Journal of Chemical Ecology, Vol. 20, No. 3, 1994

POTENTIAL ROLE OF LIPOXYGENASES IN DEFENSE AGAINST INSECT HERBIVORY

G.W. FELTON,*

J.L. BI, C . B . S U M M E R S , S.S. D U F F E Y 1

A.J. M U E L L E R ,

and

Department of Entomology University of Arkansas Fayetteville, Arkansas 72701

(Received September 20, t993; accepted November 3, 1993) Abstract--The potential role of the plant enzyme lipoxygenase in host resistance against the corn earworm Helicoverpa zea was examined. Lipoxygenase is present in most of the common host plants of H. zea, with highest activity in the leguminous hosts such as soybean and redbean. Treatment of dietary proteins with linoleic acid and lipoxygenase significantly reduced the nutritive quality of soybean protein and soy foliar protein. Larval growth was reduced from 24 to 63 % depending upon treatment. Feeding by H. zea on soybean plants caused damage-induced increases in foliar lipoxygenase and lipid peroxidation products. Larvae feeding on previously wounded plant tissue demonstrated decreased growth rates compared to larvae feeding on unwounded tissue. Midgut epithelium from larvae feeding on wounded tissues showed evidence of oxidative damage as indicated by significant increases in lipid peroxidation products and losses in free primary amines. The potential role of oxidative and nutritional stress as a plant defensive response to herbivory is discussed.

Key Words--Helicoverpa zea, Lepidoptera, Noctuidae, lipoxygenase, lipid peroxidation, resistance, herbivory, soybean, tomato, cotton, oxidative stress, induced defense. INTRODUCTION T h e phagocytic cells o f a n i m a l s produce reactive o x y g e n species and o t h e r oxidants as a defensive response to i n v a d i n g o r g a n i s m s (Baggliolini and W y m a n n , 1990). R e c e n t research indicates that plants also produce reactive *To whom correspondence should be addressed. ~Address = Department of Entomology, University of California, Davis, California 95616. 651 0898-033|/94/0300-0651507,0010 © 1994Plenum PublishingCorporation

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oxygen species and reactive oxidants in an analogous manner as a putative defense against pathogens and herbivores (Hildebrand et al., 1986a,b; Zacheo and Bleve-Zacheo, 1988; Apostol et al., 1989; Dillworth, et al., 1991; Doke et al., 1991; Montalbini, 1991; Felton et al., 1992a,b; 1994; Orlandi et al., 1992; Rubinstein, 1992; Vera-Estrella et al., 1992; Felton and Summers, 1993; Jiang and Miles, 1993; Legendre et al., 1993; Davis et al., 1993; Bi et al., 1994). The oxidative status of host-plant tissues may undergo a rapid shift from a reduced state to a more oxidative state in response to invasive pests. Oxidative shifts may result from increased activities of oxidative enzymes such as lipoxygenase (Hildebrand et al., 1989; Croft et al., 1990), peroxidase (Bronner et al., 1991), or polyphenol oxidase (Felton et al., 1992a). The plant's oxidative status is enhanced during the generation of reactive oxygen species such as hydroxyl radical, hydrogen peroxide, and superoxide anion that accompany certain plant-pathogen interactions (Doke et al., 1991; Sutherland, 1991; Popham and Novacky, 1991). Reactive oxygen species arise from the enzymatic activities of NAD(P)H oxidase, peroxidases, polyamine oxidases, uric acid oxidase, and xanthine oxidase (Choudhuri, 1988; Vianello and Macri, 1991; Montalbini, 1991). Enhanced oxidative status of plant tissues may proceed from a loss of chemical antioxidants such as carotenoids (Hildebrand et al., 1986a), ascorbate (Milo and Santini, 1966), glutathione and related thiols (Polle and Rennenberg), 1992), and/or decreases in antioxidant enzymes such as catalase, glutathione reductase, and superoxide dismutase (Choudhuri, 1988). In several instances, plant resistance to herbivores has been correlated with an enhanced oxidative state of plant tissues. The larval growth rate of the noctuid, Helicoverpa zea, was highly correlated with foliar polyphenol oxidase activity in tomato Lycopersicon esculentum (Felton et al., 1989). The mastication of foliage by larvae disrupts tissue integrity and the enzyme polyphenol oxidase is then allowed to interact with the substrate, chlorogenic acid (Felton et al., 1989). Polyphenol oxidase oxidizes chlorogenic acid to form the corresponding orthoquinone in the digestive system of lepidopteran larvae (Felton and Duffey, 1991b, Felton et al., 1989, 1992a). The quinone forms covalent bonds with proteins or amino acids, and thus limits amino acid bioavailability for the herbivore (Felton et al., 1989, 1992b; Felton and Duffey, 1991b). Spider mite resistance in soybean was highly correlated with enhanced lipid peroxidation and a loss ofcarotenoids due to lipoxygenases (Hildebrand et al., 1986b). Lipoxygenases (EC 1.13.1.12) may be important mediators of both insect and phytopathogen resistance (Shukle and Murdock, 1983; Hildebrand et al., 1988; Croft et al., 1990; Gardner, 1991; Ohta et al., 1991). Lipoxygenases are ubiquitous enzymes that catalyze the hydroperoxidation of polyunsaturated lipids possessing cis,cis-pentadiene moieties (Hildebrand et al., 1988). The major substrates in plant tissues are linoleic and linolenic acids (Hildebrand et al.,

LIPOXYGENASE DEFENSE AGAINST HERBIVORY

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1988). The hydroperoxide products may be chemically and/or enzymatically degrade to an army of reactive aldehydes, 3,-ketols, and epoxides (Gardner, 1991). Moreover, reactive oxygen species (e.g., singlet oxygen, hydroxyl radicals, superoxide anion) and peroxyl, acyl, and carbon-centered radicals are formed (Kanofsky and Axelrod, 1986; Chamulitrat et al., 1991). These reactive products may directly damage insect tissues, indirectly impair insect growth through damage to essential nutrients (e.g., linoleic acid, cholesterol, amino acids, fl-carotene), and/or act as feeding repellents (Shukle and Murdock, 1983; Mohri et al., 1990; Duffey and Felton, 1991). The aim of this investigation was to evaluate the potential for lipoxygenases to inhibit the growth of H. zea. The ability of lipoxygenase and the free fatty acid, linoleic acid, to reduce the nutritional quality of soy foliar protein was assessed. Additionally, the induction of lipoxygenase activity in soybean foliage following insect herbivory was studied. The role of dietary oxidative stress in plant defense against herbivory is discussed

METHODS AND MATERIALS

Insects and Plants. Eggs of H. zea were obtained from the University of Arkansas Insect Rearing Facility. Larvae were maintained on artificial diet (Chippendale, 1970) unless otherwise noted. Seeds of Lycopersicon esculentum (var. Castlemart), Lycopersicon hirsutum (LA 286), Glycine max (var. Forrest), Phaseolus vutgaris (a commercial redbean), and Gossypium hirsutum (var. DPL 50) were planted in one-gallon containers in the greenhouse. Assay of Lipoxygenase, Peroxidase, and Lipid Peroxidation. To assay for foliar lipoxygenase, leaf tissue was homogenized in 0.1 M potassium phosphate, pH 7.0, containing 1% polyvinylpolypyrrolidone. The resulting slurry was centrifuged for 20 min at 10,000g. The supernatant was used immediately as the enzyme source. Linoleic acid was used as a substrate and the rate of change in absorbance at 234 nm was measured (Grayburn et al., 1991). In soybean foliage, lipoxygenase was assayed at pH 5.5, 7.0, and 8.5 due to the presence of multiple lipoxygenase isozymes with different pH optima (Graybum et al., 1991). To assay for peroxidase, the supernatant prepared as described above was used as the enzyme source with the exception of adding 0.5 mM EDTA. Peroxidase was assayed with 0.4 mM hydrogen peroxide and 3 mM guaiacol and the absorbance monitored at 436 nm as described by MacAdam and Sharp (1992). To estimate lipid peroxidation in foliage, the thiobarbituric acid assay was used (Stewart and Bewley, 1980). Leaf tissue was homogenized in 0.1% TCA and 1% SDS followed by centrifugation at 5000g for 15 min. Aliquots of the supernatant were incubated in two volumes of 0.5 % thiobarbituric acid in 20%

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FELTON ET AL.

TCA at 95°C for 60 min. Samples were then centrifuged at 10,000g for 15 min. The absorbance of the supernatant was determined following Stewart and Bewley (1980). Assay of Lipoxygenase in Larval Midgut Lumen. To determine if lipoxygenase remains active in the larval midgut, a newly molted fifth instar was placed on a three-node stage plant for 24 hr. The following plant species were tested: L. esculentum, L. hirsutum, G. hirsutum, P. vulgaris, and G. max. Additionally, larvae were placed on bolls from maturing G. hirsutum. Lipoxygenase was assayed from these plant tissues at pH 7.0 as described above. Control insects were maintained on artificial diet. A total of seven replicates per plant species were tested. After 24 hr, leaflets with feeding damage were excised and assayed for lipoxygenase activity. Midguts were removed and lumen contents were separated from midgut wall and peritrophic membrane. Lumen contents were mixed with an equal weight of 0.1 M potassium phosphate buffer, pH 7.0, and centrifuged at 10,000g for 20 min. Supematant was used immediately as the enzyme preparation. Additionally, lumen activity was tested at pH 8.5. Effect of Midgut Enzymes on Lipoxygenase Activity. To determine if midgut enzymes affect foliar lipoxygenase activity, a foliar protein extract from L. hirsutum was incubated with larval midgut tissue. Midguts were pooled from 15 fifth instars and prepared as described by Felton and Duffey (1991a). Ten grams of foliage from L. hirsutum was homogenized and prepared for lipoxygenase assay as described above. Ammonium sulfate was slowly added to the enzyme preparation to 80% saturation at 4°C. The sample was centrifuged, the pellet was resuspended in H20, and the ammonium sulfate was removed from the supematant via a desalting column (PD-10; Pharmacia LKB, Uppsala, Sweden). The desalted protein extract was used immediately. Midgut preparations were mixed with the plant protein extract and immediately assayed for lipoxygenase activity at pH 8.5 (0.05 mM potassium phosphate). This pH was chosen because it is within the range normally found in the midgut lumen. The following concentrations of midgut protein were mixed with 100 ~tg of plant protein: 17, 34, 51, and 68/~g. A control containing no midgut protein was also tested. Protein was measured by the method of Stoscheck (1990) with the addition of 1% soluble polyvinylpolypyrrolidone to the Coomassie dye reagent. Bovine serum albumin was used as a standard for midgut protein and dialyzed D-ribulose 1,5-diphosphate carboxylase (Sigma Chemical. Co., St. Louis, Missouri) for a plant protein standard. To determine if protease activity in the midgut affected lipoxygenase, 50/~g soybean trypsin inhibitor (Kunitz, Type l-S; Sigma Chemical) was mixed with 51 p.g of midgut protein prior to testing its effect on lipoxygenase. The experiment was replicated three times. Induction of Lipoxygenase. To determine if feeding by H. zea larvae causes

LIPOXYGENASE DEFENSE AGAINST HERBIVORY

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an increase in the activity of foliar lipoxygenases, one fourth-instar was placed on each V3 stage soybean plant grown in the greenhouse. Each plant was covered with a screen cage to prevent larval escape. Five plants were treated with larvae and five control plants were treated identically except that larvae were excluded. After three days, fully expanded leaflets were excised from the uppermost node and assayed for peroxidase and lipoxygenase at pH 5.5, 7.0, and 8.5. Lipid peroxidation products were measured as described above following Stewart and Bewley (1980). The experiment was replicated three times. To determine if ingestion of foliage from wounded plants affected larval growth rates and midgut oxidative stress, two trifoliates also were excised from each wounded and unwounded plant and offered to a newly molted fifth-instar 1t. zea for 48 hr. Larval weight gain and amount of leaf eaten were determined and relative growth rates and consumption rates were computed following Waldbauer (1968). A total of 20 larvae was tested per treatment. After weighing larvae, midguts were extirpated, washed free of lumen contents, and homogenized. Lipid peroxidation of the midgut was determined following Stewart and Bewley (1980). Free amine content of the midgut protein was determined following the procedures of Fields (1972). Effects of Lipoxygenase on Dietary and Larval Growth. The effect of linoleic acid oxidation by lipoxygenase on protein quality was assessed in two separate experiments. In the first experiment designed to assess the effect of lipoxygenase activity on soy foliar protein quality, 600 g fresh weight leaf tissue was removed from ca. 200 V4-6 soybean plants (cv. Forrest) grown in the greenhouse. Tissue was homogenized in 2 liters of ice-cold 0.1 M potassium phosphate buffer, pH 7.0, containing 0.5 mM EDTA with 1% polyvinylpolypyrrolidone. The homogenate was filtered through Miracloth (Calbiochem, San Diego, California) and centrifuged for 30 min at 10,000g. The supernatant was removed and kept on ice while ammonium sulfate was slowly added to 80% saturation. The preparation was held on ice for 60 min and then centrifuged at 10,000g for 30 min. The pelleted protein was resuspended in 0.1 M potassium phosphate, pH 8.0, at 1 g protein/100 ml buffer and divided into six equal suspensions. To simulate the effect of linoleic acid oxidation on protein quality, three of the suspensions received 0.5 mM linoleic acid. The three linoleic acidtreated protein suspensions and three controls were stirred for 2 hr at 25°C. Following the incubation, suspensions were dialyzed at 6000-8000 mol wt cutoff for 48 hr against repeated exchanges of deionized water. The samples were frozen, freeze-dried, and incorporated into artificial diet. A 100-g preparation of artificial diet contained the following: 1 g soy foliar protein, 5.215 g cellulose, 0.685 g Vanderzant vitamins, 2.400 g agar, 0.200 g wheat germ oil, 3.370 g dextrose, 2.750 g wheat germ, 0.900 g Wesson salts, 0.425 g alginic acid, 0.365 g ascorbate, 0.180 g cholesterol, 0.090 g choline

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chloride, 0.010 g streptomycin, 0.120 g aureomycin, 0.200 g methyl paraben, 0.90 g sorbic acid, and 82 ml distilled water. The free amine content (primarily e-NH2 of lysine) of the treated proteins was determined following procedure of Fields (1972). Protein content was determined following the modification of Stoscheck (1990) described above with dialyzed D-ribulose 1,5-diphosphate carboxylase (Sigma Chemical) as a protein standard. Neonate H. zea larvae were individually placed in 18.5-ml clear plastic cups containing diet made with appropriately treated protein. Twenty larvae were tested per treatment, and the experiment was replicated three times. Larvae were weighed to the nearest 0.1 mg after 16 days. In a second experiment to assess the effect of purified soybean lipoxygenase on soybean protein quality, 1 g soybean protein (ICN Biomedicals Inc., Costa Mesa, California) was incubated in a 100-ml buffered solution (pH 8.0, 0.1 M potassium phosphate) with 2 mM linoleic acid, 100/~l Tween 20, and 100 mmol/ rain of purified soybean lipoxygenase (Type V-affinity purified; Sigma Chemicad) for 30 min with mechanical stirring. The control treatment was treated identically with the exception that lipoxygenase was not added. Following the incubation, suspensions were dialyzed at 6000-8000 mol wt cutoff for 48 hr against repeated exchanges of deionized water. The samples were frozen, freeze-dried, and incorporated into artificial diet as described above. Bioassays were conducted as described and the experiment was replicated three times. Statistics. Data were analyzed by ANOVA using CoStat Software (Berkeley, California) and means were separated using a studentized t-test.

RESULTS

Assay of Lipoxygenase in Larval Midgut Lumen. Preliminary data indicated that maximal lipoxygenase activity for most plant species tested occurred at pH 7.0, with the exception of the soybean cultivar Forrest. Subsequently all measurements of plant activity were made at pH 7.0. The levels of lipoxygenase varied considerably among the various host plants tested (Table 1). Activity ranged from a low of 5 nmol/min/g in the cotton to a high of 1458 nmol/min/ g in red bean. The activity of lipoxygenase in the wild tomato L. hirsutum (LA 286) was more than 25 times greater than the activity in commercial tomato. Lipoxygenase activity could not be detected in the midgut lumen contents of larvae regard!ess of the dietary host plant species or tissue ingested. Activity also was tested at pH 8.5, to determine if different lipoxygenase isozymes may retain activity in the midgut; however, activity also was not detectable at the higher pH.

657

LIPOXYGENASE DEFENSE AGAINST HERBIVORY

Effect of Midgut Enzymes on Lipoxygenase Activity. Midgut preparations significantly (P < 0.001) reduced lipoxygenase activity in the plant protein extracts from L. hirsutum (Figure 1). The addition of soybean trypsin inhibitor to the mixture effectively mitigated the negative effect of midgut tissue on lipoxygenase. In the absence of midgut tissue, the inhibitor had no effect on lipoxygenase activities. These data suggest that the digestive trypsinlike enzymes, are in large part responsible for digesting lipoxygenase and its subsequent loss in activity in the midgut lumen. Induction of Lipoxygenase and Effect on Larvae. Feeding by H. zea larvae on soybean foliage for 72 hr increased lipoxygenase activity by 2.27 x when assayed at pH 7.0 and a 1.72x when tested at pH 8.5 (P < 0.02) compared TABLE I. LIPOXYGENASE ACTIVITY IN MIDGUT LUMEN AND LARVAL DIET

Diet source"

Plant activity h

Lumen activity*'

L. esculentum (Castlemart) L. hirsutum (LA 286) G. hirsutum (DPL 50) Boll P. vulgaris G. max (Forrest) Artificial diet

26b 672d 5a 4a 1,458e 354c ND

ND ND ND ND ND ND ND

"Larvae were placed on the corresponding plant species for 24 hr prior to assay of lumen activity. Larvae were fed leaves or bolls of G. hirsutum. h LOX activity reported as nmol/min/g tissue. ND = not detectable. Means in columns not followed by the same letter are significantly different at P < 0.05.

105.

lOO~

-~

95~ 90.

~

• so p.gSll

85.

_~

80.

~

75.

~

to.

~

65. 60t . .

i 10

FIG.

20

30 40 50 Midgut Protein (pg)

60

70

1. Effect of midgut enzymes on foliar lipoxygenase activity in Lycopersicon hirsutum. STI = soybean trypsin inhibitor.

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FELTON El" AL.

to the undamaged control foliage (Table 2). Apparently, the lipoxygenase isozymes most active at pH 5.5 were less inducible as the difference between treatments was not significant (P > 0.05). Lipid peroxidation products in wounded foliage were 17.3% higher (P < 0.0009) than in unwounded foliage. Peroxidase activity was unaffected by larval feeding (P > 0.05). Larval growth rates were reduced by more than 27% (P < 0.01) when larvae fed on previously damaged plant foliage compared to control foliage (Table 3). H o w e v e r , relative consumption rates of larvae were unaffected by treatment (P > 0.05). Accompanying the decrease in larval growth rate was a 46% increase in lipid peroxidation in midgut tissues and a 9 . 1 % decrease in the free amine content o f midgut protein (Table 3). Both assays provided an index for oxidative stress and indicated that larvae ingesting wounded foliage are exposed to increased oxidative stress. The oxidative stress may be attributable to not only lipoxygenase activity, but also may be due to other biochemical changes in the redox status of the host plant. Effects of Lipoxygenase on Dietary Protein and Larval Growth. The isoTABLE 2. EFFECT OF WOUNDING ON FOLIAR CHEMISTRYa

LOXb Treatment Control

H. zea

pH 5.5

pH 7.0

pH 8.5

POD*

TBARSa

382a 591a

176a 400b

76a 131b

44,0a 45,0a

100.0a 117,3b

OMeans in columns not followed by the same letter are statistically different at P < 0.05. bLOX = lipoxygenase activity expressed as nmol/mintg foliage. cPOD = peroxidase activity expressed as A OD 436 nm/min/g foliage. aTBARS = foliar lipid peroxidation products measured as thiobarbituric acid reactive substances following Stewart and Bewley (1980). Expressed as relative percent. TABLE 3. EFFECTOF INGESTIONOF DAMAGEDSOYBEANFOLIAGEON LARVAEa Diet treatment

Midgut TBARS~

Free amines"

RGRd

RCRe

Undamaged Foliage Damaged Foliage

100a t46b

100b 90.9a

0.37b 0.27a

1.33a 1.55a

~Means in columns not followed by the same letter am statistically different at P < 0.05. bTBARS = lipid peroxidation products measured as thiobarbituric acid reactive substances following Stewart and Bewtey (1980). Expressed as relative percent. *Free amines = relative amount in midgut epithelium. aRGR = relative growth rate of 5th instar larvae expressed as mg/day/mg larva. eRCR = relative consumption rate expressed as mg biomass ingested/day/mg larva.

LIPOXYGENASEDEFENSEAGAINSTHERBIVORY

659

lated protein fractions had a lipoxygenase activity of 23.1 nmol/min/mg protein when tested with linoleic acid as substrate (Table 4). In the absence of the added linoleic acid, activity was not detectable, indicating that endogenous free fatty acids had been removed from the protein fraction. The oxidation of linoleic acid by lipoxygenase resulted in a decrease in the nutritional quality of foliar protein as indicated by a 4.3% loss of free amines (P < 0.01) and greater than 60% reduction in larval growth (P < 0.01) on diet containing the treated protein. It should be noted that the linoleic hydroperoxides formed by lipoxygenases during the protein incubation would likely undergo chemical and enzymatic degradation to other reactive molecules (e.g., aldehydes) that may, in large part, be responsible for the reduction in protein quality. When soybean protein was treated with purified lipoxygenase, the growth of larvae was reduced by 24.4% (P < 0.05; Table 5); however, the effect was less than the treatment with soy foliar protein. This may reflect that fewer secondary reactions of the lipid hydroperoxides may have occurred due to the absence of further enzymatic degradation.

TABLE4. EFFECTSOF LIPOXYGENASEACTIVITYON NUTRITIONALQUALITYOF SOY FOLIARPROTEINANDLARVALGROWTHa Treatment

LOX activityb

Relative free amines

Larval Growth~

Control +Linoleic acid

ND 23.1

100b 95.7a

163.2b 61.0a

~Means in columnsnot followedby the same letterare significantlydifferentat P < 0.01. bLOX activity = expressed as nmol/min/mgprotein;controlhas no detectable (ND) activityin the absence of added linoleicacid. CLarval growth = weight in mg after 16 days on artificialdiet.

TABLE 5. EFFECTSOF PURIFIEDSOYBEAN LIPOXYGENASEON NUTRITIONALQUALITYOF SOY PROTEINANDLARVALGROWTH Treatment

Larval growth~

Soy protein + 2 mM linoleicacid Soy protein + 2 mM linoleicacid + soybeanlipoxygenaseb

158.5b

°Larval growth = weight in nag after 16 days on artificialdiet. bSoy proteintreated with lipoxygenaseat 100 ~mol/min/gprotein.

119.8a

660

FELTON ET AL. DISCUSSION

Lipoxygenases may be components of induced resistance to herbivores in soybean. Larvae ingest increased amounts of lipid peroxidation products when feeding upon foliage from previously wounded plants (Table 2). The resultant increase in lipid peroxidation products also has been noted with mite damage (e.g., Hildebrand et al., 1986b). Induced resistance in soybean to Mexican bean beetle and loopers has been correlated with increased phenolic content and phenylalanine ammonia lyase and tyrosine ammonia lyase activities (Chiang et al., 1987; Nuepane and Norris, 1991a,b). These enzymes are the initial enzymes in the biosynthesis of phenolics. However, the phenolic isoflavonoids, glyceollins and coumestrol, may not be the major components of induced resistance to insects in soybean (e.g., Mexican bean beetle, velvetbean caterpillar, soybean looper). Recent tests conducted with these flavonoids indicate they are comparatively nontoxic at concentrations well above their natural occurrence (Hart et al., 1983; Rose et al., t988; Burden and Norris, 1992; Slansky and Wheeler, 1992). Thus induced resistance in soybean is most likely a multicomponent plant response and not associated with a single biosynthetic pathway. Lin and Kogan (1990) reported that prior herbivory by soybean loopers caused reductions in relative growth rates and developmental times of Mexican bean beetles and soybean loopers. They reported that induced resistance inhibited the relative growth rates of loopers by 3.4% and beetles by 10.7%. The magnitude of the induced resistance to H. z e a observed in our study was much greater (i.e., - 2 7 % reduction in relative growth rate) and may be due to plant genotypic differences and/or herbivore differences. Lin and Kogan (1990) indicated that loopers reduced leaf area by 26%, but the levels of damage in our studies were consistently less (unpublished data). Certain herbivores such as H. z e a may be more proficient at eliciting induced resistance than others. Preliminary data in our laboratory indicate that other defoliating herbivores may be less effective than 1t. z e a at eliciting lipoxygenases, despite producing comparable levels of defoliation. Lipoxygenases may function as plant defense proteins by affecting insect growth and development in a variety of direct and indirect manners. The products of lipoxygenase may be repellent to insect feeding and operate as antixenosis bases of resistance. Mohri et al. (1990) showed that the products, linoleic acid hydroperoxide and hexanal, acted as feeding repellents to several beetle species. Older soybean leaves have lower levels of lipoxygenase (Hildebrand et al., 1988; our unpublished data) and are the preferred feeding sites for H. z e a larvae throughout the growing season (Nault et al., 1992). However, in this study the effects of wounding and the associated induction of lipoxygenase did not significantly affect larval consumption rates (Table 3). The products of lipid peroxidation may be toxic and function in antibiosis-

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based resistance. The initial products of lipoxygenase (LOX) activity are fatty acid hydroperoxides; however, the hydroperoxides may chemically and/or enzymatically degrade to reactive aldehydes, 3,-ketols, and epoxides (Gardner, 1991). Many of these products can form Schiff base adducts with proteins or act as potent alkylating agents of macromolecules (Gardner, 1979). Chemical changes caused by the action of lipid hydroperoxides on proteins include lipid-protein adducts, amino acid damage, protein scission, and protein-protein cross-links (Gardner, 1979). Thus, dietary nutrients such as amino acids and carotenoids may be destroyed by LOX products (e.g., Hildebrand and Kito, 1984; Hildebrand et al., 1986a,b). Additionally, endogenous membrane proteins and lipids can be damaged by ingestion of these oxidative products as indicated in Table 3. Moreover, reactive oxygen species (e.g., singlet oxygen, hydroxyl radicals, superoxide anion) and peroxyl, acyl, and carbon-centered radicals are formed during lipoxygenase reactions (Kanofsky and Axelrod, 1986; Chamulitrat et al., 1991). Free radicals are implicated in numerous pathologies associated with protein, lipid, and DNA damage (Halliwell, 1991). Our data indicate lipoxygenase activity is rapidly degraded in the midgut (Table 1, Figure 1), and consequently, the toxic effects of lipoxygenase are likely due to the ingestion of preformed lipid peroxidation products that accumulate in wounded foliar tissue (see Table 2). This is in contrast to foliar polyphenol oxidase and ascorbate oxidase, which remain active in the midgut of H. z e a during feeding (Felton et al., 1989; Felton and Summers, 1993). Lipoxygenases also may be involved in indirect forms of pest resistance in plants such as interplant communication (Hildebrand et al., 1988; Farmer and Ryan, 1990). Lipoxygenase is required for the biosynthesis ofjasmonic acid, a compound that has been shown to promote cell senescence and rapidly induce the synthesis of new proteins (Enyedi et al., 1992). Jasmonic acid and its methyl ester, methyl jasmonate, induce the synthesis of several plant defensive proteins or chemicals including protease inhibitors, phenylalanine ammonia lyase, and alkaloids (Enyedi et al., 1992). Moreover, jasmonic acid affects its own biosynthesis because low concentrations of gaseous methyl jasmonate in soybean plants induce the accumulation of a 94-kDa storage protein, believed to be a lipoxygenase (Tranbarger et al., 1991; Enyedi et al., 1992). Grimes et al. (1992) confirmed that methyl jasmonate induces the accumulation of lipoxygenase in soybean seedlings. Recently, Hildebrand (1992) found that application of methyl jasmonate to tobacco leaves induces a 10-fold increase in lipoxygenase activity. It is becoming clear that induced resistance in soybean is a complex phenomenon and is associated with the induction and activation of several diverse biochemical pathways (Chiang et al., 1987; Kraemer et al., 1987; Hildebrand et al., 1988; Kogan and Fischer, 1991; Liu et al., 1992). Specifically, our findings provide further evidence that tipoxygenase is an important component of the induced response (Felton et al., 1994; Bi et al., 1994). The roles of

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FELTON ET AL.

lipoxygenases in wound repair and defense apparently evolved in primitive organisms and have been maintained in both higher plants and animals (Hildebrand et al., 1988). We suggest that lipoxygenases function in plant defense by initiating oxidative and nutritional stress to berbivores (Bi et al., 1994). The importance of oxidative stress in animal-parasite associations is well established (Baggliolini and Wymann, 1990). Recently, there has been a burst of research indicating that oxidative stress is an important mediator of many host plant-pathogen interactions (Zacheo and Bleve-Zacheo, 1988; Doke et al., 1991; Popham and Novacky, 1991; Sutherland, 1991; Orlandi et al., 1992; Vera-Estrella, 1992; Davis et al., 1993). However, our understanding of oxidative stress as a mediating factor in insect-plant interactions is in its infancy (Ahmad, 1992). Appreciation of oxidative stress in entomological research has been hampered by the misconception that oxidative stress is a slow-acting process and may be relatively insignificant for animals possessing short life-spans. Given the presence of elaborate antioxidant mechanisms in all aerobic organisms including tnfly short-lived microbes, it would seem that oxidative stress is an important phenomenon regardless of life-span. It is argued here that there are at least three potential roles for oxidative stress in antiherbivore defense: (1) direct oxidative injury to the herbivore; (2) indirect injury to the herbivore through oxidative damage to dietary lipids, proteins, vitamins, antioxidants, etc.; and (3) signal transduction for eliciting plant defensive systems. The most immediate research need is for confirmation that the causal bases of "oxidative" resistance is truly due to oxidative stresses rather than epiphenomena or secondary effects. Nevertheless, research studies are beginning to reveal that plants and animals share remarkably similar mechanisms of defense through production o f activated oxygen and reactive oxidants (Rubinstein, 1992). Acknowledgments--We wish to thank the USDA (89-37350-4639 and 92-34195-7162), American SoybeanAssociation(FY92-119) and the ArkansasSoybeanPromotionBoard for their support of this project. The reviews of Drs. D.T. Johnson and C.D. Steelman improved the manuscript. The manuscript was approvedby the Director of the ArkansasAgriculturalExperiment Station.

REFERENCES AHMAD,S. 1992. Biochemicaldefence of pro-oxidantplant allelochemicalsby herbivorousinsects. Biochem. Syst. Ecol. 20:269-296. APOSTOL,I., BOHLMANN,H., and REIMANN-PHILIPP,U.R. 1989. Rapid stimulationof an oxidative burst during the elicitation of cultured plant cells. Plant Physiol. 90:109-I 16. BAGGLIOLINI, M., and WYMANN,M.P. 1990. Turning on the respiratoryburst. Trends Biochem. Sci. 15:69-72. BI, J.L., FELTON, G.W., and MUELLER,A.J. 1994. Induced resistance in soybean to Helicoverpa zea: Role of plant protein quality. J. Chem. Ecol. 20:183-198. BRONNER,R., WESTPHAL,E., and DREGER,F. 1991. Enhanced peroxidaseactivity associatedwith

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