibration the protein extract was loaded on the matrix. After protein loading, the matrix was washed with 3–5 column volumes of buffer A. GST was eluted with three column volumes of buffer B (20 mM Tris– HCl pH 7.8, 1 mM EDTA and 10 mM GSH). The purified GST protein was run on the 12% polyacrylamide gel (Fig. 1). The band corresponding to GST (molecular weight approx. 27 kDa) was cut from the gel and submitted to The Centre for Genomic Applications (TCGA), Proteomics Facility, New Delhi, for identification of the protein by mass spectrometry. 2.7. Mass spectrometry The protein band corresponding to 27 kDa was cut from the gel and subjected to in-gel trypsin digestion and then MS/MS analysis. The digestion and MS/MS analysis were carried out at the proteomics facility of The Centre for Genomic Application (TCGA), New Delhi. The sample preparation was accomplished according to Shevchenko et al. [26] with slight modifications. The gel piece was first washed with 500 lL water. The silver stained gel piece was destained by immersing them in 1:1 solution of potassium ferricyanide (15 mM) and sodium thiosulphate (50 mM) for 10 min. After the destaining the gel piece was washed twice with 500 lL water to remove reducing agents. The gel piece was equilibrated with 200 mM/L of NH4CO3, and reduced using 150 ll of 10 mM DTT in 100 mM ammonium bicarbonate, and 5% acetonitrile (ACN) for 1 h at 55 °C. The gel piece was rehydrated with 100 mM NH4CO3 for 10 min. and then dehydrated in ACN for 20 min.

113

M. Basantani et al. / Pesticide Biochemistry and Physiology 99 (2011) 111–117

3.1. Growth parameters 3.1.1. Germination and survival percentage The germination percentage was high for controls in both the varieties, being highest for PDM54. It was 96.6% for PDM54, and 95% for PDM11. The treatment with glyphosate led to a decrease in germination percentage. Treatment with glyphosate decreased the germination percentage in PDM11 at all the concentrations of glyphosate. In PDM54, germination percentage decreased slightly at 2 and 4 mM treatments, was same as control at 6 mM, enhanced at 8 mM and again decreased at 10 mM (Table 1). The seedling survival showed a marked difference in the two varieties. The percentage survival of seedlings at the highest glyphosate concentration (10 mM) was 48.3% in PDM11, and 31.6% in PDM54. The percentage survival showed a gradual decrease with increasing glyphosate concentrations. In PDM11 maximum survival was in control and minimum at 10 mM with a difference of 43.3%. In PDM54, a difference of 58.4% was obtained between maximum survival in control and minimum at 10 mM.

Fig. 1. Silver stained 12% polyacrylamide gel showing GSH-agarose affinity purified GST (marked with arrow; approx 27 Kda). M = Protein molecular weight marker.

The alkylation was carried out in dark with 50 mM iodoacetamide in 100 mM NH4CO3. Subsequently, the gel piece was washed in100 mM NH4CO3 for 10 min, and ACN for 20 min. The gel piece was dried in speed vac. For trypsin digestion of the protein the gel piece was swollen in digestion buffer, 50 mM NH4CO3 with enzyme trypsin (Promega), on ice for 45 min. Thereafter the gel piece was incubated at 37 °C for 16 h in 50 mM NH4CO3. For the extraction of peptides, after 16 h the supernatant of the digestion solution was collected. The gel piece was extracted once with 20 mM NH4CO3 and the supernatant was collected. These supernatants were pooled. The gel plugs were extracted twice with 1% TFA in 50% ACN, and extracted once with ACN for 20 min. The supernatants were collected. All the supernatants were combined and concentrated in speed vac. After concentrating they were resuspended in 10–20 lL 1% TFA. For LC– MS analysis, 6 lL of the sample was injected in 2D nano LC–MS ESI ion trap (Agilent technologies, USA). Masses were scanned from m/z 300 to 3000 and the charge state of each peptide was +2 and +3. For peptide mass fingerprinting, the data was analyzed using the Mascot MS/MS ion search (http:// www.matrixscience.com). The search parameters were: fixed modifications carbamidomethyl (C), variable modifications oxidation (M), mass values monoisotopic, peptide mass tolerance ±2 Da, fragment mass tolerance ±0.8 Da and maximum missed cleavages 1. Probability based MOWSE score was calculated in terms of ion score 10Log (P), where P is the probability and observed match was considered as a random event. Protein scores were derived from ions as a non-probabilistic basis for ranking protein hits and proteins identified by LCMS/MS were in the expected size based on its position in the gel. The peptide sequences were also subjected to BLAST analysis [27]. The protein BLAST (blastn) for short, nearly exact matches was performed. 3. Results The present investigation shows the effect of glyphosate on growth and GST activity, and induction of tau-class GSTs after glyphosate treatment in two V. radiata varieties PDM11 and PDM54. Taking a cue from a handful of reports available we measured changes in GST, CAT and POD activity as well after glyphosate treatment.

3.1.2. Fresh weight The fresh weight of germinated V. radiata seedlings was taken and it was found to decrease after the treatment. The fresh weight of control seedlings was 0.22 ± 0.04 g in PDM11, and 0.18 ± 0.04 g in PDM54. It showed a gradual decrease with increasing glyphosate concentration (Table 2). At the highest glyphosate concentration, there was approximately 2-fold reduction in the fresh weight in both the varieties. It was 0.09 ± 0.02 g in PDM11, and 0.10 ± 0.02 g in PDM54. 3.1.3. Seedling root length The root length of germinated seedlings was also measured. It showed significant differences in the two varieties (Table 3). The root length of control seedlings was 11.2 ± 2.25 cm in PDM11, and 7.83 ± 1.15 cm in PDM54. But glyphosate treatment resulted in a decrease in root length. The decrease in the two varieties was not the same. The root length at 2 mM glyphosate concentration was 4.92 ± 0.79 cm in PDM11 and 4.80 ± 0.57 cm in PDM54. At the highest glyphosate concentration (10 mM), the root length of

Table 1 Effect of glyphosate on germination and survival of V. radiata Varieties. Glyphosate (mM)

Control 2 4 6 8 10

Germination percentage

Survival percentage

PDM 11

PDM 54

PDM 11

PDM 54

95 86.6 85 90 83.3 85

96.6 93.3 93.3 96.6 98.3 91.6

91.6 73.3 71.6 71.6 58.3 48.3

90 81.6 80.0 68.3 48.3 31.6

Table 2 Fresh weight of the seedlings after glyphosate treatment. Glyphosate (mM)

Control 2 4 6 8 10

Seedling fresh weight (g)AM ± SD PDM 11

PDM 54

0.22 ± 0.04 0.12 ± 0.01** 0.10 ± 0.01** 0.10 ± 0.02** 0.10 ± 0.02** 0.09 ± 0.02**

0.18 ± 0.04^^ 0.15 ± 0.03**,^^ 0.14 ± 0.02**,^^ 0.12 ± 0.01^^,** NS0.12 ± 0.02** NS0.10 ± 0.02**

NS = Not significant (p > 0.05). ** Highly significant (p < 0.01); comparison between treatment in the variety. ^^ Highly significant (p < 0.01); comparison between variety for each treatment.

114

M. Basantani et al. / Pesticide Biochemistry and Physiology 99 (2011) 111–117

Table 3 Effect of glyphosate on seedling root length. Glyphosate (mM)

Seedling root length (cm) AM ± SD PDM 11

PDM 54

Control 2 4 6 8 10

11.2 ± 2.25 4.92 ± 0.79** 4.10 ± 1.02** 3.94 ± 0.68** 3.89 ± 0.61** 3.13 ± 1.00**

7.83 ± 1.15^^,** NS4.80 ± 0.57** NS4.62 ± 0.66** NS3.89 ± 0.49** NS3.86 ± 0.66** NS2.75 ± 0.56**

NS = Not significant (p > 0.05). ⁄ Significant (p < 0.05); comparison between treatment in the variety. ** Highly significant (p < 0.01); comparison between treatment in the variety. ^^ Highly significant (p < 0.01); comparison between variety for each treatment.

PDM11 seedlings was 3.13 ± 1.00, and of PDM 54 seedlings it was 2.75 ± 0.56 cm. At 10 mM, PDM11 registered a 3.5-fold reduction in the root length as compared to control; in PDM54 it was 2.8fold. 3.2. Biochemical parameters 3.2.1. GST activity GST activity showed an increase in the glyphosate treated seedling roots in both the varieties. It was an indication that glyphosate was inducing the GST activity. However, the fold increase was found to be different in the two. This enhanced GST activity was helpful in the survival of seedlings at low glyphosate concentrations. The GST activity was lower in the PDM11 and PDM54 control seedlings (0.16 ± 0.03 lmol min 1 mg 1protein in PDM11 and 0.25 ± 0.03 lmol min 1 mg 1protein in PDM54). But glyphosate treatment led to a significant increase in the GST activity in the PDM11 and PDM54 seedling roots (Table 4). At 2 mM glyphosate concentration PDM11 showed a 3.6-fold increase, whereas in PDM54, the GST activity showed a 2-fold increase, which was lower than PDM11. This difference in the induction pattern may explain the differences observed in the survival percentage of the seedlings of the two varieties. The PDM11 seedlings were better able to survive glyphosate exposure by the induction of GST enzyme. We cannot explain through these studies whether the induction was at the transcriptional level or translational level. At the 4 mM glyphosate concentration there was a further increase in GST activity in PDM11 (0.66 ± 0.02 lmol min 1 mg 1protein) and PDM54 seedlings (0.58 ± 0.03 lmol min 1 mg 1protein) which was 4.1-fold and 2.3-fold of control, respectively. The changes were found to be significant between all the treatments, and amongst the varieties according to the Newman Keuls test. 3.2.2. CAT activity Based on the work of Sergiev et al. [9] where it was reported that glyphosate causes oxidative stress, we measured the activity of two

antioxidant enzymes, CAT and POD. In the control seedling roots, the CAT activity was 116.67 ± 25.17 mL H2O2 decomposed g 1 fresh weight in PDM11, and 136.67 ± 32.15 mL H2O2 decomposed g 1 fresh weight in PDM54 (Table 4). The CAT activity was clearly found to increase after glyphosate treatment in both the varieties. At 4 mM glyphosate dose, the activities were 223.33 ± 5.77 mL H2O2 decomposed g 1 fresh weight in PDM11, 226.67 ± 15.28 mL H2O2 decomposed g 1 fresh weight in PDM54. According to Newman Keuls test, the increase in activity across the treatments was significant in the two varieties. The fold change in the two varieties was different (Table 4). 3.2.3. POD activity POD, an antioxidant enzyme, has also been implicated in herbicide metabolism in plants. In PDM11, POD activity was 22.07 ± 3.65 DOD g 1 fresh weight in the control seedlings; in PDM54 control seedlings, peroxidase activity was 60.13 ± 5.65 DOD g 1 fresh weight. POD activity too was found to increase after glyphosate treatment in all the two varieties. The POD activity at 4 mM was 61.70 ± 5.56 DOD g 1 fresh weight in PDM11, and 105.23 ± 16.78 DOD g 1 fresh weight in PDM54. The increase in POD activities, amongst all the treatments, and across the varieties, was found to be highly significant as analysed by Newman Keuls test (Table 4). There was a 2.7-fold increase in the activity at 4 mM as compared to control in PDM11. In PDM54, the increase was 1.7-fold at 4 mM as compared to control. 3.3. GSH-agarose affinity purification of GSTs There is only a small number of reports that have shed light on the role and induction of GSTs after glyphosate applications, and none of these describe the GST class involved in glyphosate detoxification. In order to identify the GST class involved in glyphosate detoxification in V. radiata, we carried out purification of GSTs from the seedling roots by GSH-agarose affinity chromatography. The protein identity was confirmed by mass spectrometry. 3.3.1. GST peptides identified in PDM11 In PDM11, GSH-agarose affinity chromatography led to the identification of one GST protein in the control seedling roots. The protein identity was confirmed on the basis of two peptides, WASPFSNR and DELFAFFK, recognized by LCMS/MS. These two peptides showed homology with a soybean GST, GST13 (NCBI accession number AAG34803), which belongs to the tau class. Therefore, it is concluded that the GST present in the seedling roots belongs to the tau class, tentatively named VrGSTUA (Mascot score = 51). However, at 2 mM glyphosate concentration, the affinity chromatography revealed the presence of three GSTs in the seedling roots, one of them being VrGSTUA. But VrGSTUA was represented by three peptides, WASPFSNR, DELFAFFK and EGLPPRDELFAFFK, as against the two peptides in the control. Of the other two GSTs, one showed homology with soybean GST 17 (a tau GST; NCBI accession

Table 4 GST, CAT and POD activity of the seedling roots after glyphosate treatment. Glyphosate (mM)

Control 2 4

GST activity (lmol min 1 mg

1

Catalase activity (mL H2O2 decomposed g of tissue)

PDM 11

PDM 54

PDM 11

PDM 54

PDM 11

PDM 54

0.16 ± 0.03 0.58 ± 0.02** 0.66 ± 0.02**

^^0.25 ± 0.03 ^^0.50 ± 0.02** ^^0.58 ± 0.03**

116.67 ± 25.17 176.67 ± 30.55** 223.33 ± 5.77**

NS136.67 ± 32.15 NS 183.33 ± 15.28* NS 226.67 ± 15.28**

22.07 ± 3.65 40.33 ± 8.98* 61.70 ± 5.56**

^^60.13 ± 5.65 ^^74.2 ± 2.56* ^^105.23 ± 16.78**

NS = Not significant (p > 0.05). * Significant (p < 0.05); comparison between treatment in the variety. ** Highly significant (p < 0.01); comparison between treatment in the variety. ^^ Highly significant (p < 0.01); comparison between variety for each treatment.

fresh weight

Peroxidase activity (DOD g tissue)

1

)protein

1

fresh weight of

M. Basantani et al. / Pesticide Biochemistry and Physiology 99 (2011) 111–117

number AAG34807), represented by two peptides SNLLLQLNPVHK and GIEYEYIEEDIFNK; and the other showed homology with a putative A. thaliana tau GST (NCBI accession number AAF71800), represented by a single peptide TRIALEEK (The score of this peptide was below 43). These two GSTs were tentatively named VrGSTUB and VrGSTUC, respectively (Table 5). BLAST [30] search of all these peptides for short, nearly exact matches on the NCBI database showed similarity with other tau GSTs from different plant species further confirming that they are indeed tau GSTs. 3.3.2. GST peptides identified in PDM54 In PDM54, the peptides identified were different from those in PDM11. Three peptides were identified in the control: QLYEIASPNYTGK, WSQEFINHPVVK, and WSQEFVNHPVIK. QLYEIASPNYTGK showed homology with Thlaspi caerulescens GST. WSQEFINHPVVK was similar to a Glycine max tau GST, GST7 (AAG34797). WSQEFVNHPVIK was homologous to a Lotus japonicus GST. Thus, GST proteins identified in the control were tentatively named VrGSTUD, VrGSTUE, and VrGSTUF, respectively. At 2 mM glyphosate, a total of 6 peptides were identified. Three of them were the same as in control. The other 3, present at 2 mM only, were WASPFSNR, ATEAMAGEEILR, and LLAAWATMVFKGK. WASPFSNR is the same as identified in PDM11 and similar to a soybean GST, GST13 (NCBI accession number AAG34803), the GST tentatively named VrGSTUA. This is the only one similar to PDM11 peptides. The peptides ATEAMAGEEILR and LLAAWATMVFKGK were homologous to Alopecurus myosuroides GST (Table 6). This was tentatively named VrGSTUG. BLAST search of all these peptides for short, nearly exact matches on the NCBI database revealed similarity with other tau GSTs, confirming the identity of the purified GSTs as belonging to the tau class. QLYEIASPNYTGK was found to be similar to putative rice GST (NCBI accession number BAD28950). WSQEFINHPVVK showed similarity with 4 tau-class GSTs of G. max, GST13, GST6, GST8, and GST11, and one Phaseolus acutifolius GST. This peptide also showed homology with two auxin-regulated proteins, one from V. radiata (NCBI accession no AAA87183), and the other from G. max (NCBI accession number AAA33943). WSQEFVNHPVIK showed homology with G. max tau GSTs, GST13, GST7, GST8 and GST6. It was also similar to P. acutifolius GST and an auxin-regulated protein of V. radiata, and G. max like WSQEFINHPVVK. The peptides, ATEAMAGEEILR and LLAAWATMVFKGK, found in the 2 mM roots, showed similarity with A. myosuroides GST only.

Table 5 GST peptide sequences identified in PDM11 seedling root tissue protein. S. No.

Peptide sequence

Sample (mM)

1 2 3 4 5 6

WASPFSNR DELFAFFK EGLPPRDELFAFFK SNLLLQLNPVHK GIEYEYIEEDIFNK TRIALEEK

Control, 2 Control, 2 2 2 2 2

Table 6 GST peptide sequences identified in PDM54 seedling root tissue protein. S. No.

Peptide sequence

Sample (mM)

1 2 3 4 5 6

QLYEIASPNYTGK WSQEFINHPVVK WSQEFVNHPVIK WASPFSNR ATEAMAGEEILR LLAAWATMVFKGK

Control, 2 Control, 2 Control, 2 2 2 2

115

4. Discussion Many previous reports have dealt with the effects of glyphosate on plant growth, survival and changes in antioxidant enzyme activity. It was found that preharvest glyphosate applications lead to a general decrease in germination percentage. It has been earlier reported that preharvest application of glyphosate in pea reduced seed germination, seedling emergence, and shoot fresh weight [28]. Glyphosate has also been known to reduce leaf dry matter accumulation in Phaseolus vulgaris L. [29]. Our studies have also found a general decrease in germination and survival percentages, fresh weight and root length of the two V. radiata varieties, although the varieties were found to respond differently to the herbicide. PDM11 was better able to survive high glyphosate doses, as it showed 50% or less survival at 10 mM glyphosate concentration (the highest dose used in the present investigation), whereas it was 8 mM in PDM54. A good many reports have clearly demonstrated an increase in GST activity after treatment with herbicides like terbuthylazine, metolachlor, fluorodifen and fenoxaprop [30,31] and acifluorfen and fomesafen [17]. However, there are only a few instances where increase in GST activity after glyphosate exposure has been studied, and there is a scarcity of literature concerning this aspect of GST enzyme superfamily [32]. Uotila et al. [19] observed significantly increased GST activities in wheat, pea and maize tissues. Cataneo et al. [33] showed an increase in GST activity in maize shoots after glyphosate treatment. Jain and Bhalla-Sarin [20] observed differences in the GST induction patterns after glyphosate treatment in three groundnut cultivars (JL24, CO2, and TMV2). Sergiev et al. [9] reported a considerable gradual increase in GST activity in maize plants after glyphosate application. Our findings clearly show an induction of GST activity after glyphosate treatment, and further corroborate findings of other workers. Moreover, this induction was genotype dependent i.e. the two V. radiata varieties responded differently to glyphosate, and fold induction was different for the two. PDM11 showed a higher fold induction in GST activity as compared to PDM54. Similar findings demonstrating that chemical biocides viz. herbicides, pesticides, insecticides induce the activity of GST enzymes, and the in vivo amount and induction vary in different genotypes (barley, wheat, lentil, and chickpea), have been reported [34]. We also measured the activity of two antioxidant enzymes, CAT and POD, after glyphosate treatment. CATs are involved in the metabolism of oxidative stress causing herbicides and protect plants from the stress generated by herbicide overdoses. CATs involved in herbicide tolerance, or an increase in CAT activity during herbicide exposure, have been reported from several plant species [5–7]. In our experiments too, CAT activity was found to increase after glyphosate treatment. However, the fold increase was different in the two varieties. The activity increased by 1.9fold at 4 mM as compared to control in PDM11, and by 1.6-fold in PDM54. A few earlier reports too have shown an increase in CAT activity in V. radiata plants after herbicide exposure. A herbicide 2-benzoxazolinone (BOA) was found to cause oxidative stress in mung bean plants, which responded by an increase in the activity of ROS scavenging enzymes like CAT and SOD in the root and leaf tissues [8]. Sergiev et al. [9] demonstrated that CAT activity was increased after 6 and 10 days of glyphosate application in maize plants. POD upregulation after herbicide exposure has been demonstrated in wheat [35], tobacco [36] , and many other plant species. Like CAT, POD activity was also found to increase after glyphosate treatment, and the fold increase was different in the two V. radiata varieties. There was 2.7-fold increase in activity at 4 mM as compared to control in PDM11, and 1.7-fold in PDM54. POD activity

116

M. Basantani et al. / Pesticide Biochemistry and Physiology 99 (2011) 111–117 Table 7 Correlation analysis between the activities of GST, CAT and POD.

CAT POD

GST

CAT

0.95** 0.79**

– 0.58ns

NS = Not significant (p > 0.05). ** Significant (p < 0.01).

has been found to increase after glyphosate treatment in other plant species as well. Cañal et al. [10] could demonstrate the activation of different POD isozymes at different glyphosate concentrations in C. esculentus L. plants. They identified three POD species F1, F2, and F3 at pl 3.8, 4.4 and 4.8, respectively. They showed preferential expression at different glyphosate doses. The findings of the present study clearly indicate that glyphosate causes a general decrease in the growth and survival of V. radiata. It has been reported that the herbicide causes oxidative stress and to overcome this stress the enzymes of oxidative stress metabolism are induced [9]. CAT and POD, the two important antioxidant enzymes are induced following glyphosate exposure. This induction was helpful in overcoming oxidative stress caused by the herbicide, and better plant survival at low herbicide concentrations. The correlation analysis between the three enzyme activities revealed that: (i) the increase in GST and CAT activities were significantly and positively correlated with each other; (ii) the GST and POD activities were also significantly and positively correlated. However, no correlation was observed between increase in CAT and POD activities (Table 7). The most significant finding of the present study was the induction of tau-class GSTs after glyphosate exposure. This is the first report, to the best of our knowledge, where GST class involved in glyphosate detoxification has been identified. Tau GSTs involved in herbicide metabolism have been identified in other plant species too. Glyphosate has been known to cause oxidative stress in plants, and they can overcome this stress by the induction of GST activity. However, this is not due to the formation and sequestration of GSH-glyphosate conjugate [9], rather the induced GSTs could increase the accumulation of GSSG as occurs during oxidative stress tolerance [13].It is seen that in maize GSTFs are the predominant class of expressed GST, but in soybean, there is a preponderance of GSTUs and this difference accounts for the differential detoxification of diverse classes of herbicides in the two crops [37]. Our experiments demonstrate that in V. radiata the constitutively expressed GST belongs to the tau class as in soybean, also a legume. During the BLAST search it was found that WSQEFINHPVVK, EGLPPRDELFAFFK, SNLLLQLNPVHK and GIEYEYIEEDIFNK peptide sequences also showed homology with certain auxin inducible tau GSTs. A 2,4-D-inducible GST was also found to show increased accumulation after flumetsulam and metsulfuron (acetohydroxyacid synthase-inhibiting herbicides) treatment in Medicago truncatula roots [38]. On the basis of these similarities it could be proposed that perhaps similar GSTs are induced upon exposure to both glyphosate (as found in the present study) and auxins, and some components of glyphosate detoxification overlap with auxin metabolism. Auxin has been known to generate reactive oxygen species (ROS) in the quiescent centre (QC) of maize roots and these ROS are instrumental in establishment and regulation of QC; and a glutathione-dependent developmental pathway is required to mediate redox stress generated by auxin during cell division in the root meristem [38]. Therefore, considering the fact that glyphosate causes oxidative stress in plants [9], it could be proposed that glyphosate exposure might lead to upregulation of auxin-inducible GSTs. The experiments are in progress in our laboratory to elucidate these possibilities.

Acknowledgement MB is grateful to CSIR for financial assistance.

References [1] G. Coruzzi, R. Last, Amino. Acids, in: B.B. Buchanan, W. Gruissem, R.L. Jones (Eds.), Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists, 2000, p. 382. [2] J. Marc, M. Odile, R. Bellé, Glyphosate-based pesticides affect cell cycle regulation, Biol. Cell 96 (2004) 245–249. [3] J. Marc, M. Le Breton, P. Cormier, J. Morales, R. Bellé, M. Odile, A glyphosatebased pesticide impinges on transcription, Toxicol. Appl. Pharmacol. 203 (2005) 1–8. [4] E. Dallegrave, F. DiGiorgio Mantese, R.S. Coelho, J.D. Pereira, P.R. Dalsenter, A. Langeloh, The teratogenic potential of the herbicide glyphosate- RoundupÒ in Wistar rats, Toxicol. Lett. 142 (2003) 45–52. [5] S. Jung, S. Chon, Y. Kuk, Differential antioxidant responses in catalase-deficient maize mutants exposed to norflurazon, Biol. Plant 50 (2006) 383–388. [6] S. Jung, Effect of norflurazon on responses of superoxide dismutase and catalase in a standard maize inbred line and superoxide dismutase mutant, J. Pestic. Sci. 28 (2003) 281–286. [7] C.M. Radetski, S. Cotelle, J. Férard, Classical and biochemical endpoints in the evaluation of phytotoxic effects caused by the herbicide trichloroacetate, Environ. Exp. Bot. 44 (2000) 221–229. [8] D.R. Batish, H.P. Singh, N. Setia, S. Kaur, R.K. Kohli, 2-Benzoxazolinone (BOA) induced oxidative stress, lipid peroxidation and changes in some antioxidant enzyme activities in mung bean (Phaseolus aureus), Plant Physiol. Biochem. 44 (2006) 819–827. [9] I.G. Sergiev, V.S. Alexieva, S.V. Ivanov, I.A. Moskova, E.N. Karanov, The phenylurea cytokinin 4PU-30 protects maize plants against glyphosate action, Pestic. Biochem. Physiol. 85 (2006) 139–146. [10] M.J. Cañal, R.S. Tamés, B. Fernández, Peroxidase and polyphenol oxidase activities in Cyperus esculentus leaves following glyphosate applications, Physiol. Plant. 74 (1988) 125–130. [11] R. Edwards, D.P. Dixon, Plant glutathione transferases, Methods Enzymol. 401 (2005) 169–186. [12] M. Basantani, A. Srivastava, Genotypic variation in GST induction by herbicide glyphosate in Vigna radiata, in: V. Padmaja, B. Subba Rao, A. Arundhati (Eds.), Genome Characterization, Basics and Applications, Aavishkar Publishers, Jaipur, 2007, p. 333. [13] V.P. Roxas, R.K. Smith Jr, E.R. Allen, R.D. Allen, Overexpression of glutathione Stransferase/glutathione peroxidase enhances the growth of transgenic tobacco seedlings during stress, Nat. Biotechnol. 15 (1997) 988–991. [14] K.G. Kilili, N. Atanassova, A. Vardanyan, N. Clatot, K. Al-Sabarna, P.N. Kanellopoulos, et al., Differential roles of tau class glutathione s-transferases in oxidative stress, J. Biol. Chem. 279 (2004) 24540–24551. [15] A. Moons, Osgstu3 and osgstu4, encoding tau class glutathione S-transferases Are heavy metal- and hypoxic stress-induced and differentially salt stressresponsive in rice roots, FEBS Lett. 553 (2003) 427–432. [16] Q. Zhang, D.E. Riechers, Proteomic characterization of herbicide safenerinduced proteins in the coleoptile of Triticum tauschii seedlings, Proteomics 4 (2004) 2058–2071. [17] C.J. Andrews, I. Cummins, M. Skipsey, N.M. Grundy, I. Jepson, J. Townson, et al., Purification and characterisation of a family of glutathione transferases with roles in herbicide detoxification in soybean (Glycine max L.); selective enhancement by herbicides and herbicide safeners, Pestic. Biochem. Physiol. 82 (2005) 205–219. [18] R. Thom, I. Cummins, D.P. Dixon, R. Edwards, D.J. Cole, A.J. Lapthorn, Structure of a tau class glutathione S-transferase from wheat active in herbicide detoxification, Biochemistry 41 (2002) 7008–7020. [19] M. Uotila, G. Gullner, T. Komives, T, Induction of glutathione S-transferase activity and glutathione level in plants exposed to glyphosate, Physiol. Plant 93 (1995) 689–694. [20] M. Jain, N. Bhalla-Sarin, Glyphosate-induced increase in glutathione Stransferase activity and glutathione content in groundnut (Arachis hypogea L.), Pestic. Biochem. Physiol. 69 (2001) 143–152. [21] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [22] W.H. Habig, M.H. Pabst, W.B. Jakoby, Glutathione S-transferases: the first enzymatic step in mercapturic acid production, J. Biol. Chem. 249 (1974) 7130–7139. [23] H. Von Euler, K. Josephson, Uber katalase I, Leibigs Ann. 452 (1927) 158–187. [24] H. Luck, Peroxidase, in: H.U. Bergmeyer (Ed.), Methods of Enzymic Analysis, Academic Press, New York, 1963, pp. 895–897. [25] B.P. DeRidder, D.P. Dixon, D.J. Beusman, R. Edwards, P.B. Goldsbough, Induction of glutathione S-transferases in Arabidopsis by herbicide safeners, Plant Physiol. 130 (2002) 1497–1505. [26] A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Mass spectrometric sequencing of proteins silver stained polyacrylamide gels, Anal. Chem. 68 (1996) 850–858. [27] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403–410.

M. Basantani et al. / Pesticide Biochemistry and Physiology 99 (2011) 111–117 [28] M.N. Baig, A.L. Darwent, K.N. Harker, J.T. O’Donovan, Preharvest applications of glyphosate affect emergence and seedling growth of field pea (Pisum sativum), Weed Technol. 17 (2003) 655–665. [29] B.J. Brecke, W.B. Duke, Effect of glyphosate on intact bean plants (Phaseolus vulgaris L.) and isolated cells, Plant Physiol. 66 (1980) 656–659. [30] D. Del Buono, M. Micheli, L. Scarponi, A. Standardi, Activity of glutathione Stransferase toward some herbicides and its regulation by benoxacor in nonembryogenic callus and in vitro regenerated tissues of Zea mays, Pestic. Biochem. Physiol. 2 (2006) 61–67. [31] R. Edwards, D.J. Cole, Glutathione transferases in wheat (Triticum) species with activity toward fenoxaprop-ethyl and other herbicides, Pestic. Biochem. Physiol. 54 (1996) 96–104. [32] M. Basantani, A. Srivastava, Plant glutathione transferases — a decade falls short, Can. J. Bot. 85 (2007) 443–456. [33] A.C. Cataneo, G.F.G. Destro, L.C. Ferreira, K.L. Chamma, D.C.F. Sousa, Glutathione S-transferase activity on the degradation of the herbicide glyphosate in maize (Zea mays) plants, Planta Daninha 21 (2003) 307–312.

117

[34] G. Coskun, F. Zihnioglu, Effect of some biocides on glutathione S-transferase in barley Wheat, lentil and chickpea plants, Turkish J. Biol. 26 (2002) 89– 94. [35] M. Wang, Q. Zhou, Effects of herbicide chlorimuron-ethyl on physiological mechanisms in wheat (Triticum aestivum), Ecotox. Environ. Safe. 64 (2006) 190–197. [36] S. Yamato, M. Katagiri, H. Ohkawa, Purification and characterization of a protoporphyrinogen-oxidizing enzyme with peroxidase activity and lightdependent herbicide resistance in tobacco cultured-cells, Pestic. Biochem. Physiol. 50 (1994) 72–82. [37] D.P. Dixon, A.G. McEwen, A.J. Lapthorn, R. Edwards, Forced evolution of a herbicide detoxifying glutathione transferase, J. Biol. Chem. 278 (2003) 23930–23935. [38] P. Holmes, R. Farquharson, P.J. Hall, B.J. Rolfe, Proteomic analysis of root meristems and the effects of acetohydroxyacid synthase-inhibiting herbicides in the root of Medicago truncatula, J. Proteome. Res. (2006) 2309–2316.