Influence of Plant Age on Glutathione Levels and Glutathione Transferases Involved in Herbicide Detoxification in Corn (Zea maysL.) and Giant Foxtail (Setaria faberiHerrm)

JOBNAME: PBP Vol 54#3 PAGE: 1 SESS: 17 OUTPUT: Wed Aug 7 09:35:24 1996 /xypage/worksmart/tsp000/70858h/7pu

PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY ARTICLE NO. 0024

54, 199–209 (1996)

Influence of Plant Age on Glutathione Levels and Glutathione Transferases Involved in Herbicide Detoxification in Corn (Zea mays L.) and Giant Foxtail (Setaria faberi Herrm) PAMELA J. HATTON, DAVID J. COLE,*

AND

ROBERT EDWARDS

Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE United Kingdom; and *Rhône-Poulenc Agriculture Ltd., Fyfield Road, Ongar, Essex CM5 0HW United Kingdom Received November 16, 1995; accepted February 5, 1996 The influences of plant age on levels of extractable glutathione transferases (GSTs, EC 2.5.1.18) active in detoxifying atrazine, metolachlor, alachlor, and fluorodifen and on the availability of glutathione (GSH) have been determined in leaves of corn (Zea mays L. var. Artus) and the competing weed giant foxtail (Setaria faberi Herrm). Young corn plants, up to 30 days old, contained higher extractable GST activities toward atrazine, alachlor, and metolachlor than did giant foxtail plants of similar age. After 30 days growth, this difference was lost as the specific activities of the GSTs in corn declined while the GST activities in giant foxtail remained largely unchanged. Foliar applications of atrazine showed that the herbicide was only selectively phytotoxic to young giant foxtail plants, when the differences in GST activities between corn and giant foxtail were the greatest. The levels of GSH were highest in the foliage of younger plants of both species, though the levels of the thiol were always significantly higher in giant foxtail than those determined in corn. Dissection of the foliage of giant foxtail plants showed that there was an inverse correlation between the age of the leaves and the GST activity and GSH content. Increasing the GSH content of mature detached giant foxtail leaves by feeding with oxothiazolidine-2-carboxylate increased the rate of metabolism of [14C]atrazine in vivo but did not reduce steady-state chlorophyll fluorescence due to the presence of atrazine. Decreasing the GSH content in giant foxtail leaves by treatment with buthionine sulfoximine resulted in an unexpected increase in the rate of metabolism of [14C]atrazine and a reduction in the chlorophyll fluorescence caused by atrazine treatment. We conclude that GSH-mediated detoxification may be important in determining selectivity in seedlings but is less important in more mature plants. © 1996 Academic Press, Inc.

INTRODUCTION

Giant foxtail (Setaria faberi Herrm) is a major annual grass weed in U.S. corn crops where it can cause up to 25% loss of yield if uncontrolled (1). This problem is made more acute due to the wide distribution of the weed and its prolific seed production and seed longevity (1). The chloroacetanilide herbicides metolachlor, alachlor, and acetochlor and the chloro-striazine herbicide atrazine are important selective herbicides used to control giant foxtail in corn. However, control can be limited resulting in yield loss and the factors affecting the response of giant foxtail to these herbicides are therefore of interest. There are many factors affecting the selectivity of herbicides between crops and weeds including the relative sensitivities of the target site and the respective rates of uptake, translocation, and metabolism. In the case of giant foxtail, atrazine-resistant biotypes

have been isolated which show reduced target site sensitivity in the D2 protein of the photosystem II complex (2). However, this resistance mechanism has arisen as a result of mutation and does not account for the natural relative tolerance of giant foxtail to atrazine. A number of early studies suggested that the tolerance of giant foxtail to atrazine resulted from its ability to rapidly detoxify the herbicide, particularly by glutathione (GSH) conjugation (3, 4). Later studies suggested that tridiphane synergized the phytotoxicity of atrazine to giant foxtail by reducing the rate of glutathione conjugation (5), reinforcing the proposal that GSH-mediated detoxification is an important tolerance mechanism in this species. Elegant studies demonstrated that this reduction in GSH conjugation resulted from the selective inhibition by the glutathione conjugate of tridiphane of glutathione transferases (GSTs, EC 2.5.1.18) in giant foxtail which were responsible for atrazine detoxifica-

199 0048-3575/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

JOBNAME: PBP Vol 54#3 PAGE: 2 SESS: 17 OUTPUT: Wed Aug 7 09:35:24 1996 /xypage/worksmart/tsp000/70858h/7pu

200

HATTON, COLE, AND EDWARDS

tion (6). In the case of weed species, GSTs have also been implicated in the tolerance of velvetleaf (Abutilon theophrasti Medic.) (7) and fall panicum (Panicum dichotomiflorum Medic.) (8) to atrazine and we have recently determined an excellent correlation between GST activities and herbicide tolerance in a range of weed species (9). However, in the case of giant foxtail Wang and Dekker (10) were unable to demonstrate any correlations between the activities of GSTs toward atrazine and metolachlor and the apparent tolerance of accessions of the weed to the respective herbicides. There is therefore conflicting evidence regarding the importance of GSTs in conferring tolerance to a range of important herbicides in giant foxtail versus corn and this requires further critical evaluation. In particular the effect of plant development on the relative GSHconjugating capacities of corn and giant foxtail is of interest, as there is some limited evidence of differential variations in levels of GST activities and GSH content in the two species with increasing age (5, 6). We now report on the variations in GSH content and GST activities toward herbicide substrates of differing chemistries in leaves of corn and giant foxtail plants of increasing age and assess the likely importance of these two components of GSHmediated detoxification on the relative herbicide tolerance of the two species.

KW1135, and giant foxtail were obtained from Sharp International (Avonmouth, UK) and Herbiseed Ltd. (Wokingham, UK), respectively. All seeds were stored at 4°C, and plants were sown in 8-cm2 pots containing multipurpose potting compost (John Innes No. 2) and sandy loam (1/1 v/v). Plants were maintained in a glass house with a temperature of 22°C (daytime) and 16°C (night time), using a 14-hr photoperiod of light intensity 700–800 mmol sec−1 m−2 and watered daily. At 20 days growth for corn and 30 days growth for giant foxtail, plants were transferred to 18-cm2 pots and grown to maturity. The aerial parts of the plants were harvested at intervals and height, weight, and developmental stage were recorded, prior to dissection if required. Plant tissue was extracted directly for GST assays or frozen in liquid N2 and stored at −80°C pending analysis. Herbicide Spray Treatment

MATERIALS AND METHODS

Plants of varying age were treated by spray application with atrazine formulated as a suspension concentrate (Atraflow) using a laboratory track sprayer equipped with a T-jet nozzle delivering at a volume of 290 liters/ha. Plants were maintained in a greenhouse with a minimum temperature of 21°C and watered by automatic subirrigation. Herbicide damage was assessed visually at 7 and 14 days following application and plant height and scorch symptoms were recorded as a percentage compared to control plants.

Chemicals

Extraction of GST from Plant Tissue

Analytical grade herbicides were obtained from British Greyhound Chromatography and Allied Chemicals (Birkenhead, UK). Biochemicals were obtained from Sigma Chemical Company Ltd. (Poole, Dorset, UK) and BDH Lab Supplies Ltd. (Poole, Dorset, UK). [ 14 Ctriazinyl]Atrazine (288.6 MBq mmol−1) was obtained from Sigma and purified prior to use by TLC (9).

Plant tissue was frozen under liquid N2 and ground to a powder with a pestle and mortar. The powder was suspended in 0.1 M Tris–HCl, pH 7.5, containing 1 mM ethylenediamintetraacetic acid (EDTA), 14 mM 2-mercaptoethanol, and 7.5% polyvinylpolypyrrolidone. After filtering through two layers of muslin the homogenate was centrifuged (15,000g, 20 min, 4°C) and the supernatant was then treated with a 0.1 vol of 1.4% protamine sulfate and the suspension was recentrifuged. Finally, a protein precipitate was obtained by adjusting the supernatant to 80% saturation with (NH4)2SO4. This

Plant Material Seed of corn (Zea mays L. var Artus) a line arising from crossing (KW6217 × KW5120) ×

JOBNAME: PBP Vol 54#3 PAGE: 3 SESS: 17 OUTPUT: Wed Aug 7 09:35:24 1996 /xypage/worksmart/tsp000/70858h/7pu

EFFECT OF PLANT AGE ON HERBICIDE DETOXIFICATION

was centrifuged as above and the protein pellet obtained was stored at −20°C. Protein extracts were taken up and desalted in 2 mM potassium phosphate buffer (pH 6.8) and then desalted on a Sephadex G-25 column (PD-10 Pharmacia) prior to use. Protein content was measured using the Bio-Rad dye-binding reagent according to the manufacturer’s instructions using g-globulin as the reference protein. All extracts were adjusted to 10 mg protein ml−1 for the assay of the enzyme. Enzyme Assays and GSH Determination Enzyme activity was measured spectrophotometrically at 340 nm by using 1-chloro-2,4dinitrobenzene (CDNB) as the substrate (11). The final concentrations of reagents in a 3-ml assay were 0.1 M potassium phosphate buffer, pH 6.8, containing 3 mM reduced GSH and 1 mM CDNB. GST activities toward the herbicide substrates were determined by adding the enzyme extract (120 ml) to herbicide dissolved in acetone (10 mM; 10 ml), 10 mM GSH (20 ml adjusted to pH 7.0), and either 0.1 M potassium phosphate buffer (pH 6.8; 50 ml) for the assay of triazine and chloroacetanilide substrates or 50 mM glycine–NaOH buffer (pH 9.5; 50 ml) for the assay with fluorodifen. The mixtures were incubated at 37°C for 60 min and the reactions were terminated by the addition of 0.6 M hydrochloric acid (10 ml). The assay mixtures were then frozen at −20°C and the precipitated protein was removed by centrifugation for 5 min at 12,000g, and the supernatants (50 ml) were analyzed by HPLC as described (9). Glutathione content was determined using a glutathione reductase coupled assay and the efficiency of the determination was monitored by spiking the extract with GSH as described previously (9). Manipulation of GSH Levels in Giant Foxtail and the Effects on Atrazine Metabolism and Phytotoxicity Giant foxtail seedlings (14 days old) were excised at the base of the stem and transferred to either water or aqueous solutions of 300 mM buthionine-[S,R]-sulfoximine (BSO) or 1 mM

201

oxathiazolidine-2-carboxylate (OTC) for 48 hr. The seedlings were then transferred to a solution (1.5 ml) of [14C-triazinyl]atrazine (2.43 mM, 288.6 MBq mmol−1) and incubated in darkness for 18 hr at room temperature. The seedlings were then analyzed for [14C]atrazine and its metabolites by TLC as described previously (9). To determine the effects of modified GSH content on the inhibition of photosynthesis by atrazine, larger 50-day-old plants were treated with BSO and OTC as above, except that the feeding treatment was restricted to 48 hr. Atrazine was dissolved in acetone and added to the feeding solution at 2% v/v giving final concentrations of either 10 or 20 mM. For control treatments an identical volume of acetone was added to the feeding solution. Photosynthetic activity of intact leaves was analyzed by a Modulated Fluorescence Measurement System (Hansatech Ltd., King’s Lynn, UK) used to determine chlorophyll fluorescence kinetics. Steady-state modulated fluorescence yield in the presence of low-intensity modulated light was measured after 3-min exposure to white actinic light to drive photosynthesis. Enhanced steady-state fluorescence is indicative of inhibition of photosynthetic electron transport (12). The GSH content of the leaf tissue used in all these studies was determined prior to use. RESULTS

Postemergence Herbicide Treatment of Corn and Giant Foxtail Corn and giant Foxtail were grown under glasshouse conditions (Fig. 1) and assayed for their sensitivity to atrazine. Herbicides were applied postemergence at timed intervals and the injury to the seedlings was assessed 14 days after treatment. At all application rates of atrazine, corn seedlings remained undamaged (i.e., 0% phytotoxicity, data not shown). Giant foxtail seedlings showed between 70 and 80% damage at the higher application rates (Table 1). This selectivity was lost after 35 days growth, after which no phytotoxic effects were observed toward either corn or giant foxtail. The effect of using alternative formulations of atrazine to improve penetration into the older plants was not

JOBNAME: PBP Vol 54#3 PAGE: 4 SESS: 18 OUTPUT: Wed Aug 7 09:35:24 1996 /xypage/worksmart/tsp000/70858h/7pu

202

HATTON, COLE, AND EDWARDS

FIG. 1. The relative growth of corn (u) and giant foxtail (■) plants. Data are mean values of seven determinations.

investigated. However, the results are in agreement with earlier studies which have shown that control of giant foxtail with atrazine declines rapidly with increasing plant size and maturity (13). Variations in Extractable GSTs with Plant Age Prior to initiating a comparative study on the GSTs of corn and giant foxtail, the activities present in crude extracts from both species were characterized briefly using CDNB, atrazine, and metolachlor as substrates. Enzyme activities from both sources had identical pH optima (pH 6.8) with all substrates and the formation of glutathione conjugates was directly proportional to protein content over the range of 0.05– 1.5 mg protein per assay. Crude enzyme preparations from both plants were highly stable having a half-life at 4°C in excess of 72 hr and less than 10% of the activity toward CDNB was lost following heating to 50°C. Crude desalted protein extracts from the entire foliage of corn and giant foxtail plants of increasing age were incubated with the herbicide substrates atrazine, alachlor, metolachlor, and fluorodifen and the amount of GSHconjugated product formed (pkats mg−1 protein) was determined by HPLC analysis (9). In the absence of any enzyme the conjugation rates with the herbicides were in the order alachlor (0.14 pkats) >; atrazine (0.06 pkats) >; metolachlor (0.03 pkats) >; fluorodifen (0.01 pkats) (9).

After correcting for the respective nonenzymic reaction rates GST activities toward the herbicide substrates in the foliage of corn plants were generally in the order atrazine >; alachlor 4 metolachlor >; fluorodifen (Fig. 2). These substrate preferences were in contrast to those determined in the same line of corn seedlings grown in an environmental growth room, where the activities were in the order atrazine 4 alachlor >; metolachlor 4 fluorodifen (10). In addition, while the specific activities of the GSTs with activity toward fluorodifen and the chloroacetanilides declined steadily with increasing plant age the activity toward atrazine increased nearly 10-fold between 7 and 14 days, suggesting that the regulation of this GST activity is more sensitive to environmental and developmental factors than the other activities. To confirm the increase in atrazine conjugating activity, the activities of GST were monitored in a repeated time–course study conducted in a growth chamber. In the second experiment the GST activity toward atrazine activity increased eightfold from 0.2(0.002) nkats g−1 protein at Day 5 to 1.73 (0.25) nkats g−1 protein at Day 10 before declining again, confirming the apparent control of this activity by plant growth in this species. In the glasshouse study between 14 and 30 days the activity of the GST responsible for conjugating atrazine declined from 21 to 1 nkats g−1 protein and then remained constant (Fig. 2). The influence of plant age on GST activities in giant foxtail was less pronounced than that in corn. Thus, the GST activity toward atrazine

TABLE 1 The Phytotoxicity of Atrazine Applied to Giant Foxtail Plants of Various Ages Plant age (days)

Atrazine application rate

18

27

45

55

(kg ai/ha) 4 2 1 0.5

80 40 10 10

Phytotoxicity % 80 30 0 30 20 0 10 5 0 10 5 0

0 0 0 0

35

Note. Values refer to the average percentage damage to four plants compared with untreated controls.

JOBNAME: PBP Vol 54#3 PAGE: 5 SESS: 18 OUTPUT: Wed Aug 7 09:35:24 1996 /xypage/worksmart/tsp000/70858h/7pu

EFFECT OF PLANT AGE ON HERBICIDE DETOXIFICATION

203

FIG. 2. The effect of plant age on GST activity in protein extracts of corn (u) and giant foxtail (■) aerial tissue toward four herbicide substrates. Data are mean values of triplicate readings.

also increased between 7 and 14 days, but in the weed this only represented a fivefold increase in specific activity. With the exception of this transient change, specific activities of the GSTs which catalyzed the conjugation of atrazine, alachlor, and metolachlor remained unchanged with increasing age, while minor differences were observed with the activity toward fluorodifen. When compared with the corn plants of similar age, the GST activities toward alachlor, metolachlor, and atrazine were significantly lower in giant foxtail plants which were less than 30 days old. However, after 30 days these differences were lost due to the age-dependent decline in the specific activities of the corresponding GSTs in corn. With fluorodifen as substrate there were no significant interspecies differences in GST activities with increasing plant age. GSH Availability In order to determine the likely availability of

GSH for herbicide detoxification, the total GSH content in the foliage of corn and giant foxtail plants was determined using a glutathione reductase-coupled assay (9). GSH content was highest in the youngest plants and declined with age with giant foxtail having appreciably higher levels compared to corn at all time points (Fig. 3). These results were at variance with previous reports of young corn plants containing more of the thiol than the weed (6, 14). The levels of GSH in giant foxtail were of an order similar to those reported in young plants in an earlier study where the total thiol content was determined (5). However, our results differed from those which determined GSH using either a GST-coupled assay (6) or HPLC (14) which both reported that corn contained higher levels of GSH than giant foxtail. The reasons for the high concentration of GSH determined in giant foxtail relative to corn are unknown. All of the determinations were carried out in triplicate and previous experiments have shown the glutathione reductase-coupled assay to be reliable (9).

JOBNAME: PBP Vol 54#3 PAGE: 6 SESS: 18 OUTPUT: Wed Aug 7 09:35:24 1996 /xypage/worksmart/tsp000/70858h/7pu

204

HATTON, COLE, AND EDWARDS

FIG. 3. The effect of plant age on GSH content of corn (u) and giant foxtail (■) plants. Data are mean values of triplicate readings.

The consistency and replication of the GSH determinations in both species suggested that the results were valid, though unexpected. GSH Content and GST Activities in the Different Leaves of Giant Foxtail Plants Although GST activities toward herbicide substrates appeared to be largely uninfluenced by plant age in giant foxtail, previous studies with the nonherbicide substrate CDNB had suggested that GST activities and GSH content increased in giant foxtail leaves as they grew older while the opposite was the case in corn (6). Since at any given time mature giant foxtail plants consist of many leaves of differing age, it was of interest to determine the respective GSH contents and GST activities toward herbicides of the individual leaves of 21-day-old (4-leaf stage) plants. Plants were dissected into their four component leaves with leaf 1 (5.5 cm) being the oldest and leaf 4 (18.0 cm) the youngest (Fig. 4). The GSH content was the highest in leaf 3 which also contained the highest activity toward the general substrate CDNB. In contrast, in previous studies the concentration of GSH was reported to be similar in the older and younger leaves of giant foxtail, though in these earlier studies the total thiol content rather than

GSH was determined (5). GST activities toward all herbicide substrates were inversely proportional to leaf age in the order leaf 1 < leaf 2 < leaf 3 < leaf 4. The specific activities of the GSTs in the largest leaves, leaves 3 and 4, were significantly higher than those determined for whole giant foxtail plants of similar age in which the combined GST activities of all the leaves, but not stems, had been determined (Fig. 2). However, the relative preference for the substrates was similar to that determined in the whole plant studies. The same seed was used for both studies, though the experiments were conducted 3 months apart. Observations regarding seasonal variations in the GST activities of giant foxtail have been reported in earlier studies (5). Similarly, levels of GSH in the dissected plant parts were considerably lower than those determined in the corresponding whole plants in the earlier study (Fig. 3). Similar large variations in the thiol content of giant foxtail seedlings have also been reported to occur between experiments in previous studies (5). The Effect of Altering GSH Content on the Detoxification and Phytotoxicity of Atrazine in Giant Foxtail Studies with inhibitors of GSH biosynthesis

JOBNAME: PBP Vol 54#3 PAGE: 7 SESS: 18 OUTPUT: Wed Aug 7 09:35:24 1996 /xypage/worksmart/tsp000/70858h/7pu

EFFECT OF PLANT AGE ON HERBICIDE DETOXIFICATION

205

FIG. 4. GST activities and GSH content in the different leaves of 21-day-old giant foxtail plants. Data are mean values of triplicate readings with the standard deviation in parentheses.

in corn have demonstrated that limiting GSH availability can enhance the phytotoxicity of the herbicide metolachlor, which is detoxified by GSH conjugation (15). These studies suggested that GSH availability, rather than the respective GST activity, was a major determinant of herbicide tolerance and it was therefore of interest to determine whether this was also the case in a weed species, such as giant foxtail. For our studies atrazine was used because the herbicide was available in radiolabeled form, so that its metabolism could be monitored. In addition, unlike metolachlor, the mode of action is well defined and can be monitored by noninvasive methods such as determining the increase in chlorophyll fluorescence (12). Buthionine-

[S,R]-sulfoximine, a specific inhibitor of g-glutamylcysteine synthetase, was used as an inhibitor of GSH biosynthesis (15). Oxothiazolidine 2-carboxylate (OTC), an oxoproline analog which has been used to increase the GSH levels in plants by supplementing the cysteine pool (16), was also used. The detoxification of atrazine in giant foxtail was monitored by feeding plants through the cut stems and then analyzing the radiolabeled metabolites by TLC (9). As demonstrated in previous studies over an 18-hr incubation period, the only major radioactive metabolites present in the leaves of both 19- and 36-day-old giant foxtail plants were atrazine and glutathione conjugates of atrazine (9, 17) (Table 2).

JOBNAME: PBP Vol 54#3 PAGE: 8 SESS: 18 OUTPUT: Wed Aug 7 09:35:24 1996 /xypage/worksmart/tsp000/70858h/7pu

206

HATTON, COLE, AND EDWARDS

TABLE 2 Effect of Manipulating GSH Content on the Detoxification of [14C-triazinyl]Atrazine in Seedlings of Giant Foxtail Plant age (days)

Leaf stage

Treatment

GSH content (nmol g−1)

% Radioactivity absorbed

% Conjugated

36 19 14 14 14

5 4 2 2 2

None None None OTC BSO

410 (30) 200 (20) 615 (15) 2792 (424) 437 (84)

21.06 (0.5) 3.14 (0.12) 3.6 (1.5) 2.7 (1.3) 3.6 (0.9)

25 (12) 14 (3) 14 (4) 24 (2) 34 (10)

Note. Data are presented as mean values of triplicate readings with the standard deviation given in parentheses.

Young giant foxtail plants (14-day-old, second leaf stage) were used for the study of the manipulation of GSH synthesis. While OTC increased GSH content over fourfold, BSO had only a minor effect on GSH levels (Table 2). The OTC-mediated increase in GSH levels was coupled with an increase in the rate of atrazine conjugation. Surprisingly, the BSO-treated plants which contained less GSH than the untreated plants conjugated the atrazine most efficiently. The inhibitors did not affect the uptake of atrazine into the leaves and the GSH conjugation remained the major route of metabolism with all treatments. To determine how modifying GSH levels affected the phytotoxicity of atrazine, modulated chlorophyll fluorescence was determined as an indication of the herbicidal inhibition of photosynthetic electron transport in vivo. It was necessary to use older plants (50-day-old) with larger leaves to enable the modulated fluorescence probe to be attached and fluorescence

measurements to be made. Following pretreatment with water, OTC, or BSO, the GSH contents of the leaves were 194, 754 and 124 nmol g−1, respectively. The leaves were then placed into the above test solutions in the presence or absence of 20 mM atrazine and the steady-state chlorophyll fluorescence was determined 2, 4, 8, and 48 hr after herbicide treatment (Table 3). As anticipated, atrazine treatment alone increased the steady-state chlorophyll fluorescence at all time points. In the absence of herbicide, pretreatment with OTC or BSO alone had no significant effect upon chlorophyll fluorescence, suggesting that the changes in GSH content had not perturbed photosynthetic electron transport. Pretreatment with OTC had very little effect on the atrazine-mediated increase in chlorophyll fluorescence. In contrast, BSO treatment reduced the chlorophyll fluorescence due to the action of atrazine at the earlier time points, but this protective effect was lost after 8 hr of herbicide treatment.

TABLE 3 Effects of BSO and OTC Pretreatment on Steady-State Chlorophyll Fluorescence in Mature Leaves of Giant Foxtail Fed with Atrazine Time after treatment (hr) Treatment None BSO OTC

± atrazine

2

− + − + − +

29 54 25 34 29 45

4

8

Steady-state chlorophyll fluorescence (arbitrary units) 30 25 59 42 21 24 47 52 24 29 57 49

48 27 59 21 52 30 65

Note. Values refer to means of duplicate determinations. The errors of the points determined were within 10% of the mean.

JOBNAME: PBP Vol 54#3 PAGE: 9 SESS: 18 OUTPUT: Wed Aug 7 09:35:24 1996 /xypage/worksmart/tsp000/70858h/7pu

EFFECT OF PLANT AGE ON HERBICIDE DETOXIFICATION

DISCUSSION

In this study we have investigated the effect of plant age and development on GSH availability and the activity of GSTs involved in the metabolism of the herbicide substrates atrazine, alachlor, metolachlor, and fluorodifen in corn and the associated weed giant foxtail. Although the GSTs of corn are relatively well characterized (18, 19, 20), their regulation during plant growth and development has been largely ignored. Our results would suggest that the GSTs involved in herbicide metabolism are most highly expressed in young, rapidly growing plants, and then decline. In particular the activity of the GST involved in detoxifying atrazine was highly influenced by plant growth and development, suggesting that GST isoenzymes specific for this herbicide may have an important role in endogenous metabolism. Significantly, it was this same GST activity which was selectively lost when corn tissues underwent dedifferentiation (21) and our recent studies have shown that the expression of this enzyme is increased by exposure to light (22). In giant foxtail the activities of herbicide-detoxifying GSTs were also higher in young developing leaves than in fully expanded mature leaves. However, the GST activities of the whole plant remained largely unaffected by increasing plant age. Previous studies suggested that GST activity toward CDNB increased with age in giant foxtail leaves (6), while the activity toward atrazine underwent a similar modest decline at the later time points to that determined in our study (5). The ranges of GST activities in corn and giant foxtail were qualitatively similar with atrazine being the preferred substrate followed by the chloroacetanilides and fluorodifen. In a recent study an accession of giant foxtail was shown to have GST activity toward atrazine but none with metolachlor as substrate, though the activity was present in other Setaria species (10). In the giant foxtail plants tested here, activity toward metolachlor could be readily determined, suggesting that within differing biotypes of giant foxtail there may be variation in GST activities similar to that determined for other Setaria species (10).

207

The observation that giant foxtail contained significantly higher concentrations of GSH than did corn was surprising and would suggest that GSH availability is not a major determinant in the detoxification and selectivity of either the chloroacetanilides or atrazine in these two species. Earlier studies using specific assays for reduced GSH (6, 14) showed corn seedlings to contain levels of the thiol similar to those reported in our study. However, the concentrations of GSH previously reported in giant foxtail were lower than those observed in the current study and consistently lower than the concentration of the thiol in corn. The reasons for this variation in GSH content in giant foxtail are unclear, but would seem to be unlikely to be due to inaccuracies in the assay procedure, which gave measurements of GSH content in corn consistent with those reported earlier (6, 14). In addition, we have always determined similar or higher concentrations of GSH in giant foxtail compared with corn in this and earlier studies (9). We can only conclude that the GSH content of giant foxtail is highly variable, depending on environmental factors or possibly the varying biotypes used in different studies. Boydston and Slife (5) observed up to sixfold variation in GSH content in giant foxtail seedlings in experiments spaced over 6 months. Similarly, we observed large differences in GSH content in the weed over the experimental period (3 months). Such seasonal and environment-dependent variations in GSH content are not restricted to giant foxtail and have been reported in a number of plant species grown under glasshouse conditions (23). In the case of atrazine, elevating GSH levels in giant foxtail with OTC did increase the conjugation of the herbicide in vivo, but an even greater increase in the conjugation of atrazine was observed following BSO treatment which actually reduced GSH content. Similarly, BSO gave some protection from the atrazinemediated inhibition of photosynthesis without increasing GSH levels, while OTC caused an accumulation of GSH without alleviating toxicity. These results suggest that total GSH levels are unlikely to be a factor in determining the

JOBNAME: PBP Vol 54#3 PAGE: 10 SESS: 18 OUTPUT: Wed Aug 7 09:35:24 1996 /xypage/worksmart/tsp000/70858h/7pu

208

HATTON, COLE, AND EDWARDS

tolerance of giant foxtail to atrazine. The results with BSO are unexpected and suggest that the use of this inhibitor in determining the role of GSH in herbicide susceptibility may be limited. It is possible that the enhanced GSH conjugation of atrazine and alleviation of inhibition of photosynthesis may result from a selective redistribution of the GSH pool, such that there is more GSH available for herbicide detoxification even though overall levels decline. Alternatively, the BSO may be acting to elevate the GST active in detoxifying atrazine in a manner analogous to that reported with GST with activity toward metolachlor in BSO-treated corn seedlings (15). Interpretation of the importance of GSTs and detoxification in the relative tolerances of corn and giant foxtail of varying age to herbicides is not straightforward. The results of this study would indicate that a role for GSTs in selectivity could only be inferred for young plants. The fact that the tolerance of giant foxtail plants to herbicides increases with age (13) even though GSTs with activities toward these substrates remain at similar levels and GSH availability does not appear to be limiting at any stage of growth would argue against the involvement of GSH conjugation in the natural herbicide tolerance of this weed to atrazine and the chloroacetanilides. Other factors such as the reduced uptake or bioavailability of the herbicide are more likely to confer herbicide tolerance in the older giant foxtail plants. As such, our results support the conclusions of Wang and Dekker (10) who could find no relationship between herbicide tolerance and GSH-mediated detoxification systems in accessions of Setaria of varying susceptibilities to herbicides. Rather it is the unusually high activities of these GSTs in young corn plants which protects the crop from injury and allows the herbicides to be used selectively at the seedling stage. We have also shown that young corn plants have much higher GST activities toward a range of selective herbicides than competing weeds of similar age (9). The use of young plants in determining the mechanisms of herbicide selectivity may bias our thinking regarding the importance of metabolism in herbicide se-

lectivity and the relative importance of detoxification in the tolerance of older plants to herbicides may require reevaluation in some instances. It was also of interest to note the similarity in the range of GST activities present in corn and giant foxtail such that in the older plants the portfolios of detoxifying activities were virtually indistinguishable. Under these circumstances the difficulties of controlling selectively giant foxtail in corn using s-triazine or chloroacetanilides is understandable (1). However, the selective inhibition by tridiphane of the GSTs responsible for atrazine detoxification in giant foxtail (6) does suggest that there must be significant differences in the active sites of these enzymes in the crop and the weed. To this end we are currently purifying the major GSTs from giant foxtail and intend to study how the enzymes differ from those in corn. ACKNOWLEDGMENTS Pamela Hatton acknowledges the CASE studentship funded by the Biotechnology and Biological Sciences Research Council and Rhône-Poulenc Agriculture Ltd.

REFERENCES 1. E. L. Knake and F. W. Slife, , Competition of Setaria faberii with corn and soybean, Weeds 10, 26 (1962). 2. R. L. Ritter, L. M. Kaufman, T. J. Monaco, W. P. Novitzky, and D. E. Moreland, Characterization of triazine-resistant giant foxtail (Setaria faberi) and its control in no-tillage corn (Zea mays), Weed Sci. 37, 591 (1989). 3. L. Thompson, Jr, Metabolism of chloro s-triazine herbicides by Panicum and Setaria, Weed. Sci. 20, 584 (1972). 4. K. I. N. Jensen, G. R. Stephenson, and L. A. Hunt, Detoxification of atrazine in three Gramineae subfamilies, Weed. Sci. 25, 212 (1977). 5. R. S. Boydston and F. W. Slife, Alteration of atrazine uptake and metabolism by tridiphane in giant foxtail (Setaria faberi) and corn (Zea mays), Weed. Sci. 34, 850 (1986). 6. G. L. Lamoureux and D. G. Rusness, Tridiphane [2-(3,5-Dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane] an atrazine synergist: Enzymatic conversion to a potent glutathione S-transferase inhibitor, Pestic. Biochem. Physiol. 26, 323 (1986). 7. M. P. Anderson and J. W. Gronwald, Atrazine resistance in a velvetleaf (Abutilon theophrasti) biotype

JOBNAME: PBP Vol 54#3 PAGE: 11 SESS: 18 OUTPUT: Wed Aug 7 09:35:24 1996 /xypage/worksmart/tsp000/70858h/7pu

EFFECT OF PLANT AGE ON HERBICIDE DETOXIFICATION

8.

9.

10.

11.

12.

13.

14.

15.

due to enhanced glutathione S-transferase activity, Plant Physiol. 96, 104 (1991). R. DePrado, E. Romera, and J. Menendez, Atrazine detoxification in Panicum dichotomiflorum and target site Polygonum lapathifolium, Pestic. Biochem. Physiol. 52, 1 (1995). P. J. Hatton, D. P. Dixon, D. J. Cole, and R. Edwards, Glutathione transferase activities and herbicide selectivity in maize and associated weed species, Pestic. Sci. 46, 267 (1996). R. Wang and J. Dekker, Weedy adaptation in Setaria spp. III. Variation in herbicide resistance in Setaria spp, Pestic. Biochem. Physiol. 51, 99 (1995). R. Edwards and W. J. Owen, Comparison of glutathione S-transferases of Zea mays responsible for herbicide detoxification in plants and suspension cultured cells, Planta 169, 208 (1986). M. F. Hipkins and N. R. Baker, Spectroscopy, in “Photosynthetic Energy Transduction: A Practical Approach” (M. F.Hipkin and N. R.Baker, Eds.) IRL Press, Oxford, pp. 51–101, (1986). R. S. Boydston and F. W. Slife, Postemergence control of Giant Foxtail (Setaria faberi) in corn (Zea mays) with tridiphane and triazine combinations, Weed. Sci. 35, 103 (1987). E. J. Breaux, J. E. Patanella, and E. F. Sanders, Chloroacetanilide herbicide selectivity Analysis of glutathione and homoglutathione in tolerant, susceptible, and safened seedlings, J. Agric. Food. Chem. 35(4), 474 (1987). S. Farago, K. Kreuz, and C. Brunold, Decreased glutathione levels enhance the susceptibility of maize

16.

17.

18.

19.

20.

21.

22.

23.

209

seedlings to metolachlor, Pestic. Biochem. Physiol. 47, 199 (1993). R. Edwards, J. W. Blount, and R. A. Dixon, Glutathione and the elicitation response in legume cell cultures, Planta 184, 403 (1991). P. J. Hatton, D. J. Cole, and R. Edwards, Glutathione transferase activities in major weed species, Pestic. Sci. 42, 173 (1995). K. P. Timmermann, Molecular characterisation of corn glutathione S-transferase isozymes involved in herbicide detoxification, Physiol Plant. 77, 465 (1989). G. P. Iryzk and E. P. Fuerst, Purification and characterisation of a glutathione S-transferase from benoxacor-treated maize (Zea mays), Plant Physiol. 102, 803 (1993). D. C. Holt, V. J. Lay, E. D. Clarke, A. Dinsmore, I. Jepson, S. W. J. Bright, and A. J. Greenland, Characterisation of the safener-induced glutathione Stransferase isoform II from maize, Planta 196, 295 (1995). R. Edwards and W. J. Owen, Regulation of glutathione S-transferases of Zea mays in plants and cell cultures, Planta 175, 99 (1988). P. J. Hatton, R. Edwards, D. J. Cole, and L. J. Price, Glutathione transferases responsible for herbicide metabolism in Setaria faberi, Proc. Brighton Crop Protest. Conf.-Weeds. 1, 281 (1995). P. Broadbent, G. P. Criessen, B. Kular, A. R. Wellburn, and P. M. Mullineaux, Oxidative stress responses in transgenic tobacco containing altered levels of glutathione reductase activity, Plant J. 8, 247 (1995).