Gary W. Felton

Foliar oxidative stress and insect herbivory: Primary compounds, secondary metabolites, and reactive oxygen species as components of induced resistance

Oxidative responses of plants to pathogens and other environmental stresses have received considerable recent attention. We propose that an oxidative response also occurs following attack by herbivores. Our data strongly indicate a shift in the oxidative status of soybean following herbivory by the insectHelicoverpa zea. Herbivory caused significant increases in lipid peroxidation and ·OH radical formation. The activity of several oxidative enzymes including lipoxygenases, peroxidase, diamine oxidase, ascorbate oxidase, and NADH oxidase I increased after herbivory on soybean. The enhanced production of phenolic compounds is indicated by an increase in the activity of phenylalanine ammonia lyase in wounded tissues. On the other hand, the level of soybean foliar antioxidants such as ascorbic acid, total carotenoids, nonprotein thiols, and catalase decreased significantly following herbivory. These results implicate primary compounds (e.g., ascorbic acid, proteins), secondary metabolites (e.g., phenolics), and reactive oxygen species (e.g., hydroxyl radical, hydrogen peroxide) as multiple components of induced resistance. The oxidative changes in the host plant correspond with increased oxidative damage in the midgut of insects feeding on previously wounded plants. Decreases in nonprotein thiols and reduced ascorbic acid occurred in midgut epithelial tissue from insects feeding on wounded plants compared to the insects on control plants. In contrast, midgut hydroperoxides and dehydroascorbic acid concentrations were greater in insects on wounded plants compared to their counterparts on control plants. We conclude that oxidative responses in soybean may have both positive and negative effects upon the host plant: a decrease in herbivory and an increase in oxidative damage to the plant. The salient benefit to the plant, in terms of insect resistance, is the relative balance between these opposing effects.

Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory

Pre-inoculation of plants with a pathogen that induces necrosis leads to the development of systemic acquired resistance (SAR) to subsequent pathogen attack [1]. The phenylpropanoid-derived compound salicylic acid (SA) is necessary for the full expression of both local resistance and SAR [2] [3]. A separate signaling pathway involving jasmonic acid (JA) is involved in systemic responses to wounding and insect herbivory [4] [5]. There is evidence both supporting and opposing the idea of cross-protection against microbial pathogens and insect herbivores [6] [7]. This is a controversial area because pharmacological experiments point to negative cross-talk between responses to systemic pathogens and responses to wounding [8] [9] [10], although this has not been demonstrated functionally in vivo. Here, we report that reducing phenylpropanoid biosynthesis by silencing the expression of phenylalanine ammonialyase (PAL) reduces SAR to tobacco mosaic virus (TMV), whereas overexpression of PAL enhances SAR. Tobacco plants with reduced SAR exhibited more effective grazing-induced systemic resistance to larvae of Heliothis virescens, but larval resistance was reduced in plants with elevated phenylpropanoid levels. Furthermore, genetic modification of components involved in phenylpropanoid synthesis revealed an inverse relationship between SA and JA levels. These results demonstrate phenylpropanoid-mediated cross-talk in vivo between microbially induced and herbivore-induced pathways of systemic resistance.

Trade-offs between pathogen and herbivore resistance

During the past year genetic and pharmacological experiments have revealed a molecular basis for the cross-talk between signaling pathways mediating pathogen and herbivore resistance. These findings provide considerable insight into the apparently contradictory results reported for trade-offs between pathogen and herbivore resistance.

Salivary Glucose Oxidase: Multifunctional Roles for Helicoverpa zea?

Labial glands of Helicoverpa zea produced a glucose oxidase (GOX), which was present in the saliva and midgut lumen. We purified GOX 23-fold by isoelectric focusing of labial gland homogenates and investigated physical and kinetic properties of the enzyme. D-glucose and 6-deoxy-D-glucose were the optimal substrates of 22 carbohydrates tested with GOX. The enzyme was not inhibited by several inhibitors of fungal GOX but was sensitive to HgCl(2). Labial gland GOX activities varied daily during larval development with highest activities found when larvae were actively feeding. Based on pH optimum, pI, molecular weight estimate and K(m(glucose)), the insect enzyme is not derived from fungal GOXs but appears to have similar kinetic and physical attributes to other insect GOXs. Some possible functions are discussed, including antimicrobial properties, manipulating midgut O(2) levels, altering host plant defense responses, and metabolizing carbohydrates. Arch. Copyright 1999 Wiley-Liss, Inc.

Oxidative responses in soybean foliage to herbivory by bean leaf beetle and three-cornered alfalfa hopper.

Variation in induced responses in soybean is shown to be dependent, in part, upon herbivore species. Herbivory by the phloem-feeding three-cornered alfalfa hopper caused increases in the activities of several oxidative enzymes including lipoxygenases, peroxidases, ascorbate oxidase, and polyphenol oxidase. Bean leaf beetle defoliation caused increased lipoxygenase activity, but had little effect upon peroxidase, polyphenol oxidase, ascorbate oxidase, or trypsin inhibitor levels in either field or greenhouse studies. In one field experiment, prior herbivory by the bean leaf beetle subsequently reduced the suitability of foliage to the corn earwormHelicoverpa zea. The contribution of these findings to emerging theories of insect-plant interactions is discussed.

Antinutritive and Oxidative Components as Mechanisms of Induced Resistance in Cotton to Helicoverpa zea

Induced resistance in cotton (Gossypium hirsutum) foliage and squares to herbivory by Helicoverpa zea (Lepidoptera: Noctuidae) is reported in this study. Induced resistance was indicated by decreased larval growth when larvae fed on previously damaged foliage or squares compared to the controls. Herbivory caused a significant decline in host nutritional quality as shown by a reduction in protein and most amino acids in both foliage and squares. Peroxidase, ascorbate oxidase, and diamine oxidase activities increased in both damaged foliage and squares, whereas levels of the nutritional antioxidant, ascorbate, were depressed after larval feeding. Larval feeding also markedly enhanced lipoxygenase activity and lipid peroxides in square tissues. Moreover, feeding damage altered the quantitative levels of phenolic compounds in foliage and squares. These results indicate a significant shift in the oxidative status of cotton plants following herbivory as indicated by increased oxidative enzyme activity, decreased levels of the nutritional antioxidant ascorbate, and increased levels of phenolic prooxidants (i.e., chlorogenic acid) and lipid peroxides.

NUTRITIVE QUALITY OF PLANT PROTEIN : SOURCES OF VARIATION AND INSECT HERBIVORE RESPONSES

Protein quality has received comparatively little attention as a factor in host plant suitability for insects. It is argued here that plant protein quality is subject to considerable variation from genetic and environmental influences and thus may significantly impact herbivore performance. Furthermore, other phytochemicals that are ingested with protein may negatively impact protein utilization. There is a wide distribution of alkylating agents found in plants (e.g., quinones, phenolics, aldehydes, pyrrolizidine alkaloids, sesquiterpene lactones, isothiocyanates) that form covalent bonds with nucleophilic side chains of proteins (e.g., -SH, -NH, -NH2) and potentially limit amino acid availability. The behavioral and physiological adaptations of insects to variation in protein quality are also discussed. Finally, preliminary evidence for physiological adaptation to low protein quality in Helicoverpa zea is provided. The potential role of protein quality in host plant specialization is summarized.

Ascorbate peroxidase: A novel antioxidant enzyme in insects

larvae. We thank T. Meade for his thorough and invaluable editing. We also thank D. Johnson,and P. Mathews for their efforts as reviewers. This work was approved for publication by theDirector, Arkansas Agricultural Experiment Station, manuscript 95140.Sponsored by USDA-NRICGP (93-37302-9571).*Correspondence to: M. Claravon Mathews, Department of Entomology, 321 Agriculture Bldg.,University of Arkansas, Fayetteville AR 72701.Received 16 November 1995; accepted 29 May 1996.Abbreviations used: AFR = ascorbate free radical; AFRR = ascorbate free radical reductase; APOX= ascorbate peroxidase; ASC = ascorbate; DHA = dehydroascorbate; DHAR = dehydroascorbatereductase; EDTA = ethylenediaminetetraacetic acid; GPOX = glutathione peroxidase; GSH =glutathione; H

Potential role of lipoxygenases in defense against insect herbivory

The potential role of the plant enzyme lipoxygenase in host resistance against the corn earwormHelicoverpa zea was examined. Lipoxygenase is present in most of the common host plants ofH. 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 byH. 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.

Induced resistance in soybean toHelicoverpa zea: Role of plant protein quality

Resistance in soybean toHelicoverpa zea is comprised of both constitutive and inducible factors. In this study, we investigated the induction of resistance byH. zea in both greenhouse and field studies. In a greenhouse experiment, fourth-instarH. zea growth rates were reduced by 39% after 24 hr feeding and by 27% after 48 hr when larvae fed on previously wounded V3 foliage (cv. Forrest) compared with undamaged foliage. In a field study, the weight gain by larvae was more than 52% greater when larvae fed for 72 hr on undamaged R2/R3 soybean plants (cv. Braxton) compared to those that fed on previously wounded plants. A significant component of the induced resistance is due to a decline in the nutritional quality of foliar protein following foliar damage byH. zea. Foliar protein was extracted from damaged and undamaged foliage and incorporated into artificial diets. Larval growth was reduced 26% after four days and 49% after seven days on diets containing protein from damaged plants compared to larvae feeding on foliar protein from undamaged plants. Chemical analyses of protein quality also indicated a decline in quality in damaged plants compared to unwounded plants. Increases in lipoxygenase activity (53%), lipid peroxidation products (20%), and trypsin inhibitor content (34%) were observed in protein from wounded plants. Moreover, a 5.9% loss in free amines and 19% loss in total thiols occurred in protein from wounded plants. Larval feeding causes a significant increase in foliar lipoxygenase activity that varied among genotypes. Lipoxygenase isozymes were measured at pH 5.5, pH 7.0, and pH 8.5 in V3 stage plants of Forrest, Hark, D75-1069, and PI 417061 genotypes. Lipoxygenase activity in each genotype was significantly increased after 72 hr of larval feeding at each pH level tested, with the exception of lipoxygenase isozymes at pH 5.5 in genotype PI 417061. Larval feeding on R2/R3 stage plants (field-grown cv. Braxton) for six days also increased foliar lipoxygenase activity.