ISSN 10214437, Russian Journal of Plant Physiology, 2010, Vol. 57, No. 1, pp. 131–136. © Pleiades Publishing, Ltd., 2010. Published in Russian in Fiziologiya Rastenii, 2010, Vol. 57, No. 1, pp. 139–145.
RESEARCH PAPERS
Alterations in Glutathione Pool and Some Related Enzymes in Leaves and Roots of Pea Plants Treated with the Herbicide Glyphosate1 L. PE. Mitevaa, S. V. Ivanovb, and V. S. Alexievac a
Resbiomed EOOD, 4A Simeonovsko Shouse Blvd. Sofia, Bulgaria; fax: +35929522407; email:
[email protected] b Centre of Food Biology, 1592 Sofia, Bulgaria c Acad. M. Popov Institute of Plant Physiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Received July 29, 2008
Abstract—Our previous studies have demonstrated that application of glyphosate caused oxidative events in young pea and wheat plants. In this work, the changes in the endogenous level of glutathione (total and oxi dized) and the activities of glutathione reductase (GR) and glutathione Stransferase (GST) after treatment with glyphosate were studied in pea plants (Pisum sativum L., cv. Skinado). Glyphosate was applied in two ways: (1) by leaf spraying with 10 mM solution; and (2) in nutrient medium as 0.01 mM solution. Measure ments were made in both leaves and roots. Root and leaf treatments provoked the increase in both total and oxidized glutathione contents. Both types of herbicide application caused activation of GR in treated organs. Slight increase was detected also in untreated roots. It was found that glyphosate application to leaves pro voked strong enhancement in the GST activity in leaves, while its root application stimulated the enzyme activity in the roots. We observed the higher GST activity in the organ directly treated with herbicide. Fur thermore, we suggested that the activated isoforms of GST(s) participated in detoxification of hydrogen per oxide and lipid peroxides. Key words: Pisum sativum glutathione glutathione reductase glutathione Stransferase glyphosate oxida tive stress DOI: 10.1134/S1021443710010188 1
INTRODUCTION
Glutathione ([γ]GluCysGly) is a multifunc tional tripeptide found in plants and animals. It is the main nonprotein, lowmolecular thiol in most organ isms [1, 2]. Glutathione participates in a variety of detox ification, transport, and metabolic processes [3–5]. It is a donor of reducing equivalents in the glutathione– ascorbate shuttle (Halliwell–Asada cycle) [2]. In this process, a reduced form of glutathione becomes oxi dized in order to reduce dehydroascorbate (which is transformed into ascorbate). Restoration from the oxi dized form of glutathione back to its reduced form is cat alyzed by glutathione reductase (GR, EC. 1.8.1.7). Glu tathione participates also in direct peroxide detoxifi cation. In this reaction, reduced glutathione (GSH) reacts with hydrogen peroxide (or another organic peroxide) to yield water (or water and alcohol) and glutathione dimer (GSSG). The process is accom 1 This text was submitted by the authors in English.
Abbreviations: CDNB—1chloro2,4dinitrobenzene; DTNB— 5,5'dithiobis(2nitrobenzoic acid); GR—glutathione reduc tase; GSH—reduced glutathione; GSSG—oxidized glutathione; GST—glutathione Stransferase; PVP—polyvinylpyrrolidone; SOD—superoxide dismutase; TG—total glutathione.
plished by glutathione peroxidase (EC 1.11.1.9) or glutathione Stransferase (GST, EC. 2.5.1.18). Important function of glutathione is its ability to maintain sulfhydryl groups of intracellular proteins in the correct oxidation states [2]. The TG/GSSG ratio is essential for the cell homeostasis and provides infor mation regarding the capability of plants to withstand the oxidative stress [2]. Some authors suggest that the glutathione redox state can be a valuable stress marker in plant ecophysiological studies [6]. Glutathione is responsible for detoxification of potentially harmful molecules, such as pesticides or heavy metals [4]. The process of conjugation can be accomplished spontaneously or in the presence of GST. The important role of GST for detoxification of many herbicides is well known [5, 7]; furthermore, some authors suggest that GST may play a significant role in the process of phytoremediation [8]. Glu tathione also participates in the metabolism of various compounds, including the aromatic organic mole cules responsible for plant color, flavor, and fragrance, storage form of reduced sulfur, etc. [2, 3, 7]. Glyphosate is a nonselective, postemergence her bicide, widely used to eliminate unwanted plants both in agricultural and nonagricultural landscapes [9]. It is
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used in circumstances where total control of vegeta tion is required [10]. Glyphosate penetrates in the plants mainly through their green parts, but it can also be absorbed by the soil colloids for further intake by the plant roots. Glyphosate persistence in the soil var ies widely, from 55 days to 1–3 years, depending on the soil characteristics [9]. In plants, glyphosate inhibits the shikimic acid pathway, preventing the synthesis of three aromatic amino acids. In 1980, Steinrucken and Amrhein [11] discovered that the key enzyme inhib ited by glyphosate is 5enolpyruvylshikimic acid3 phosphate synthase (EC.2.5.1.19). Glyphosate can also affect plant enzymes, which are not related to the shikimic acid pathway. For example, it inhibits some major detoxification enzymes in plants, such as cyto chrome P450 reductase [12]. Moreover, previous experiments have shown that glyphosate caused oxi dative events in pea, wheat, and maize plants [13, 14]. An increase in the MDA and hydrogen peroxide con tents and activation of antioxidant enzymes (SOD, catalase, and guaiacol peroxidase) were observed. The present work studied in details the impact of glypho sate on the glutathione pool and on the activities of GST and GR in various organs (second leaves and roots) of pea plants. MATERIALS AND METHODS Plant material and growth conditions. The experiments were carried out with pea plants (Pisum sativum L., cv. Skinado) purchased from the local market. Seeds were surfacesterilized for 15 min with KMnO4 and soaked in water during 4 h. After germination in dark thermostatic chamber for 3 days, the plants were transferred into a growth chamber (a 12h photope riod, photon flux density of 70 μmol/(m2 s), and tem perature of 25 ± 1°C) and grown in water cultures on the Hoagland–Arnon nutrient medium. The plants were treated at the stage of the third leaf development. Second leaf spraying was performed with 10 mM gly phosate (Roundup, produced by Monsanto, United States). Root treatment was made with 0.01 mM solu tion of glyphosate. The concentration used for the leaf treatment was calculated on the basis of the field rate of the herbicide [15]. The concentration applied to the roots was selected on the basis of our preliminary experiments. Plant material was pea second leaves and roots. All measurements were made on the 2nd, 5th, and 9th days after treatments. Determination of glutathione content. Endogenous glutathione levels were detected according to the enzymatic recycling method of Gronwald et al. [16]. Briefly, 0.3 g of leaves and roots was homogenized in 3 ml of 5% TCA and centrifuged at 15000 g for 15 min to sediment insoluble material. An 1ml aliquot of the supernatant was neutralized with 1 ml of 0.5 M potas sium phosphate buffer (pH 7.5). The standard incuba tion mixture for total glutathione quantification con sisted of sodium phosphate buffer (pH 7.5) containing
EDTA, DTNB, NADPH, 1 units of GR type III (Ald rich Chemical), and the neutralized plant extract. The change in absorption at 412 nm was followed with a Shimadzu UVvisible spectrophotometer (Shimadzu, Japan). Oxidized glutathione was determined through a similar procedure after the trapping of GSH by N ethylmaleimide. The amounts of total glutathione (GSH + GSSG) and GSSG were determined from standard curves prepared with GSH and GSSG. Determination of enzyme activities. Plant material was homogenized in 50 mM potassium phosphate buffer (pH 7.0) with 1% watersoluble PVP and cen trifuged 30 min at 15000 g. Glutathione Stransferase (GST) activity was mea sured spectrophotometrically with the artificial sub strate CDNB (1chloro2,4dinitrobenzene) with glutathione, according to Gronwald et al. [16]. The reaction mixture contained in the volume of 1.5 ml 1.2 ml of 100 mM potassium phosphate buffer (pH 7.0) and 100 μl of the enzyme extract. The reac tion was started by the addition of reduced glutathione (GSH) and CDNB (dissolved in 96% alcohol). The change in absorbance due to GSH–CDNB conjugate formation was spectrophotometrically measured at 25°C for 1 min at 340 nm. A molar extinction coeffi cient of 9.6/(mM cm) [17] was used to calculate enzyme activity, which was corrected for nonenzy matic conjugation. The rate of nonenzymatic conju gation was determined by using the same reaction mixture without the crude plant extract. Glutathione reductase (GR) activity was deter mined by monitoring the reduction of DTNB (5,5' dithiobis(2nitrobenzoic acid)), according to the method described by Smith et al. [18]. The method is based on the increase in the absorbance at 412 nm when DTNB is reduced by GSH. The reaction mix ture contained 50 mM potassium phosphate buffer (pH 7.5), 1 mM EDTA, 50 mM DTNB (dissolved in 96% methanol), 1.5 mM NADPH, and 150 μl of the enzyme extract. Reaction was initiated by adding 7.5 mM GSSG (oxidized glutathione). The increase in the absorbance at 412 nm was recorded at 25°C over a period of 1 min with an UVvisible spectrophotom eter. Activity was calculated using an extinction coef ficient of 13.6/(mM cm). All the spectrophotometri cal assays were conducted with a Shimazu UVVIS spectrophotometer. The protein content was determined by the method of Bradford [19] using BSA as a standard. Statistical analysis. All the experiments were repeated twice with three replicates (n = 6); the data presented are means and their standard errors. RESULTS The total concentration of glutathione in plants varies from 0.1 to 10 mM [1]. Similar concentration range was found in the experiments of Smith [20] and Uotila et al. [21] with pea and other legumes. In our
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Fig. 1. Effect of glyphosate treatment on total glutathione content in (a) leaves and (b) roots of pea plants. Glutathione levels were measured on the 2nd, 5th, and 9th days after the treatment. (1) Untreated plants; (2) leaf application of herbicide; (3) Root application of herbicide.
experiments, total glutathione (TG) content on the second day after treatment in leaves of control plants was up to 800–1000 nmol/g fr wt and less than 100 nmol/g fr wt in the roots (Figs. 1a, 1b). We observed that the percentage of oxidized of total glu tathione in control plants, was higher in roots, as com pared to that in the leaves of pea plants (table). During the entire experimental period, the content of TG in leaves was higher in plants treated with gly phosate as compared to the control leaves and more pronounced was the effect of leaf treatment with the herbicide. In the roots, the detected TG amounts were much lower than in leaves (Figs. 1a, 1b). Application of glyphosate provoked a tendency toward the increas ing of TG in roots, since, by the end of the experimen tal period, the values exceeded control values by three times. The effect of both types of herbicide application was more pronounced in roots than in leaves. Oxidized glutathione content in the leaves of leaf treated plants was about two times above the control in two days after treatment (Fig. 2a). In 5 and 9 days after treatment, a decrease was observed. During the entire RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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Fig. 2. Effect of glyphosate treatment on oxidized glu tathione content in (a) leaves and (b) roots of pea plants. (1) Untreated plants; (2) leaf application of herbicide; (3) root application of herbicide.
experimental period, GSSG content in the leaves of roottreated plants showed minor differences vs. the control. When calculated as percent of GSSG of TG (table), after leaf application this percent initially increased in leaves, but by the end of the experimental period, a decrease was detected as compared with con Effect of pea leaf and root treatment with glyphosate on the per cent of GSSG of TG Days after treatment
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Fig. 3. Effect of leaf and root treatment with glyphosate on GR activity in (a) leaves and (b) roots of pea plants. (1) Untreated plants; (2) leaf application of herbicide; (3) root application of herbicide.
Fig. 4. Effect of leaf and root glyphosate application on GST activity in (a) leaves and b) roots of pea plants. (1) Untreated plants; (2) leaf application of herbicide; (3) root application of herbicide.
trol. Root treatment induced similar trend on the 5th and 9th days (table). Initially both types of herbicide application decreased the oxidized glutathione con tent in the roots of pea plants (Fig. 2b). In 5 and 9 days after treatment, strong accumulation of GSSG amount in the roots was observed. The highest GSSG levels were detected in the roots in 9 days after treat ment, when values in treated plants were four times above the control. This trend has kept also in the per centage of GSSG of TG, which was slightly above the control in the roots on the 9th day after treatment (table). Glutathione reductase plays a key role in the response to oxidative stress by maintaining the intrac ellular glutathione pool primarily in the reduced state [1, 3]. Treatment with glyphosate increased GR activ ity during the entire experimental period in both leaves and roots (Figs. 3a, 3b). However, this increase in most cases was not great and varied from 10 to 50% above the control. In both organs, the highest GR activity was detected in 9 days after treatment and was pro voked by both types of application of the herbicide. Glutathione Stransferase is an enzyme known to be activated by xenobiotics under stress conditions [2]. Glyphosate application provoked enhancement in the
GST activity (Figs. 4a, 4b). Activation of GST was more pronounced in the treated organ. In leaves, leaf treatment led to more than twofold increase above the control in 5 and 9 days after treatment. Root treatment also brought about twofold enhancement of GST activity in leaves on the 5th day, which remained stable until the 9th days after treatment. However, direct exposure of roots to 0.01 mM glyphosate resulted in substantially enhanced GST activities in 2 and 9 days after treatment, while leaf spraying was not able to induce an appreciable increase in the GST activity in the roots. DISCUSSION Earlier experiments of Smith [20] did not show sig nificant changes in TG and GSSG contents after application of glyphosate to barley, tobacco, soybean, and maize. Later, Uotila et al. [21] observed an eleva tion in the glutathione levels in roots and leaves of wheat plants treated with glyphosate. In our model system, the tendency towards increased TG and GSSG content (with respect to the control) was very clear, especially in the roots of pea plants (Figs. 1, 2). A key parameter for the antioxidant defense potential
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is the GSH/GSSG redox status (presented as percent age of GSSG of TG), which under normal conditions is markedly shifted in favor of its reduced form [1]. The general internal thiol–disulfide balance has a great influence on biochemical processes, including photo synthesis, photorespiration [22], and gene expression in the plant cell [2]. The redox status of glutathione is closely related to the general thiol status, as GSH is the most abundant nonprotein thiol in most plant species [1–3]. We observed that in leaves of pea plants both treatments with herbicide caused an oxidation of glu tathione, which was reflected in the increasing of the GSSG percentage on the 5th days after treatment. However, on the 9th day, the percentage of GSSG was below the control values. In roots, leafapplied gly phosate increased the GSSG/TG ratio, while root application did not change markedly this ratio on the 5th and 9th days, although markedly reduced it on the 2nd day. GR activation in the leaves of herbicideaffected plants corresponded to a decrease in the GSSG/TG ratio; however, this tendency was manifested only on the 9th day after treatment. GR is the enzyme that catalyses the NADPHdependent reduction of oxi dized glutathione and maintains the intracellular glu tathione pool primarily in the reduced state [2, 3]. It was shown that GR in plants is induced under various stress conditions [23, 24]. In the experiments of Tsai et al. [25], enhanced GR activity in rice roots was observed in response to NaCl. The experiments of Dixit et al. [26] showed similar tendencies, following treatment of pea plants with cadmium at different concentrations. The treated plants registered augmen tation of the activity of GR and a decline (below the control) in the level of GSSG. Such results suggest that different stress factors may cause similar effects in plants. In our study, we observed elevated GR activity dur ing the entire experimental period (Fig. 3). However, a decrease in the GSSG content was found only on 9th day (in leaves, see table). Therefore, a decrease in the GSSG content was delayed. It occurred few days later than the changes in GR activity. Usually, the increase in the GSH content under stress condition is assumed as a positive adaptive response, whereas its decrease and a rise in the GSSG percent leads to the inhibition of antioxidant defense and plant death [14, 27, 28]. The increased GR activ ity, the higher total GSH content, and a decreased per cent of GSSG clearly show activation of the antioxi dant defense. With slight discrepancies in the dynam ics of some of the parameters (e.g. delay in alteration of GSSG percent), these results fit into the stress response concept of the glutathione system. Hitherto there are few reports concerning the effect of glyphosate treatment on GST activity and the level of lowmolecular thiols in plants [14, 20, 21, 29]. The experiments of Uotila et al. [21] showed that glypho sate stimulated GST activity in various plant species, RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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such as pea, wheat, and maize. In addition, Jain and BhallaSarin [29] demonstrated that glyphosate treat ment resulted in a significant and concentration dependent acceleration of the GST activity and in an increase of GSH levels in the three groundnut culti vars. The data presented in this research also con firmed that glyphosate enhanced GST activity. Leaf application of herbicide provokes an elevation of the GST activity in leaves, while root treatment activated the root GST. Apparently, the herbicide influence on GST activity was more pronounced in directly treated organs. The physiological role of GST induction is of interest. The GST(s) may participate in the detoxifica tion of glyphosate through conjugation with glu tathione. For example, this reaction is the basic way for the elimination of atrazine in maize and sorghum [12]. On the other hand, the increased GST activity can be due to the de novo expression of the isoforms, which take part in the removal of the ROSinducible hydrogen peroxide. Previous experiments have dem onstrated that glyphosate causes oxidative events in pea, wheat, and maize plants [13, 14] and activates antioxidant enzymes: SOD, peroxidase, and catalase. Most probably, oxidative stress is a secondary effect of the blocked shikimate pathway, because direct mecha nism, by which glyphosate induces ROS generation, is unidentified. Additionally, Sergiev et al. [14] observed an increase in the GST activity in maize treated with glyphosate. The GST induction can be related to the elevated levels of the oxidative injuries and the amount of hydrogen peroxide. Based on these studies, we pre sume that the increment in GST activity was provoked by the development of oxidative processes in the plant cells. Most probably, the enzyme participates in the detoxification of glyphosateinducible hydrogen per oxide and lipid hydroperoxide generation. Supporting this hypothesis is the fact, that there is no experimental data about detoxification of glyphosate by conjugation with glutathione. Moreover, based on our results, we connected the changes in the glutathione levels and GR activity observed with the progress of the oxidative stress in plants. Apparently, the inhibiting of the shikimic acid pathway by glyphosate induces nonspe cifically the oxidative stress. Despite the activation of the antioxidant system, oxidative stress appears to be the major reason for the injuries of the plants. Experiments regarding the nature of glyphosate injuries in other plant species are in progress. Such studies will provide us with more information regard ing the role of oxidative events and the role of antioxi dants for the sustainability of plants. ACKNOWLEDGMENTS The authors wish to thank to Mr. Alfred Behar for his constructive and valuable remarks. This study was partly supported by the projects CC1413 and MUB 1505 (Ministry of Education and Science of Bulgaria). No. 1
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