Activity of stress-related enzymes in the perennial weed leafy spurge (Euphorbia esula L.)

Environmental and Experimental Botany 46 (2001) 95 – 108 www.elsevier.com/locate/envexpbot

Activity of stress-related enzymes in the perennial weed leafy spurge (Euphorbia esula L.) David G. Davis *, Harley R. Swanson USDA-ARS Biosciences Research Laboratory, PO Box 5674, State Uni6ersity Station, Fargo, ND 58105 -5674 USA Received 11 October 2000; received in revised form 27 February 2001; accepted 28 February 2001

Abstract The activities of several enzymes involved in plant protection against stress were assayed to determine physiological aspects of the perennial noxious weed leafy spurge (Euphorbia esula L.) that might render the plant vulnerable to integrated pest management procedures. Stresses imposed on leafy spurge plants were heat (41°C up to 48 h), cold (5°C up to 25 days), drought (up to 5 days) and feeding by a flea beetle (Aphthona lacertosa), a biocontrol insect used for control of leafy spurge (1- and 2-day feedings). The effects varied with the stress imposed and the times of exposure. The effects on the specific activity of gluthathione S-transferase in plants exposed to the four stresses were: more than doubled in heat, remained at essentially control levels in the cold, increased by 50% during drought, and increased by 20% or less in flea beetle-fed plants. Glutathione reductase specific activity decreased slightly with heat, nearly doubled with cold, increased almost 60% during drought, and remained essentially unchanged in beetle-fed plants. Catalase-specific activity decreased in plants under all four stresses. The specific activities of superoxide dismutase remained essentially constant in plants exposed to heat, increased in the cold, increased very slightly during drought, and increased in beetle-fed plants. Ascorbate peroxidase specific activity increased with the high temperature, was significantly higher only at 3 days during drought but returned to control levels or below by 5 days, and was greatly inhibited in flea beetle-fed plants. The effects of cold on ascorbate peroxidase-specific activity are not well defined. Initial experiments indicated little change up to 24 days, but subsequent experiments resulted in a significant decrease at 25 days, with recovery closer to control levels in plants returned to 25°C for 1 day. Published by Elsevier Science B.V. Keywords: Glutathione S-transferase; Glutathione reductase; Ascorbic acid peroxidase; Catalase; Superoxide dismutase

1. Introduction

* Corresponding author. Tel.: + 1-701-2391250; Fax: +1701-2391252. E-mail address: [email protected] (D.G. Davis).

Leafy spurge (Euphorbia esula L.) is a perennial weed that causes economic losses in rangelands, pastures, recreational areas and other non-crop lands (Bangsund et al., 1996). The weed continues to spread in spite of a long history of control

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measures with varying degrees of success. The increasing discoveries of weeds becoming resistant to present-day control methods is an economic and social problem due to expense of its control and encroachment upon grazing and recreational areas. Considerable effort is being put forward to find new and innovative methods to use in integrated pest management systems for leafy spurge. Enzyme systems that have evolved for the plant’s protection against stresses might be exploited in as yet unknown ways to devise innovative approaches to overcome inherent resistance to stress. All plants are subjected to stresses of various kinds throughout their lives. Four of the important stresses that were used in these experiments are high and low temperatures, insufficient water, and defoliation by flea beetles (Aphthona spp.). The latter are currently being used as a biocontrol agent against leafy spurge (Nelson and Hirsch, 1999; Kirby et al., 2000). Both abiotic and biotic stresses are known to induce plants to produce the reactive oxygen species (ROS) that can cause damage to the tissues and/or signal the start of physiological defense responses (Dat et al., 2000). Kubo et al. (1999) found that the response of antioxidant enzymes in Arabidopsis thaliana differ with the environmental stress imposed. Glutathione (GSH) appears to be one of the most important antioxidants that occurs in biological systems (Alscher, 1989; Dalton, 1995; Noctor and Foyer, 1998) so that changes in its content or metabolism is important in the protection of the plant. GSH and H2O2 have been thought to act alone or together as signaling mechanisms for acclimation or tolerance to stress (Foyer et al., 1997). Glutathione S-transferase (GST) has been among the heavily studied enzymes in plants and catalyzes conjugation of GSH to xenobiotics (Alscher, 1989; Lamoureux and Rusness, 1989; Marrs, 1996), and therefore is of great interest to pesticide producers and, ultimately, to some weed control experts. It is ubiquitous and is reported to play a role in protecting plants from some stresses (Droog et al., 1995; Gronwald and Plaisance, 1998), and it is sometimes considered among the antioxidant enzymes (Flury et al., 1998; Polidoros and Scandalios, 1999). Presumably, GST exists to

help the plant ward off harmful events that occur when pesticides (primarily herbicides) are sprayed on them, but the list of substrates also undoubtedly must include a variety of endogenously produced unwanted chemicals. Therefore, it seems logical that, if harmful stress-induced products other than ROS are produced, GST may be involved in ridding the plant of them by rendering them inactive through conjugation and/or sequestration in the vacuoles (Marrs, 1996; Alfenito et al., 1998). The GSTs in most organisms consist of isozymes; some of which may possess peroxidase activity (Hausladen and Alscher, 1993; Marrs, 1996), and leafy spurge also contains multiple isoforms of GST (J.V. Anderson, unpublished data). McGonigle and O’Keefe (1998) reference work on cloning of a heat shock protein by Czarnecka et al. (1984) that later proved to be a GST. Sitbon and Perrot-Rechenmann (1997) present arguments by some workers that the GST enzyme system might best be described as stress responsive rather than hormone inducible. Plants have a large battery of enzymes that aid in their defense against adverse environmental conditions and attack by other organisms. The enzymes chosen for this report are part of an array of protective enzymes in plants. They are gluthathione S-transferase, glutathione reductase (GR), ascorbic acid peroxidase (APOX), catalase (CAT) and superoxide dismutase (SOD). APOX and CAT are both involved in regulating H2O2 concentrations, and SOD scavenges superoxide radicals, resulting in protection of the plant against those chemical species, and are included as part of an ‘antioxidant network’ (Chaudier and Ferrari-Iliou, 1999). This research was undertaken to test the hypothesis that the activity of one or more enzymes involved in protecting leafy spurge against stress will change in the same general direction (i.e. increase or decrease) when leafy spurge is subjected to stress, whether environmental or biological. If true, the same, or similar, reactive oxygen species may be produced in response to different stresses so that it may be possible to interfere with a generalized response mechanism in such a way as to weaken the plant’s defense and reduce its competitiveness. Perhaps a plasmid could be in-

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troduced in the field that would disrupt the plant’s normal response to stress, rendering it incapable of surviving stress of any kind. This might then result in leafy spurge becoming only a very minor constituent of the forage and minimize the chance of contact with the plant, so that grazing animals might continue to graze the infested acreage and/or humans can use the land for recreational purposes.

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2.3. Cold treatment

2. Materials and methods

Plants were moved from the greenhouse at approximately 25°C and placed into an environmental growth chamber in the Cone-tainers® at 5°C for 2, 4, 8, 16 and 24–25 days. At the indicated times, the upper one-third of each plant was harvested from four plants per treatment, frozen in liquid nitrogen maintained at − 80°C until they were ground and the enzymes extracted, and assayed as indicated later.

2.1. Growth of plants

2.4. Drought treatment

Leafy spurge (E. esula L.) plants were grown in a greenhouse or growth chambers using cuttings from greenhouse-grown plants. The apical 5-cm portion of the shoots were stripped of the lower leaves, dipped into Rootone® rooting hormone, inserted into Sunshine Mix c 1 in Ray Leach Cone-tainers® and exposed to occasional mist. Cuttings were transplanted to greenhouses for 1–2 months and were usually 15- to 20-cm tall at the time of the study. Plants were watered with tap water and fertilized twice weekly with Peters® 20–20–20 (N – P– K) fertilizer. Temperatures were maintained at approximately 25°C, 16 h/8 h day/ night cycles, with daylight supplemented by 400 W high-pressure sodium lamps in the greenhouses, and using 60 W cool white high-output fluorescent lamps supplemented with 60 W incandescent bulbs in the growth chamber. Light fluences were approximately 350 mmol m − 2 s − 1 in the greenhouses and approximately 80 mmol m − 2 s − 1 in the growth chambers (LiCor-185 photometer; LiCor, Inc., Lincoln, NE, USA).

Plants were grown in a greenhouse at approximately 25°C and in the Cone-tainers® for 1, 2, 3, 4 and 5 days. The Cone-tainers were plastic containers, 4-cm diameter at the top, tapering to 2.5 cm diameter 18 cm from the top, with further tapering to a 1-cm diameter hole at the bottom (20.5 cm total length). Because the Sunmix growth medium and the plant dried out very rapidly due to the rapid air movement in the environmental chambers available, the drought experiments were carried out in the greenhouse where moisture loss was considerably slower. This allowed a more moderate drought condition to occur. The Sunmix medium in the Conetainer was quite dry by 5 days, the leaves were wilted and many of the lower leaves were chlorotic, as opposed to the controls that were watered daily, and retained their turgor and green color. At the indicated times, leaves from the upper one-third of each plant were harvested from three plants per treatment at each time period, frozen in liquid nitrogen, maintained at −80°C until they were ground and the enzymes extracted and assayed as indicated later.

2.2. Heat treatment 2.5. Flea beetle collection and feeding Plants were moved from the greenhouse at approximately 25°C and placed into an environmental growth chamber in the Cone-tainers® at 41°C for a total of 48 h. At the indicated times, the upper one-third of each plant was harvested from four plants per treatment at 0, 4, 8 and 24 h, frozen in liquid nitrogen maintained at −80°C until they were ground and the enzymes extracted, and assayed as indicated later.

Twenty soil cores (10.2 cm diameter× 15.4 cm deep) were dug from the east side of Lake Ashtabula, north of Valley City, North Dakota on 21 April 1999, and were put into cardboard containers with a plastic trap to capture emerging insects. The soil cores were placed into an environmental growth chamber at 25°C, 8 h/16 h light cycle, 350 mm m − 2 s − 1 cool white fluorescent lights. Flea beetles

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that began to emerge on 8 May 1999 were collected and placed onto leafy spurge plants contained in a plastic containment chamber to maintain them until they could be used for the feeding experiments. The beetles were presumed to be mostly Aphthona lacertosa, since they have been the predominant species recovered at this site (Don Mundal, personal communication). Leafy spurge plants were from the experimental plants grown in the greenhouse in Conetainers® with Sunmix c 2, watered and fertilized as already described. Plants were moved from the greenhouse at approximately 25°C, transplanted into the same medium in 7.5-cm diameter plastic pots and placed into Rubbermaid® transparent plastic 3.8-l jars, covered with cheesecloth under cool white fluorescent light at 70 mMm − 2 s − 1, 16 h/8 h light dark cycle and 28°C. Fifty beetles were placed on each plant. At the indicated times, the upper one-third of each plant that contained both intact leaves and those partially eaten by the flea beetles were harvested from eight plants with beetles and four control plants. The tissue was frozen in liquid nitrogen and maintained in a freezer at −80°C until they were ground and the enzymes were extracted as indicated later. Only three experiments were run using the flea beetles due to a relatively short time between emergence of the adults and their disappearance; presumably to lay eggs in the rooting medium followed by death of the adults. In Experiment 1, 50 insects were placed onto the plants for 24 h, at which time the apex of the plants were removed and discarded, and the upper one-third of the leaves were frozen in liquid nitrogen with no attempt to remove the insects from the tissues. It was felt that few beetles remained on the leaves since they tended to jump away quickly when the plants were handled. Because it appeared that the insects preferred to eat the mature leaves, in Experiment 2 the lower one-half of the leaves (oldest leaves) were removed to see if the flea beetles were discouraged from eating the young leaves, and the feeding time was extended to 48 h. In this experiment, efforts were made to get rid all of the insects prior to freezing the tissue to avoid any possible contamination by the insects. In

Experiment 3, all of the leaves were left on the plant prior to insect application. After 48 h, the insects were removed from the plants and the tissue was frozen as already described.

2.6. Protein isolation Approximately 0.5 g tissue was ground in a small motor-driven ground glass grinder in 4 ml extraction buffer (0.05 M phosphate buffer (pH 7.5) containing 10 mM KCl, 1 mM ethylenediamine tetraacetic acid (EDTA), 5 mM dithiolthreitol, 0.5 mM Pefabloc, and 1/4 (w/w) polyvinylpolypyrrolidone). The extract was centrifuged for 20 min at 17500×g. The supernatant was given a second centrifugation for 90 min at 100000× g. The resulting supernatant was passed through a 0.45 mm filter. Three milliliters of the extract was then desalted using a Bio Rad PD10 column equilibrated with 0.05 M tris-acetate buffer (pH 7.0) and the protein was eluted with 4 ml equilibration buffer. Protein concentrations were determined with a coomassie blue reagent (Pierce Chemical Co., Rockford, IL, USA) using bovine serum albumin as a standard. Unless otherwise designated, all chemicals were from Sigma (St. Louis, MO, USA).

2.7. Enzyme assays The activity of GST was determined by minor modifications of the specrophotometric method of Habig et al. (1974). All GST assays were carried out with 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione as substrates. The reaction medium contained 50 mM potassium phosphate buffer and 10 mM tris-acetate buffer (pH 7.0), 5 mM GSH, 0.4 mM CDNB, 1% ethanol and 25–50 mg protein in a final volume of 1.0 ml. The reaction was initiated at room temperature (nominally 25°C) by adding CDNB substrate. The change in absorbance at 340 nm was monitored for 5 min with a Beckman DU7400 spectrophotometer. All initial rates for all enzymes were corrected for nonenzymatic activity. One unit of activity for GST, GR, APOX and CAT is defined as 1 nmol product min − 1 per mg protein.

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The activity of GR was determined by the modification of the specrophotometric method of Smith et al. (1988). The reaction medium contained 50 mM potassium phosphate buffer and 10 mM tris-acetate buffer (pH 7.0), 0.5 mM EDTA, 1.0 mM DTNB (5,5%-dithio-bis(2-nitrobenzoic acid)), 0.1 mM NADPH, 1.0 mM GSSG and 25– 50 mg protein in a final volume of 1.0 ml. The reaction was initiated at 25°C by adding GSSG (oxidized glutathione) substrate. The change in absorbance at 412 nm was monitored for 5 min with a Beckman DU 7400 spectrophotomer. The activity of APOX was determined by a modification of the spectrophotometric method of Asada (1984). The reaction medium contained 50 mM tris-acetate buffer (pH 7.0), 0.5 mM ascorbic acid, 0.5 mM H2O2 and 50 – 100 mg protein in a volume of 1.0 ml. The reaction was initiated by addition of H2O2. The change in absorbance at 290 nm was monitored for 0.5 min with a Beckman DU 7400 spectrophotometer. The activity of CAT was determined by a modification of the spectrophotometric method of Aebi (1984). The reaction medium contained 50 mM tris-acetate buffer (pH 7.0), 20 mM H2O2 and 50 –100 mg protein in a volume of 1.0 ml. The reaction was initiated by addition of H2O2. The change in absorbance at 240 nm was monitored for 0.5 min with a Beckman DU 7400 spectrophotometer. Activity of SOD was determined by modification of the method of Beauchamp and Fridovich (1971). The reaction mixture contained 100 mM potassium phosphate buffer and 5 mM tris-acetate buffer (pH 7.0), 0.3 mM xanthine, 0.1 mM EDTA, 50 mM nitroblue tetrazolium (NBT), 0.02 U xanthine oxidase and 10– 25 mg protein in a volume of 1.0 ml. For cyanide inhibition experiments, 0.1 ml of 20 mM NaCN was added to the reaction mixture for 2 min at 25°C and then xanthine oxidase was added to initiate the reaction. The change in absorbance at 570 nm was monitored for 8 min with a Beckman 7400 DU spectrophotometer. All rates were corrected for non-enzymatic activity. One unit of SOD activity is defined as the inhibition of NBT reduction by 50%.

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2.8. Malondialdehyde (thiobarbituric acid reacti6e substances) estimation Malondialdehyde (MDA) concentration is frequently used to quantify the level of lipid peroxidation in tissues due to a fairly simple colorimetric procedure, but is probably best to report the results as thiobarbituric acid reactive substances (TBARS) due to the possibility of the presence of substances (e.g. pigments and sugars) either produced in vivo or in vitro during tissue work-up that interfere with the analyses (Smirnoff, 1993; Cherife et al., 1996) or from oxidized products of amino acids and carbohydrates in water-stressed plants (Halliwell and Gutteridge, 1993). MDA (TBARS) also may not be present in many oxidized lipids, and often are minor or secondary oxidation products (Frankel, 1998). However, because of the simplicity of the assay, MDA was used to estimate the amount of ROS produced in the tissues of the stressed leafy spurge plants as a general indicator of the level of stress imposed. Malondialdehyde (MDA or TBARS; Cherife et al., 1996) was estimated by method 1 in Draper et al. (1993). Frozen tissue (0.25 g) was ground with 2.5 ml of 10% (v/v) aqueous trichloroacetic acid and 0.25 ml of 50 p.p.m. (w/v) methanolic butylated hydroxytoluene, and heated for 40 min at 90°C. The mixture was centrifuged at 10000×g for 10 min and the supernatant was reacted for 30 min at 90°C with an equal volume of 0.3% (v/v) aqueous thiobarbituric acid. The change in absorbance at 400–600 nm was monitored for 1 min with a Beckman DU7400 spectrophotometer.

2.9. Statistical analyses Experiments were repeated three or more times. Data are two or more replicates9standard errors, from three or four plants per treatment, except eight plants were fed to the flea beetles per experiment. Statistical analyses were conducted using paired t-tests with SIGMASTAT (SPSS, Inc., Chicago, IL), with a 95% level of confidence (PB 0.05) or greater between treatments and controls.

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3. Results The changes in specific activities of all five enzymes resulting from the different stresses imposed upon leafy spurge plants are shown in Figs. 1–8. All data except those in Figs. 4 and 8 are shown relative to the control values to compare the extent of the changes that occurred because the actual specific activities in units for each enzyme differ considerably in control plants. The control data are included in Figs. 4 and 8 because the figures are from three separate experiments that were conducted differently (see Section 2).

3.1. Changes in enzyme specific acti6ities due to stress 3.1.1. GST and GR The patterns of the induction of GST- and GR-specific activities varied with the stress imposed (Figs. 1– 4). With heat stress (41°C; Fig. 1), the GST-specific activity was increased to more than 100% greater (P B 0.05) than the controls within 48 h, but under cold stress (5°C; Fig. 2) there was no significant change within 24 days (PB0.05). The reverse situation was true for GR. At 41°C (Fig. 1), the GR-specific activ-

Fig. 1. Relative specific activities ( 9 standard error) of GST and GR extracted from leafy spurge plants exposed to 41°C up to 48 h. Control specific activities for GSTand GR were 76 92 and 186 9 6 U, respectively. n =2 replicates representing four plants per treatment. * Differs from 0 h (PB 0.05).

Fig. 2. Relative specific activities ( 9 standard error) of soluble enzymes extracted from leafy spurge plants exposed to 5°C up to 24 days. Control specific activities for GST and GR were 110 92 and 114 9 4 U, respectively. n = 2 replicates representing three plants per treatment. * Differs from day 0 (P B0.05).

ity was reduced by approximately 20%, but at 5°C (Fig. 2) the activity increased to 86% greater (PB 0.05) than the controls. In droughtstressed leafy spurge (Fig. 3), the activities of both GST and GR increased at approximately the same rates to maximum levels that were more than 50% greater than controls.

Fig. 3. Relative specific activities ( 9 standard error) of soluble enzymes extracted from leafy spurge plants without watering up to 5 days. Controls were plants 1 day after water was removed since the plants were not stressed at that time. Control specific activities for GSTand GR were 76 92 and 167 97 U, respectively. n = 2 replicates representing three plants per treatment. * Differs from day 1 (P B0.05).

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Fig. 4. Relative specific activities ( 9standard error) of soluble enzymes extracted from leafy spurge plants on which were placed 50 flea beetles per plant. Left to right: Experiment 1, 24 h; Experiment 2, all leaves were left intact, 48 h; Experiment 3, older leaves removed prior to the flea beetles being applied, 48 h. n = 2 replicates representing eight plants. * Differs from controls (P B0.05).

Fig. 5. Relative specific activities ( 9standard error) of APOX, CAT and SOD extracted from leafy spurge plants exposed to 41°C up to 48 h. Control specific activities for APOX, CAT and SOD were 944 99, 64 93 and 44 92 U, respectively. Four plants per treatment, with four replicates for APOX and CAT and two replicates for SOD. * Differs from 0 h (PB 0.05).

Because of the short time the adult insects were available for feeding, only three flea beetle experiments were run. The results from the three experiments that are not exact replicates are included in Fig. 4, so the actual values (in units) for specific activity are shown rather than the relative values, and the controls are also included for reference. Experiments 1 and 2 (Fig. 4, left to right) were 24 and 48 h feedings, respectively. In Experiment 3 (Fig. 4, extreme right), the older leaves were removed from the plants (see Section 2) and a 48 h feeding time was used. In flea beetle-stressed plants, GST-specific activity increased in all three experiments (Fig. 4). The differences from the controls were less than 20%, and the increase was significantly higher (P B 0.05) than the controls only when the intact plants were used. The conclusion is that there appears to be a fairly small increase in GST-specific activity caused by feeding flea beetles. The specific activity of GR did not change greatly in flea beetle-fed plants (Fig. 4). Only in Experiment 3, where the older leaves were removed mechanically prior to the experiment, was the activity significantly greater (P B0.05) than in the controls, but similar to the results for GST the difference was small; in this case, only 5%.

3.1.2. Ascorbic acid peroxidase In heat-treated plants (41°C; Fig. 5), APOXspecific activity increased within 8 h, reaching 117% greater than that of the controls (PB 0.05) by 48 h. In cold-treated plants (5°C; Fig. 6), APOX-specific activity was not significantly dif-

Fig. 6. Relative specific activities ( 9 standard error) of soluble enzymes extracted from leafy spurge plants exposed to 5°C for 24 days. Control specific activities for APOX, CAT and SOD were 688 941, 78 9 3 and 9 91 U, respectively. n = 2 replicates representing three plants per treatment. * Differs from day 0 (P B0.05).

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Table 1 APOX-specific activity in leafy spurge plants exposed to 5°C for 25 days and returned to 25°C for 1 daya Experiment

1 2 3

a b

APOX-specific activity (U) Control

5°C 25 days

5°C 25 days to 25°C 1 day

180993 (100) 1315955 (100) 13059 74 (100)

426954 (24)b 11649 35 (88) 9359 21 (57)b

1396 9 26 (77)b 1589925 (121)b 1535954 (88)b

Data presented as average 9S.E. (% of control). Differs from controls (PB0.005).

ferent from the controls at any time, although at day 4 it was 20% greater (P B0.05) than the controls. However, in three other experiments (Table 1) in which APOX was not assayed until day 25 (assuming that the APOX-specific activity was relatively constant based on the first experiment), the specific activity decreased to 24, 89 and 72% of controls, i.e. a large variation in response. Some plants were removed from the cold chamber and returned to the greenhouse (at approximately 25°C) for 1 day to determine the ability of the plant to restore homeostasis (Table 1). The APOX-specific activity increased to 77, 121 and 118% of the controls in Experiments 1, 2 and 3, respectively. The numerical values for APOX-specific activity are quite large (Table 1) due to a strong absorbance of the product formed, but the sensitivity of the instrumentation was close to the lower limits of the sensitivity of the instrumentation used. Because of the large variation in average values among the experiments, no well-defined conclusions can be drawn at this time about the effects of cold on APOX-specific activity. The APOX-specific activities of flea beetle-fed plants are shown in units in Fig. 8 (as in Fig. 4) for GST and GR. The APOX-specific activities were significantly lower (P B 0.05) in all three experiments, ranging from only 11 to 43% of controls (Fig. 8).

3.1.3. Catalase The specific activity of CAT declined over time in all abiotically stressed plants. In heat-treated plants (41°C; Fig. 5), the activity was 66 and 23% of controls by 24 and 48 h, respectively. At 5°C (Fig. 6), the decline was slower than at 41°C, but CAT-specific activity decreased to approximately 26% of the controls by 24 days. In droughttreated plants (Fig. 7), the day 1 plants were considered the controls because the growth medium was very moist at harvest time so the plants were not under moisture stress. In droughttreated plants, the CAT-specific activity was quite erratic in that it was not significantly different (PB0.05) from day 1 to day 3, but at days 4 and 5 it was 52 and 25%, respectively, of the activity at day 1. The specific activity of CAT at day 2 in Fig. 7 appears to be greater than for day 1, but the variation was great and it was not significantly different (PB0.05) from that of day 1. In three other experiments, the CAT-specific activity at day 2 was slightly less than the activity at day 1 but, again, it was not significantly different (PB 0.05). Flea beetle-fed plants also had reduced CAT relative specific activity by 2 days, ranging from 67 to 76% of controls. The flea beetle experiment in which the older leaves had been removed

Fig. 7. Relative specific activities ( 9 standard error) of soluble enzymes extracted from leafy spurge plants without watering up to 5 days. Controls were plants 1 day after water was removed, specific activities for APOX, CAT and SOD of 1355 915, 78 910 and 51 90 U, respectively. n =2 replicates representing three plants per treatment. * Differs from day 1 (P B0.05).

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Fig. 8. Relative specific activities ( 9standard error) of soluble enzymes extracted from leafy spurge plants on which 50 flea beetles were eating. Experiment 1, 24 h; Experiment 2, all leaves were left intact, 48 h; Experiment 3, older leaves removed prior to the flea beetles being applied, 48 h. n =2 replicates representing eight plants. * Differs from controls (P B0.05).

(Fig. 8, right) was terminated at 48 h because essentially all of the leaves would likely have been totally consumed by the next day, leaving none for analyses.

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3.1.4. Superoxide dismutase The specific activity of SOD was not significantly (PB 0.05) affected by exposure of the plants to heat (41°C; Fig. 5). The SOD-specific activity in plants exposed to cold (Fig. 6) increased nearly 70% above control levels on days 4 and 8, and then declined slightly to approximately 50% above controls by 24 days. At both 16 and 24 days, the SOD-specific activity remained well above the control levels, but the average values were only significantly different from the control values at the 94 and 93% levels of confidence, respectively. In drought-treated plants (Fig. 7), the SOD-specific activity increased 12% or less in 5 days. In the flea beetle-fed plants (Fig. 8), the SOD-specific activity increased by 26 and 46% above controls at 24 and 48 h, respectively. In Experiment 3, where the older leaves were removed before the beetles were placed on them, the SODspecific activity increased 79% over the controls in 24 h (Fig. 8). 3.1.5. Summary table of enzyme responses A much simplified, general summary of the responses of all five enzymes from plants sub-

Table 2 MDA levels in stressed leafy spurge plants Stress

Number of experiments

Treatment

MDA range (nmol gFW−1)

Average 9 S.E. (nmol gFW−1)a

Relative value

Heat (41°C)

2

Cold (5°C)

Drought (−H2O)

4 1 1 1 1 4 4 4

Beetles

3

Control 8h 24 h 48 h Control, 0 days 2 days 4 days 8 days 16 days 25 days 5°C 25 days, 25°C 1 day 1 day 2 days 3 days 4 days 5 days −beetles +beetles

108–169 158–168 144–249 164–249 72–124 76 85 76 73 70–104 79–114 60–125 78–164 91–245 109–279 115–295 79–122 197–220

138 9 30A 163 95A 196 96A 206 942A 94 911B 90 101 90 86 96 9 8B 102 96B 117 9 28C 116 9 18C 168 943C 159 933D 189 9 38D 98 9 13E 205 98E

100 922 118 94 142 94 149 9 30 100 912 96 107 96 91 102 99 108 96 100 924 99 9 15 144 937 136 928 162 9 32 100 9 13 209 98

a

Average values followed by the same letter are not statistically different at PB0.05.

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Table 3 General summary of the responses of some antioxidant enzymes in leafy spurge to various stresses Enzyme

Heat

Cold

Withdrawal of H2O

Flea beetles

GST GR APOX CAT SOD

2× increase Slight decrease \2× increase Decrease No change

Little change 2× increase No change Decrease Increase

1.5× increase 1.5× increase Transient increase Decrease after 1 day Very slight increase

520% increase No change Decrease Decrease Increase

jected to all four stresses is compiled in Table 3. It can be seen that only CAT responded consistently to stress in that its activity decreased in response to all four stresses. GST increased to all stresses except cold. GR increased in cold and drought, but little change when exposed to heat or flea beetles. APOX responded differently to all four stresses. SOD activity ranged from no change to some increase with all stresses except heat.

3.2. MDA (TBARS) concentrations in the stressed plants Malondialdehyde (TBARS) concentrations were quite variable in plants stressed with heat, cold or drought, but were very consistent in plants being eaten by flea beetles. Table 2 presents the ranges of MDA (TBARS) concentration in plants from the various experiments, and also average values if all of the available data from duplicate experiments and treatments are combined. Even in control tissues, the range in MDA (TBARS) concentrations was large. Coldstressed plants had MDA (TBARS) levels approximately equal to controls. In plants stressed with heat, drought or flea beetles, the trends increased with time of exposure. The heat- and drought-treated plants had MDA (TBARS) concentrations 49 and 62% greater than controls, respectively. However, because of the large variations in responses in duplicate experiments, a statistical analysis has little meaning except to state that, if the data for the same treatments are combined and treated as single experiments for each of the stresses, there are no statistical

differences between controls and treated plants exposed to heat, cold or drought. Only the flea beetle-fed plants differed significantly (P B 0.05) from the controls, with the average MDA (TBARS) levels more than 100% greater than the controls.

4. Discussion The role of GR in biological systems is to maintain glutathione in the reduced state (Beutler and Dale, 1989) making GSH available for conjugation of xenobiotics by GST resulting in plant protection (Lamoureux and Rusness, 1989; Dalton, 1995; Marrs, 1996), or as part of an oxidation/reduction mechanism of the Foyer/ Halliwell pathway (Foyer and Halliwell, 1976). It seems logical that the activity of GST should rise and GR activity might also increase to maintain homeostasis in the internal pool of GSH by the reduction of oxidized glutathione. However, in heat-stressed leafy spurge plants, GST-specific activity more than doubled by 48 h, yet GR-specific activity actually declined slightly. Some of the GST in heat-induced plants may be diverted to functions other than simply inactivation of xenobiotics or other similar compounds. Cold treatment induced the GRspecific activity but not that of GST. Therefore, the reduction (conversion) of oxidized glutathione to GSH may increase for at least 24 days since an increase in GR specific activity occurred during that time. If so, it may be that the level of GSH by itself does not induce a signal for GST activity.

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In flea beetle-fed plants, the specific activities of both GST and GR were not altered greatly, increasing within 48 h by only 10– 20% for GST and 5% or less for GR. There may have been insufficient time for a build-up of substrates in 24 or 48 h to induce large amounts or activity of either enzyme. Longer times were not possible to test in these experiments since the leaves would likely have been consumed totally by 72 h. The fate of these plants is unique among the four stresses used for this report since the leaves are consumed by the stressor, in contrast to the abiotic stresses in which the plants essentially remain intact. The heat-treated plants had increased APOX activity up to 48 h, and it is unknown if the activity would decrease with time. At least a transient increase in APOX-specific activity occurred in cold- and drought-treated plants. In the plants fed to flea beetles, the APOX-specific activity decreased greatly. The implication is that ascorbate may not be heavily involved in the defense mechanism of leafy spurge under insect attack, and therefore may vary greatly in its participation, depending on the particular type of stress imposed on the plant. Heat may induce different mechanisms of protection from those induced by the other stresses in leafy spurge. Ascorbate is reported to occur at concentrations greater than 20 mM in chloroplasts (Smirnoff and Wheeler, 2000), but it may be somewhat limited or not readily available in leafy spurge for use as a substrate for APOX, or the levels of H2O2 may not have been sufficient to induce the enzyme during those stresses. The CAT-specific activity was not induced by any of the stresses used here, but instead declined continuously over time to less than 25% of control levels during exposure to heat, cold and drought; in plants fed to flea beetles, the activity decreased by between 24 and 35%. This contrasts some reports where CAT activity was stimulated or remained unchanged in plants under stress. In cultivars of winter rye (Secale cereale L.), both the CAT activity and photosystem II were strongly inhibited at 4°C in plants that were not hardened to the cold, and both were unchanged in hardened plants (Streb et al., 1999). Presumably, leafy

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spurge contains more than one form of CAT, as do several plant species (Frugoli et al., 1996; Scandalios et al., 1997). Three genes that express CAT have been shown to be dependent on H2O2 concentration to be expressed; low concentrations inhibited their expression in corn, but high concentrations induced the genes (Scandalios et al., 1997; Polidoros and Scandalios, 1999). Possibly the levels of H2O2 in leafy spurge under stress may not be high enough to stimulate CAT activity due to the presence of other efficient scavengers that may have been induced or stimulated by the stressful conditions. The interactions of the APOX, CAT and SOD and their involvement in scavenging ROS is very complex, and also involves other peroxidases (Scandalios, 1997a,b; Noctor and Foyer, 1998). The effects of the ROS that are known to cause membrane and cellular damage are likely to differ depending on the stress imposed on them and the molecular environment encountered by them. This seems apparent in leafy spurge as judged by the results obtained. Others have found CAT is sensitive to light, turns over rapidly (Hertwig et al., 1992) and is photoinactivated under low and high temperature stress, and varies with the plant species (Feierabend et al., 1992). Prasad (1997) found that, in cold-acclimated (4°C) maize seedlings, SOD and APOX activities did not change, but CAT, GR and guaiacol peroxidase activities increased. Decreases in both CAT and APOX, as well as monodehydroascorbate reductase activity, have been implicated in chilling tolerance at early stages of development in corn (Hodges et al., 1997). Moran et al. (1994) found activities of GR, CAT, APOX and monodehydroascorbaste reductase and dehydroascorbate reductase decreased in water-stressed pea (Pisum sati6um L.), while SOD activity increased. Both low temperature and flea beetle-feeding resulted in increased SOD-specific activity in leafy plants, indicating that the plants may be responding to the induction of the superoxide radical produced by these two stresses. In contrast, both high temperature and drought had only minor effects on SOD-specific activity, implying that the superoxide radical may not be produced in significant amounts during those two stresses or that the

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radical was rapidly scavenged by other means. Apparently, somewhat different physiological processes are induced in leafy spurge by these two stresses. Perhaps different forms of SOD in leafy spurge can be induced by stress. In heat-shocked plants recovering from chilling stress, the mRNA for Cu/Zn/SOD was the only form of mRNA that was strongly induced by the stresses that were imposed (Tsang et al., 1991). In chloroplasts, SOD, APOX and monodehydroascorbate reductase activities increased due to cold acclimation, while dehydroascorbate reductase did not (Schoner and Krause, 1990). Similar interactions, or some even more complex, may occur in leafy spurge and other perennial plants. For a recent review on SOD responses to several stresses, see Raychaudhuri and Deng (2000). Although each of the three flea beetle experiments was conducted slightly differently, the general responses of the five enzymes were quite consistent. It appeared in Experiment 1 that the beetles preferred the older leaves and might be slow to feed on the younger leaves. However, this did not seem to be the case because, in plants where the older leaves were removed in Experiment 3, the beetles had no choice and they readily consumed the young leaves. In that experiment, APOX-, CAT- and SOD-specific activities in the flea beetle-stressed plants were all somewhat higher (relative to the controls) than in the other two experiments, and this may be related to the age and physiological condition of the young leaves remaining. The measurements of MDA (TBARS) in leafy spurge were inconsistent so that it is not possible to make definitive conclusions regarding the value of such measurements. The level of MDA (TBARS) appears to remain essentially constant in cold-treated plants, and to increase in plants exposed to heat, drought and the flea beetles. Further research is needed to confirm the presence of MDA, using more definitive techniques such as high-performance liquid chromatography, and its relevance in stress on leafy spurge and other perennial weeds. The basic hypothesis that leafy spurge may generate the same or very similar ROS under stresses of all types does not appear to be sup-

ported by the data presented in the present study. Catalase was the only one of the five enzymes to behave consistently under stress, and it decreased upon exposure to all four of the stresses used. It may be more difficult to control an enzyme whose activity is shut down than to inhibit an enzyme that is turned on. This, coupled with the fact that several isoforms of CAT and all of the other enzymes tested here are likely to exist, may preclude them from serving as unique target sites for control mechanisms. Other antioxidant enzymes, such as monodehydroascorbate reductase and dehydroascorbate reductase, might be investigated also, along with detailed analyses of the changes in concentrations of the substrates of the enzymes and their products. Identifying the genes that regulate some of the enzymes involved in stress and finding ways to interfere with gene regulation may prove useful to aid in an economically viable control of persistent perennial weeds in difficultto-reach locations or along waterways where the use of herbicides is undesirable or illegal.

Acknowledgements The authors wish to thank the following members of the Entomology Department, North Dakota State University for their help with the flea beetle part of the research: Don Mundal for collecting the flea beetles, Dr Denise Olson and Dr Robert Carlson for advice and expertise in the habits of the beetles; and Dr Kristi Biewer for maintaining the beetles and the plants for those experiments.

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