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Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2004? 2004 27911351148 Original Article 2,4-D and ROS metabolism M. C. Romero-Puertas et al.

Plant, Cell and Environment (2004) 27, 1135–1148

Reactive oxygen species-mediated enzymatic systems involved in the oxidative action of 2,4dichlorophenoxyacetic acid* M. C. ROMERO-PUERTAS,1 I. MCCARTHY,1 M. GÓMEZ,2 L. M. SANDALIO,1 F. J. CORPAS,1 L. A. DEL RÍO1 & J. M. PALMA1 1

Departamento de Bioquímica, Biología Celular y Molecular de Plantas and 2Departamento de Agroecología y Protección Vegetal, Estación Experimental del Zaidín, CSIC, Apartado 419, 18080 Granada, Spain

ABSTRACT

INTRODUCTION

2,4-dichlorophenoxyacetic acid (2,4-D) is an analogue compound to the plant hormone indole-3-acetic acid (IAA), which is used either as a growth-promoting substance or as a herbicide, depending on its concentration. In this work, the effect of 2,4-D on the growth and ROS metabolism of pea (Pisum sativum L.) leaves is reported. The herbicide considerably reduced the plant growth and negatively influenced several physiological parameters in a dosedependent manner. At structural level, damage of the mesophyll cells and the enlargement and dilation of thylakoids were observed in 2,4-D-treated plants. 2,4-D notably affected xanthine oxidase and superoxide dismutase activities, as well as the activity and transcript levels of the ascorbate–glutathione cycle enzymes, ascorbate peroxidase, monodehydroascorbate reductase, and glutathione reductase. Furthermore, in herbicide-treated plants, an increase in the H2O2 production, levels of lipid peroxidation, endopeptidase activity and oxidatively modified proteins took place. Results obtained showed that an overproduction of superoxide radicals (O2-) and hydrogen peroxide (H2O2) could take place in plants treated with 2,4D, thus contributing to the generation of oxidative stress, with the concomitant degradation of proteins. A model of the role of ROS-mediated enzymatic systems in the oxidative mode of action of 2,4-D and other auxinic herbicides is proposed.

Since their discovery, auxinic herbicides have been among the most successful compounds used to control weeds in agriculture. The most common abnormalities induced by auxinic herbicides are leaf epinasty, stem curvature, growth inhibition of roots and shoots, and intensified green leaf pigmentation. These effects are concomintant with reduction in stomatal aperture, accelerated foliar senescence, progressive chlorosis, destruction of membranes and vascular system integrity, dessication, and localized cell death (Grossmann 2000; Grabi n¢ ska-Sota, Wi s¢ niowska & Sota 2003). 2,4-dichlorophenoxyacetic acid (2,4-D) is a phenoxycarboxylic acid belonging to this kind of herbicides, together with other substances such as naphthalene acids, benzoic acids, and pyridine- and quinoline-carboxylic acids. At high concentrations, 2,4-D acts as a herbicide on dicotyledoneous plants, and it has been used for several decades in cereal crops (Aberg & Eliasson 1978; Heering & Peeper 1991; Johnson & Murphy 1991; Sterling & Hall 1997). However, at low concentrations, is broadly used in in vitro plant tissue culture for induction of calli formation and as a somatic embryogenesis stimulating substance due to its similarity to auxins (Bronsema et al. 1998; Kitamiya et al. 2000; Wei, Zheng & Hall 2000; Ramanayake & Wanniarachchi 2003). Unlike other herbicides that have specific targets in the cell – by interacting with some metabolic pathways – the intimate mode of action of herbicide 2,4-D is less known. During the last decades, different effects were reported for 2,4-D on the physiology and cellular biochemistry of plants, such as growth inhibition (Weaver & Derose 1946; Cárdenas et al. 1968), decrease of carbohydrate content (Mitchell & Brown 1946; Rasmussen 1947), stimulation of photorespiration (Rasmussen 1947; Kelly & Avery 1949), increase of nucleotides and RNA content (Key & Hanson 1961), and inhibition of CO2 fixation (Malakondaiah & Fang 1979). More recently, some authors described a mutagenic and genotoxic potential for 2,4-D which increases the mutation frequency in a concentration-dependent mode (Kumari & Vaidyanath 1989; Pavlica, Pape s˘ & Nagy 1991). However, in mammalian cell cultures, other authors did not find evidence

Key-words: 2,4-dichlorophenoxyacetic acid (2,4-D); ascorbate–glutathione cycle; oxidative stress; proteolysis; reactive oxygen species; superoxide dismutase; xanthine oxidase.

Correspondence: José M. Palma. Fax: +34 958129 600; e-mail: [email protected] *This article is dedicated to the loved and esteemed memory of Professor Dr Julio López-Gorgé, Estación Experimental del Zaidín, CSIC, Granada, who died of a stroke on 7 June 2004, at the age of 69. © 2004 Blackwell Publishing Ltd

1135

1136 M. C. Romero-Puertas et al. of genotoxicity for this herbicide (Gollapudi et al. 1999). In plants, 2,4-D stimulates some enzymatic activities such as phospholipase A2 (Scherer & André 1989), cytochrome P450 and several hydroxylases (Mougin et al. 1991; Topal et al. 1993), and an NADH oxidase localized on the external cell surface (Hicks & Morré 1998). In studies carried out with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in Norway spruce seedlings, it was found that its main mode of action was the damage of thylakoid membranes and the inhibition of photosystem II activity, thus resulting in oxidative stress (Segura-Aguilar, Hakman & Rydström 1995). Recently, Grossmann and colleagues (Grossmann, Kwiatkowski & Tresch 2001) reported that the application of auxinic herbicides, together with an abscisic acid (ABA)and ethylene-mediated effect, led to an overproduction of H2O2 which was involved in the induction of tissue damage and cell death. However, in plants there is very little biochemical and molecular information on the effect of 2,4-D on different ROS-mediated enzymatic systems, proteolytic activity, and lipid peroxidation, as well as on the oxidative modification of proteins and its further degradation by herbicide-induced proteases. In this work, using pea plants the global effect of 2,4-D on the growth, ultrastructure, antioxidative enzymes, and proteolysis, was studied. On the basis of the results obtained, a possible enzymatic mechanism involved in the reactive oxygen species (ROS)-mediated effect of 2,4-D and other analogue herbicides is proposed.

MATERIALS AND METHODS Plant material and growth conditions Pea (Pisum sativum L., cv. Lincoln) seeds, obtained from Royal Sluis (Enkhuizen, Holland), were sown in distilled water and germinated in vermiculite for 14 d under greenhouse conditions (28–18 ∞C, day–night temperature; 80% relative humidity). The most healthy seedlings were selected and grown in aerated optimum-nutrient solutions for 21–28 d under the greenhouse conditions indicated above. The herbicide was applied to the plants once, by two different ways: (1) by spraying leaves with 2,4-D and growing plants under the above conditions for 4 d; (2) by adding 2,4-D to the nutrient solutions and collecting leaves after 4 d of growing in the greenhouse. In control and 2,4-Dtreated plants, the following physiological parameters were determined according to Sandalio et al. (2001): number of leaflets per plant (NL), total leaf area (LA), leaf fresh weight (LFW), shoot fresh weight (SFW), root fresh weight (RFW), leaf area index (LAI), transpiration rate (TR), stomatal conductance (SC), CO2 exchange rate (CER), water use efficiency (WUE), and intercellular CO2 concentration (ICC).

Light and electron microscopy For microscopy studies, leaves of control and 2,4-D-treated plants were cut in 1 mm2 segments and fixed in 2.5% glut-

araldehyde solution in 50 mM potassium phosphate, pH 6.8, for 2.5 h at room temperature. Samples were post-fixed for 30 min in 1% OsO4 in 50 mM cacodylate buffer, pH 7.2, dehydrated in a graded ethanol series (30–100%; v/v), and embedded in Spurr resin. For light microscopy, semi-thin sections were stained with methylene blue, and for electron microscopy ultra-thin sections were stained with uranyl acetate and lead citrate and examined in a Zeiss (Oberkochen, Germany) EM 10C transmission electron microscope (Sandalio et al. 2001).

Preparation of crude extracts Pea leaves were homogenized in 0.1 M Tris-HCl, pH 8.0, 10% (v/v) glycerol, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2% (v/v) Triton X-100, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulphonyl fluoride (PMSF). Homogenates were filtered through two layers of nylon cloth and were centrifuged at 27 000 g for 20 min. The supernatants were used for further assays.

Enzyme assays Xanthine oxidase (XOD; EC 1.1.3.22) activity was assayed spectrophotometrically by the method of Rajagopalan (1985), and isoforms were individualized by non-denaturing polyacrylamide gel electrophoresis (PAGE) in 6% acrylamide gels and stained for superoxide as described earlier (López-Huertas et al. 1999). XOD protein levels were analysed by western blotting. After native PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membranes and a polyclonal antibody against rat XOD (Moriwaki et al. 1996) was then used. The secondary antibody and the detection method were those reported elsewhere (Corpas et al. 1998). Superoxide dismutase (SOD; EC 1.15.1.1) activity was determined by the ferricytochrome c method, using xanthine/xanthine oxidase as the source of superoxide radicals, and a unit of activity was defined according to McCord & Fridovich (1969). For the separation of the SOD isozymes, non-denaturing PAGE was performed on 10% acrylamide gels, and SOD activity in gels was detected by the photochemical nitroblue tetrazolium (NBT) reduction method (Beauchamp & Fridovich 1971). Catalase (CAT; EC 1.11.1.6) was determined according to Aebi (1984), and guaiacol peroxidase (GPX) was measured by the method of Quessada & Macheix (1984). AcylCoA oxidase (ACOX; EC 1.3.3.6) and glycolate oxidase (GOX; EC 1.1.3.1) were assayed by the method of Gerhardt (1983) and Kerr & Groves (1975), respectively, and for the hydroxypyruvate reductase (HPR; EC 1.1.1.29) activity, the method of Schwitzguébel & Siegenthaler (1984) was followed. Glutathione reductase (GR; EC 1.6.4.2) and glutathione S-transferase (GST; EC 2.5.1.18) were monitored by following the method of Jiménez et al. (1997) and Habig, Pabst & Jacoby (1974), respectively. For the determination of ascorbate peroxidase (APX; EC 1.11.1.11), crude extracts were prepared in the same buffer

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1135–1148

2,4-D and ROS metabolism 1137 indicated above but 2 mM ascorbate was added, and the activity was assayed by measuring the ascorbate oxidation (Jiménez et al. 1997).

H2O2 localization in situ Leaves from control and 2,4-D-treated plants were excised and immersed in a 1% (w/v) solution of 3,3¢-diaminobenzidine-HCl (DAB) in 10 mM MES buffer (pH 6.5), vacuuminfiltrated for 2 h and then incubated at room temperature for 8 h in the dark. Leaves were illuminated until appearance of brown spots characteristic of the reaction of DAB with H2O2. Leaves were bleached by immersing them in boiling ethanol to visualize brown spots with a higher contrast (Romero-Puertas et al. 2004).

RNA isolation and northern blot analysis Total RNA was extracted from leaves of control and 2,4-Dtreated pea plants using the Trizol method, following the manufacturer’s instructions (GibcoBRL, Life Technologies, Carlsbad, CA, USA). Samples (15 mg) of total RNA were subjected to electrophoresis in 1.2% (w/v) agarose-MOPS gels under denaturing conditions (Sambrook, Fritsch & Maniatis 1989). RNA was transferred to nylon membranes (Bio-Rad Laboratories, Richmond, CA, USA) by capillarity overnight, and was cross-linked under UV light. The transfer efficiency was checked by staining membranes with 0.04% (w/v) methylene blue (Sambrook et al. 1989). Hybridization was carried out in Na-phosphate buffer 0.5 M, pH 7.1, 2 mM EDTA, 7% (w/v) sodium dodecyl sulphate (SDS), 0.1% (w/v) Na-pyrophosphate at 42–65 ∞C for 2 h (Church & Gilbert 1984), using the following 32Plabelled probes: APX from cotton (Bunkelmann & Trelease 1996), and Mn–SOD (del Río et al. 2003b), CuZn– SOD (Isin, Burke & Allen 1990), CAT (Isin & Allen 1991), MDHAR (Murthy & Zilinskas 1994), and GR (Stevens, Creissen & Mullineaux 1997) from pea. Analyses of membranes were performed by autoradiography with a X-ray film (Hyperfilm MP; Amersham Pharmacia Biotechnology, Uppsala, Sweden).

Determination of oxidatively modified proteins The method used was based on the determination of carbonyl groups by reaction with 2,4-dinitrophenylhydrazine (DNPH) (Levine et al. 1990). For the identification of oxidized proteins, SDS-PAGE was performed on 12% acrylamide gels, and proteins were transferred to PVDF membranes (Millipore Corp., Bedford, MA, USA) and detected by Western blotting, as indicated above. A polyclonal antibody against DNPH (Sigma Chemical Co., St Louis, MO, USA) was used. Several controls were performed by incubating samples with different H2O2 concentrations as described by Romero-Puertas et al. (2002).

Proteolytic activity and degradation of oxidized proteins Endoproteolytic activity was determined spectrophotometrically by the method of azocasein degradation, and the different isozymes were identified by PAGE on 8% acrylamide gels containing 0.1% (w/v) SDS and 0.05% (w/v) gelatin (Distefano et al. 1997). For the analysis of proteolytic degradation of oxidized proteins, crude extracts of leaves from control and 2,4-D-treated plants were incubated at 37 ∞C for 16 h, and then detection of oxidized proteins was carried out as indicated above.

Other assays Lipid peroxidation was measured by the thiobarbituric acid-reacting substances (TBARS) method (Buege & Aust 1978), and protein concentration was determined according to Bradford (1986), using bovine serum albumin as standard. All results are, at least, means of four replicates. Data were subjected to ANOVA, and means were compared by either LSD or Duncan’s multiple range test (P < 0.05).

RESULTS In order to select the appropriate herbicide concentrations for further experiments, different levels of 2,4-D (0.22– 45.2 mM) were applied to pea leaves, and the effect of the herbicide on different plant physiological parameters was studied (Tables 1 & 2). Treatment with 2,4-D produced a rapid and significant growth inhibition of pea plants, even at the lowest 2,4-D concentration (0.22 mM). Net photosynthesis (CER) and stomatal conductance were also inhibited at that herbicide concentration, but transpiration rates, water use efficiency and mesophyll CO2 concentration were more adversely affected at higher 2,4-D levels (Table 2). On the basis of these physiological results and those preliminarily obtained on some ROS-related enzymes using the same range of herbicide concentrations (Romero-Puertas et al. 1999), a threshold concentration of 22.6 mM 2,4-D was chosen to apply on leaves (treatment D1). Similarly, other experiments were carried out in parallel where 2,4-D was added to nutrient solutions for 4 d, and the same parameters were analysed (Romero-Puertas et al. 1999). As a result of these last experiments, a threshold concentration of 45.2 mM was selected for the root application of 2,4-D (treatment D2). In both treatments (D1 and D2), visible symptoms of toxicity were observed which were characterized by a thickening of shoots and leaf and fruit epinasty (Fig. 1). The analysis of leaves by light microscopy showed that the main differences between control and 2,4-D-treated plants were found in the mesophyll cells, where visible damage was observed (Fig. 2a & b). Under the electron microscope, chloroplasts from plants subjected to 2,4-D application showed an enlargement and dilation of thylakoids and an increase in the number of plastoglobuli (Fig. 2d–f).


1138 M. C. Romero-Puertas et al. Table 1. Effect of different 2,4-D concentrations on the growth of pea plants 2,4-D (mM)

NL

LA (cm2)

LFW (g plant-1)

SFW (g plant-1)

RFW (g plant-1)

LAI

0 0.22 2.26 4.52 22.6 45.2

530.3 227.1 247.3 180.9 161.2 163.4

3800 1725 1746 1288 1245 1167

62.43 30.40 28.52 22.31 18.64 16.46

71.98 53.59 52.02 43.65 41.40 31.46

124.3 105.2 95.31 83.21 74.04 66.63

60.88 56.75 61.08 57.73 67.27 70.05

P< LSD

0.001 62.8

0.001 328

0.001 4.54

0.001 7.04

0.001 8.71

0.001 6.76

Plants were treated once with 22.6 mM 2,4-D by foliar application and then were grown for 4 d in the greenhouse. After this time the different growth parameters were determined. NL, total number of leaflets. Only healthy leaflets with no visual damages were considered to determine this parameter. LA, total leaf area; LFW, leaf fresh weight; SFW, shoot fresh weight; RFW, root fresh weight; LAI, leaf area index; LSD, least significant difference (Duncan, P < 0.05).

In pea leaves, the activity of the O2·– generating enzyme xanthine oxidase (XOD) was enhanced more than 25-fold by treatment with 2,4-D, either by foliar spraying (D1) or root application (D2) (Fig. 3a). This behaviour was confirmed by non-denaturing PAGE, where the only XODstained band, corresponding to the native enzyme, was considerably more intense in 2,4-D-treated plants than in control ones (Fig. 3b). Besides, the XOD protein level, detected by western blot analysis using an antibody against rat XOD, was also higher in treatments D1 and D2 than in control plants (Fig. 3b). In the preliminary experiments (Romero-Puertas et al. 1999), a gradual enhancement of XOD was observed at increasing 2,4-D concentrations (results not shown). Due to the notable increase of XOD activity, in vitro enzyme assays in the presence of different 2,4-D concentrations were performed. However, the activity of XOD remained unchanged (results not shown). With regard to superoxide dismutase (SOD), which catalyses the disproportionation of O2·– into H2O2 and molecular oxygen, this enzyme activity also was significantly higher in leaves treated with 2,4-D either via foliar application or root absorption. These results were con-

firmed by studying the SOD insoenzymatic pattern in all treatments by native PAGE (Fig. 4). Nevertheless, densitometric analysis of SOD-stained gels showed that the percentages of each isozyme were similar in control and 2,4D-treated plants. Mn-SOD ranged between 22 and 29% and percentages of CuZn–SOD I and CuZn–SOD II were 37–43 and 38–44%, respectively, in the three treatments (control, D1, and D2). Two additional H2O2-generating enzymes were also analysed, acyl-CoA oxidase (ACOX), involved in the first step of the peroxisomal fatty acid b-oxidation, and the photorespiratory enzyme glycolate oxidase (GOX). ACOX activity was about 35% higher in treatment D1 than in control plants, but no changes were observed in treatment D2. GOX activity did not show any significant alteration due to 2,4-D, and the same pattern was found with another photorespiratory enzyme, hydroxypyruvate reductase (HPR) (Table 3). The activity of the H2O2-scavenging enzyme catalase (CAT), determined in crude extracts, was not affected by treatment D1, but a slight increase in roottreated plants (treatment D2) was observed (Table 3). These results were confirmed by analysis of the catalase

Table 2. Effect of different 2,4-D concentrations on some physiological parameters of pea plants 2,4-D (mM)

TR (M H2O m-2 s-1)

SC (M m-2 s-1)

CER (mM CO2 m-2 s-1)

WUE (mM CO2 m-2 s-1/M H2O m-2 s-1)

ICC (mM M-1)

0 0.22 2.26 4.52 22.6 45.2

0.217 0.192 0.161 0.168 0.151 0.155

0.230 0.174 0.104 0.150 0.089 0.092

32.79 22.81 15.22 13.98 10.23 5.72

15154 12227 9647 8546 6864 3691

206.2 204.3 217.3 252.7 280.2 371.4

P< LSD

0.01 0.037

0.01 0.0075

0.001 3.98

0.001 2442

0.01 101.8

Plants were treated once with 22.6 mM 2,4-D by foliar application and then were grown for 4 d in the greenhouse. After this time the different physiological parameters were determined. TR, transpiration rate; SC, stomatal conductance; CER, CO 2 exchange rate; WUE, water use efficiency; ICC, intercellular CO2 concentration. LSD, minimal significant difference (Duncan P < 0.05). © 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1135–1148

2,4-D and ROS metabolism 1139 Table 4. Activity of the ascorbate–glutathione cycle enzymes, ascorbate peroxidase (APX) and glutathione reductase (GR), and glutathione S-transferase (GST) in leaves of pea plants treated with 2,4-D

a

Treatment

APX

GR

GST

C D1 D2

125.2 ± 23.5 278.3 ± 33.8 183.5 ± 30.0

76.17 ± 7.77 143.1 ± 3.91 127.8 ± 9.28

43.74 ± 9.49 140.50 ± 14.36 84.27 ± 15.40

Plants were treated either with 22.6 mM 2,4-D by foliar application or with 45.2 mM 2,4-D through the nutrient solution, as described in Materials and Methods. After 4 d growth, leaves were collected and the different enzymatic activities determined Activities are expressed as nmol min-1 mg-1 protein. Values are the mean ± SEM. C, control plants; D1, plants treated with 2,4-D by foliar application; D2, plants treated with 2,4-D through the nutrient medium.

b

Figure 1. Effect of 2,4-D on pea plants grown under greenhouse conditions. Plants were treated once by foliar application of 22.6 mM 2,4-D and then were grown for 4 d in the greenhouse and analysed. (a) Control plants and 2,4-D-treated plants. (b) Aerial organs of plants treated with 22.6 mM 2,4-D.

isozyme pattern by isoelectric focusing, which was similar in control, D1 and D2 plants (results not shown). Nevertheless, the activity of another enzyme that uses hydrogen peroxide as substrate, guaiacol peroxidase (GPX), increased significantly (about two-fold) in crude extracts of D1 and D2 plants (Table 3). Two enzymes of the ascorbate–glutathione cycle were studied, ascorbate peroxidase (APX) and glutathione reductase (GR). The activity of these enzymes was significantly increased in treatment D1 and, to a lesser extent, in

D2 compared to control plants. Furthermore, the glutathione S-transferase (GST) activity was also remarkably higher in plants treated with 2,4-D than in control plants (Table 4). The histochemical detection of H2O2 by the DAB staining method in whole leaves showed a higher labelling in the central vein and in peripheral areas of 2,4-D-treated plants than in control ones (Fig. 5). The H2O2 staining was prevented by incubation of leaves with 1 mM ascorbate prior to DAB (results not shown). The expression of six antioxidative enzymes was studied in leaves of control and 2,4-D-sprayed plants (treatment D1) using different probes. The transcript levels of catalase and, to a lesser extent, of Mn-SOD were increased by the herbicide, whereas the CuZn–SOD levels decreased (Fig. 6). The transcripts of the ascorbate–glutathione cycle enzymes studied, APX, GR and monodehydroascorbate reductase (MDHAR), were all enhanced in 2,4-D-treated plants (Fig. 6). Proteolytic activity of leaves, determined by the azocasein digestion method, was notably enhanced in plants treated with 2,4-D (Fig. 7a), and this was confirmed by analysis of endoprotease activity in SDS-gels containing gelatin (Fig. 7b). In control plants, only one endopeptidase (EP) isozyme, of 93 kDa, was detected (EP1), and this pattern was also found in D1 plants, although the activity of EP1

Table 3. Activity of ROS-related enzymes in leaves of pea plants treated with 2,4-D Treatment

CAT

GPX

ACOX

GOX

HPR

C D1 D2

173.7 ± 11.7 175.0 ± 19.6 216.7 ± 3.5

40.02 ± 2.58 85.20 ± 11.99 85.12 ± 12.16

510 ± 39.2 685 ± 54.7 453 ± 29.6

82.94 ± 7.71 77.46 ± 12.31 89.03 ± 2.57

968 ± 9.30 901 ± 81.2 986 ± 34.1

Plants were treated either with 22.6 mM 2,4-D by foliar application or with 45.2 mM 2,4-D through the nutrient solution, as described in Materials and Methods. After 4 d growth, leaves were collected and the different enzymatic activities determined. Catalase (CAT), acylCoA oxidase (ACOX) and glycolate oxidase (GOX) are expressed as mmol min-1 mg-1 protein, whereas guaiacol peroxidase (GPX) and hydroxypyruvate reductase (HPR) are expressed as nmol min-1 mg-1 protein. Values are the mean ± SEM. C, control plants; D1, plants treated with 2,4-D by foliar application; D2, plants treated with 2,4-D through the nutrient medium. © 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1135–1148

1140 M. C. Romero-Puertas et al.

a

b

c

d

f

e

was higher than in control plants. However, in pea plants where the herbicide was applied through the roots, a new endopeptidase isozyme was found (EP2) with a molecular mass of 86 kDa (Fig. 7b). The lipid peroxidation, measured as TBARS, was enhanced by treatment with 2,4-D, specially in those plants in which the herbicide was applied through the leaves (Fig. 8a). The proteolytic degradation of oxidatively modified proteins was also studied in the three treatments. Plants sprayed with 2,4-D showed a higher content of carbonyl groups than control plants, as demonstrated by western blotting with an antibody against DNPH (Fig. 8b). The intensity and number of oxidized polypeptides was even greater in plants treated with 2,4-D through the roots (treatment D2). On the other hand, the oxidatively modified proteins were susceptible to protein degradation, since most of them disappeared when samples were previously incubated at 37 ∞C for 16 h and then analysed by SDSPAGE and western blotting (Fig. 8b).

DISCUSSION The results presented in this work clearly show that 2,4-D, applied either to leaves or roots, affected severely the growth of pea plants, particularly at the highest concentra-

Figure 2. Effect of 2,4-D on the structure and ultrastructure of pea leaves. Plants were treated once with 22.6 mM 2,4-D by foliar application and then were grown for 4 d in the greenhouse and analysed. (a) and (b), Light microscopy of leaves from control and 2,4-Dtreated plants, respectively. (c)–(f), electron micrographs of leaves from control (c) and 2,4-D-treated plants (d– f). Ec, epidermal cells; Pc, palisade cells; Mc, mesophyll cells; Ch, chloroplast; M, mitochondrion; P, peroxisome. The arrow indicates plastoglobuli and the asterisk mesophyll damaged cells. Bars represent 50 mm in (a) and (b), and 1 mm in panels (c)–(f).

tions used in our experimental conditions. Pea plants developed the typical visual symptoms produced by auxinic herbicides such as leaf epinasty, stem curvature, and inhibition of root and shoot growth (Young, Evans & Hertel 1990; Sterling & Hall 1997; Grossmann 2000; Grabi n¢ skaSota et al. 2003). In fruits, an epinasty-like twisting and a development arrest were observed. At structural level, the 2,4-D effects were similar to those reported elsewhere, characterized by a breakdown in the cell membrane structure of the epidermis, palisade, and mesophyll tissues, with concomitant plasmolysis and chloroplast damage (Hallam 1970; Grossmann 2000). The increase found in the CO2 concentration of mesophyll cells from 2,4-D-treated plants might be due to a higher respiration rate of this tissue, as was reported several decades ago (Kelly & Avery 1949), but also to the continuous supply of CO2 through the photorespiration pathway. In fact, the photosynthesis and photorespiration processes appear to be affected in a different way. Whereas the photosynthesis rate is diminished, photorespiration seems to be functional, since the activity of GOX and HPR, two enzymes participating at the beginning and the end of the pathway, respectively, were maintained at their original values. This suggests a possible regulation point at the Rubisco level, with a predominance of the oxygenase activity of this


2,4-D and ROS metabolism 1141

Figure 3. Xanthine oxidase in leaves of plants treated with 2,4D. (a) XOD activity was determined spectrophotometrically and is expressed as nmol of uric acid mg-1 min-1. (b) Non-denaturing PAGE of XOD. Activity was detected by staining gels (100 mg lane-1) with 1 mM xanthine NBT (López-Huertas et al. 1999), and XOD protein content was studied by western blotting with an antibody against rat XOD (Moriwaki et al. 1996). C, control plants; D1, plants treated with 22.6 mM 2,4-D by foliar application; D2, plants treated with 45.2 mM 2,4-D through the roots.

enzyme versus its carboxylase activity. An inhibition of the Rubisco carboxylase activity might be due to a partial oxidation of the protein, as it has been observed in other abiotic stresses (Kingston-Smith & Foyer 2000; RomeroPuertas et al. 2002). The ROS metabolism was substantially altered by 2,4-D, favouring the generation of O2·– and possibly H2O2. Xanthine oxidoreductase (XOR) is an enzyme that can be found as two interconvertible forms in eukaryotes. The form D (xanthine dehydrogenase, XDH) is reversibly converted into the form O (xanthine oxidase) by oxidation, whereas is irreversibly transformed into the form O by proteolytic cleavage. XOD has been located in the cytosol and in peroxisomes from plant cells, and is the predominant form of XOR under stress conditions (Palma et al. 2002; del Río et al. 2003a). Xanthine oxidase (XOD) oxidizes xanthine/hypoxanthine to uric acid with the concomitant production of superoxide radicals (O2·–). The dramatic increase of XOD in 2,4-D-treated plants supports the idea that the O2·– levels are notably enhanced in those plants. Considering that superoxide radicals are the substrate of SOD, with production of H2O2, the higher activity of SOD isozymes in herbicide-treated plants would bring about an augmentation of the H2O2 levels in pea leaves. In fact, a higher H2O2 histochemical labelling was observed in plants treated with 2,4-D. Interestingly, this labelling was coincident with leaf zones which showed clear epinastial symptoms. Other enzymatic sources of hydrogen peroxide were studied in this work, mainly ACOX and GOX. ACOX has been proved to

Figure 4. Effect of 2,4-D on the superoxide dismutase activity of pea leaves. (a) Total activity was determined by the ferricytochrome c method. (b) SOD isozymes were characterized by native PAGE (100 mg lane-1) and staining the gels in the presence of specific inhibitors (2 mM CN– and 5 mM H2O2). C, Control plants. D1, plants treated with 22.6 mM 2,4-D by foliar application. D2, plants treated with 45.2 mM 2,4-D through the roots. Mn-SOD, manganese-containing superoxide dismutase; CuZn–SOD, copper-zinccontaining superoxide dismutase.

be stimulated by xenobiotics, and has been selected as a biomarker in environmental pollution assessments (Cajaraville et al. 2003), and GOX was similarly enhanced in pea plants subjected to cadmium stress (McCarthy et al.

Figure 5. Histochemical localization of H2O2 in leaves of plants treated with 2,4-D. The herbicide was applied by spraying leaves with a solution of 22.6 mM 2,4-D as indicated in Materials and Methods. H2O2 labelling was mainly detected as brown spots (arrows) in the central vein and in peripheral zones of 2,4-Dtreated plants.



Figure 6. Northern blot analysis of antioxidative enzymes in leaves from control (C) and 2,4-D-treated plants by foliar application (2,4-D). Pea 18S rRNA was used as internal control to assess RNA loading. Mn-SOD, manganese-containing superoxide dimutase; CuZn–SOD, copper-zinc-containing superoxide dismutase; CAT, catalase; APX, ascorbate peroxidase; GR, glutathione reductase; MDHAR, monodehydroascorbate reductase.

2001). Nevertheless, these enzymes did not change significantly in plants treated with 2,4-D. Superoxide radicals and H2O2 can react, by a metal-catalysed Haber–Weiss reaction, producing the powerful oxidizing species hydroxyl radical (·OH). The overproduction of ROS is possibly responsible for the lower photosynthesis rate observed, since these reactive species are able to inhibit some of the carbon reduction cycle enzymes such as fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase (Halliwell & Gutteridge 2000). Furthermore, ·OH radicals can rapidly attack biological membranes and all types of biomolecules, including DNA and other proteins, leading to irreparable metabolic dysfunction and cell death (Halliwell & Gutteridge 2000). In our experimental conditions, the changes produced in the ROS metabolism might be responsible for the enhanced lipid peroxidation and protein oxidation and degradation found in pea plants treated with 2,4-D. Protein degradation in plants is a complex process involving many proteolytic pathways that can be carried out in several cell compartments. The role of proteases in some developmental stages, such as germination, senescence and programmed cell death, is essential (Palma et al. 2002). Among the different types of proteases, the serineand cysteine-proteinases are the most associated to these physiological situations, as well as to several stresses (Vierstra 1996; Palma et al. 2002). In fact, the appearance of new isozymes belonging to both the serine- and the cysteineproteinase classes has been reported in peroxisomes from senescent and cadmium-stressed pea leaves (Distefano et al. 1997; McCarthy et al. 2001). In this sense, the changes observed in the endopeptidase isoenzyme pattern, specially in root-treated plants, reveal that proteolysis plays an important role in the mode of action of 2,4-D. However,

the identity of both EP1 and EP2 is not yet known, and further studies using different inhibitors/activators are needed. The enhanced proteolytic activity found in 2,4-Dtreated plants might be partly responsible for the increased XOD activity, by favouring the conversion of XDH into XOD. This regulatory mechanism for EPs has been proposed to occur in peroxisomes from senescent pea leaves (Distefano et al. 1999). In plants, as in other eukaryotes, cleavage of oxidatively modified proteins is usually linked to oxidative stress situations induced by biotic and abiotic stress and senescence (Palma et al. 2002). The degradation pattern of oxidized proteins reported in this work has been also observed in pea plants exposed to toxic levels of Cd and in pea leaf extracts incubated with different H2O2 concentrations (Romero-Puertas et al. 2002). On the other hand, the increase observed in XOD activity might be also an index of a higher nucleic acid degradation produced by the herbicide, since this enzyme is involved in the catabolism of purines. However, this effect is under study in our laboratory and needs to be confirmed. Several papers have previously reported a relationship between auxinic herbicides and ROS metabolism in plants.

Figure 7. Effect of 2,4-D on the endoproteolytic activity of pea leaves. (a) Total activity determined by the azocasein digestion method (Distefano et al. 1997). (b) EP activity (150 mg protein per lane) detected in gelatin-containing SDS gels (Distefano et al. 1997). Pea endopeptidase isozymes are designated as EP1 and EP2. Molecular mass markers are indicated on the left. C, control plants. D1, plants treated with 22.6 mM 2,4-D by foliar application. D2, plants treated with 45.2 mM 2,4-D through the roots.


2,4-D and ROS metabolism 1143

Figure 8. Oxidative stress parameters in pea plants treated with 2,4-D. (a) Lipid peroxidation determined by the TBARS method, using malondialdehyde (MDA) as standard. (b) Degradation of oxidatively modified proteins. Oxidized proteins were detected by SDS-PAGE and western blotting using an antibody against DNPH. Previous to electrophoresis, samples were incubated at 37 ∞C for 0 and 16 h. Under these conditions endogenous proteases are able to degrade sample proteins (Romero-Puertas et al. 2002), but only those which were oxidized and recognized by the antibody against DNPH were detected. Molecular mass markers are indicated on the right. C, control plants. D1, plants treated with 22.6 mM 2,4-D by foliar application. D2, plants treated with 45.2 mM 2,4-D through the roots.

A plasma membrane superoxide-producing NADH oxidase that is stimulated by 2,4-D was described by Morré & Brightman (1991). However, more recent references evidenced two components of this system: a constitutive NADH oxidase which reduces oxygen, and an auxinresponsive NADH oxidase that transfers electrons to disulfide bonds of proteins (Chueh et al. 1997; Hicks & Morré 1998). Other reports showed that auxin promoted a O2·– release and subsequent generation of ·OH radicals, thus inducing the elongation of maize cells, what supported the hypothesis of a role for ROS in the auxin-mediated signalling pathway (Schopfer et al. 2002). Besides elongation, auxin also caused gravitropic curvature in an ROS-mediated manner (Joo, Bae & Lee 2001). According to this, the increases in XOD and SOD activity found in 2,4-D-treated plants, might be partly responsible for the visible symptoms observed in the different aerial organs and the slight increase of cell size. In the mid-1990s, studies carried out in Norway spruce revealed that an oxidative stress took place after treatment

with 2,4,5-T (Segura-Aguilar et al. 1995). By contrast, in monocots, which are tolerant to 2,4-D, this compound seems to prevent the generation of ROS by diclofop-methyl (DM), another herbicide which causes senescence and abscission of old leaves (Shimabukuro et al. 1999). More recently, it has been found that auxinic herbicides stimulated the H2O2 generation in shoots of cleavers, and an ABA-mediated stomatal closure leading to an overproduction of ROS has been proposed. This effect seems to be involved in the induction of tissue damage and cell death favoured by auxinic herbicides (Grossmann et al. 2001). However, the ROS-generating systems which may participate in the mechanism of action of these herbicides still remain unknown. In fact, no relationships among the auxinic herbicides and neither chloroplastic nor mitochondrial ROS-producing mechanisms have been yet reported. To our knowledge, this is the first time that a XOD-mediated effect of a herbicide has been investigated in plants. The only reports on this effect have been described in animals treated with paraquat (Kitazawa et al. 1991; Sakai et al. 1995; Ali, Diwakar & Paxa 2000), an unrelated herbicide to those referred in this work. The possible role of XOD and SOD in the mode of action of 2,4-D is depicted in Fig. 9. Our model clearly confirms that reactive oxygen species are involved in the mechanism by which auxinic herbicides exert their activity through an oxidative stress. Besides, XOR-dependent nitric oxide (NO) production has recently been reported (Harrison 2002; del Río, Corpas & Barroso 2004). Thus, XOR might also participate in the mechanism of action of 2,4-D through its potential to generate NO. In fact, NO mediates several abscisic acid (ABA) plant responses (Neill, Desikan & Hancock 2003), and ABA is considered a secondary messenger additional to ethylene in the auxinic signalling pathways (Grossmann et al. 2001). With respect to the SOD isozymes, the increased activity of Mn-SOD was mirrored by an enhanced expression of Mn-SOD, but there was no correlation between the activity and the transcript levels of CuZn–SODs, which showed an opposite behaviour. The lower level of the CuZn–SOD mRNA in plants treated with 2,4-D, might be due to a higher instability of the mRNA favoured by the herbicide. Similar discrepancies between SOD transcript levels and activities have been also found in pine tree, maize and tobacco plants exposed to different stress conditions (Williamson & Scandalios 1992; Karpinski et al. 1992; Zhu & Scandalios 1994; Savouré et al. 1999). Regarding other antioxidative systems, catalase (CAT) activity in pea leaves was not changed significantly by the herbicide treatment, but its specific mRNA content was notably enhanced by 2,4-D. An induction of the genes Cat1, Cat2 and Cat3 was found in maize immature embryos treated with auxins – either IAA or 2,4-D -, and a parallel increase in the activity of the respective isozymes was also detected (Guan & Scandalios 2002). This suggests that, in pea plants, changes in the translation efficiency or at post-translational level might be induced by 2,4-D, as it was proposed for the CAT synthesis in rice (Schmidt, Dehne & Feierabend 2002). Another



Figure 9. Model proposed for the action of 2,4-D in the oxidative metabolism of pea leaves. The excess O 2·– and H2O2, generated by the enhanced xanthine oxidase (XOD) and superoxide dismutase (SOD) activities, respectively, can lead to an overproduction of hydroxyl radicals (·OH) by a Haber–Weiss-type reaction in the presence of certain heavy metals (Cu2+, Fe3+, etc.). Under these conditions, epinasty and gravitropism occur, but oxidative stress, characterized by growth and photosynthesis inhibition, lipid peroxidation, protein oxidation, proteolysis, and cell death, also takes place. The ascorbate–glutathione cycle, whose enzyme levels are enhanced by the 2,4-D treatment, participates in the removal of the excess H2O2 and in providing reduced glutathione (GSH) for the glutathione S-transferase (GST) activity to form the complex GSH-2,4-D.

H2O2-consuming enzyme is GPX. Many peroxidases are localized in vacuoles, the cell wall and the cytosol (Asada 1992). In our study, the GPX activity was two times higher in plants treated with the herbicide in comparison with control plants, which could indicate that cell wall-bound peroxidases might be involved in the cell growth characteristic of the auxin-mediated response. Schopfer et al. (2002) found that peroxidase inhibitors suppressed the auxin-promoted cell growth, although they proposed a peroxidasecatalysed ·OH production as the mechanism to facilitate the extension of cells. The ascorbate–glutathione cycle, one of the most important antioxidative systems for H2O2 scavenging in plant cells (Noctor & Foyer 1998; Asada 2000), was clearly influenced by the herbicide treatment. The activity of two enzymes of this cycle, APX and GR, were higher in treated plants than in control ones, which indicates that the excess H2O2 is not only removed by unspecific peroxidases, as

mentioned above, but also by this cycle (Fig. 9). The expression of APX and GR correlated the activity values, and this provides evidence for an induction of APX and GR genes by 2,4-D. In higher plants, the APX activity increases along with the activities of other antioxidant enzymes under environmental stress (Shigeoka et al. 2002). However, in spinach leaves, the analysis of the steady-state mRNA of each APX isoenzyme revealed that only the cytosolic APX was expressed in response to high light intensity and methyl viologen treatment, whereas the other isoenzymes were constitutively expressed under normal and stress conditions (Yoshimura et al. 2000). Moreover, another gene of the ascorbate–glutathione cycle, MDHAR, was also induced in 2,4-D-treated plants. Similar MDHAR induction was observed in pea plants treated with 50 mM CdCl2 and during natural senescence of leaves (Leterrier et al. 2003). These data indicate that MDHAR could be also the target of different types of


2,4-D and ROS metabolism 1145 stresses and changing physiological situations of the plant. Little is known on the effect of xenobiotic compounds on the ascorbate–glutathione cycle, and the results reported in this work suggest that this pathway is a target for 2,4-D. Therefore, the antioxidative ascorbate–glutathione cycle seems to be modulated at the gene level by 2,4-D. Glutathione S-transferases (GSTs) are a family of enzymes with the ability to conjugate xenobiotics to glutathione. The complex, thus formed, is translocated to the vacuole, and the potential damaging effect of such compounds is therefore diminished (Marrs 1996; Fujita, Adachi & Sakato 1998; Edwards, Dixon & Walbot 2000). In Hyoscyamus muticus a model has been proposed in which 2,4-D is a substrate for GST (Bilang & Sturm 1995). The increased GST activity found in pea plants treated with 2,4D, reported in this work, suggests that this enzyme is most likely involved in the herbicide detoxification by the binding of 2,4-D to reduced glutathione (GSH). Considering this mechanism, an additional role of the ascorbate–glutathione cycle in this detoxification process by providing reduced glutathione (GSH), the substrate of GSTs, cannot be ruled out (Fig. 9). GST could also protect against oxidative stress induced by 2,4-D, similarly as has been reported in black grass for other herbicides (Cummins, Cole & Edwards 1999; Edwards et al. 2000) and in auxin-treated Arabidopsis (Laskowski et al. 2002). In conclusion, our results show that in pea plants 2,4-D produces similar effects independently of its application through the roots or leaves. This is in accordance with the polar transport of auxinic compounds reported in plants (Jones 1998; Rosen, Chen & Mason 1999; Muday & DeLong 2001). Taking into account this property of 2,4-D and that both aerial and root treatments trigger the same response in pea leaves, the mechanism proposed in this work appears to be directly responsible of the action of 2,4D, and not an indirect consequence of the herbicide. 2,4-D seems to induce severe oxidative stress in pea plants, although a detoxifying mechanism involving the glutathione S-transferase system also appears to contribute to the plant defence response against 2,4-D (Fig. 9). The oxidative stress, favoured by an enhancement of O2·– and H2O2 levels, could be responsible for important adverse effects such as the inhibition of plant growth and photosynthesis rate, protein oxidation and further proteolysis of oxidatively modified proteins, and cell death.

ACKNOWLEDGMENTS M.C.R-P and I.McC., who equally contributed to this work, acknowledge their Ph.D. fellowships from the Junta de Andalucía and Ramón Areces Foundation (Spain), respectively. This work was supported by grants PB98-0493–01 and AGL2002-00988 from the CICYT and by the Junta de Andalucía (Research Group CVI 0192), Spain. The generous donation of antibodies against rat xanthine oxidase by Dr Yuji Moriwaki, Hyogo College of Medicine, Japan, cotton APX cDNA by Dr Richard N. Trelease, Arizona State University, USA, pea CuZn–SOD cDNA by Dr Randy

Allen, Texas Tech University, USA, pea GR cDNA by Dr Phil M. Mullineaux, John Innes Centre, Norwich, UK, and pea MDHAR and Mn-SOD cDNA by Dr Barbara A. Zilinskas, Rutgers University, NJ, USA, is appreciated. The light and electron microscopy assays were carried out at the Centre of Scientific Instrumentation of the University of Granada.

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