Sean S. Duffey

Plant proteinase inhibitors: Mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exiqua

Abstract We compared soybean trypsin inhibitor and potato proteinase inhibitor II in relation to their effects on the growth and digestive physiology of larval H. zea (Boddie) and S. exiqua (Hubner). When incorporated into an artificial diet, both proteinase inhibitors significantly reduced the growth and development of the larvae. However, when the artificial diet containing the proteinase inhibitor was supplemented with methionine, the reduction of growth did not occur. The proteinase inhibitors had no effect on the in vivo digestion of protein, indicating that they do not reduce the level of digested protein. However, when the larvae chronically ingest the proteinase inhibitors, there was significant elevation of the level of tryptic activity (the primary alimentary protease for both species of insect). Thus, we conclude that the mode of action of proteinase inhibitors is to cause the pernicious hyperproduction of trypsin. This, coupled with insufficient dietary availability of sulphur-containing amino acids (i.e. methionine) needed for enzyme synthesis, results in inhibition of growth. Thus, protein quantity/quality is very important in dictating the anti-nutritional effects of plant proteinase inhibitors on herbivorous insects.

Activation of plant foliar oxidases by insect feeding reduces nutritive quality of foliage for noctuid herbivores

The foliage and fruit of the tomato plantLycopersicon esculentum contains polyphenol oxidases (PPO) and peroxidases (POD) that are compartmentally separated from orthodihydroxyphenolic substrates in situ. However, when leaf tissue is damaged by insect feeding, the enzyme and phenolic substrates come in contact, resulting in the rapid oxidation of phenolics to orthoquinones. When the tomato fruitwormHeliothis zea or the beet army-wormSpodoptera exigua feed on tomato foliage, a substantial amount of the ingested chlorogenic acid is oxidized to chlorogenoquinone by PPO in the insect gut. Additionally, the digestive enzymes of the fruitworm have the potential to further activate foliar oxidase activity in the gut. Chlorogenoquinone is a highly reactive electrophilic molecule that readily binds cova-lently to nucleophilic groups of amino acids and proteins. In particular, the —SH and —NH2 groups of amino acids are susceptible to binding or alkylation. In experiments with tomato foliage, the relative growth rate of the fruitworm was negatively correlated with PPO activity. As the tomato plant matures, foliar PPO activity may increase nearly 10-fold while the growth rate of the fruitworm is severely depressed. In tomato fruit, the levels of PPO are highest in small immature fruit but are essentially negligible in mature fruit. The growth rate of larvae on fruit was also negatively correlated with PPO activity, with the fastest larval growth rate occurring when larvae fed on mature fruit. The reduction in larval growth is proposed to result from the alkylation of amino acids/protein byo-quinones, and the subsequent reduction in the nutritive quality of foliage. This alkylation reduces the digestibility of dietary protein and the bioavailability of amino acids. We believe that this mechanism of digestibility reduction may be extrapolatable to other plant-insect systems because of the ubiquitous cooccurrence of PPO and phenolic substrates among vascular plant species.

Exogenous jasmonates simulate insect wounding in tomato plants (Lycopersicon esculentum) in the laboratory and field.

Wounding increases the levels and activities of several defense-related proteins in the foliage of the tomato plant,Lycopersicon esculentum Mill. Evidence indicates that two of these responses, the systemic increases in polyphenol oxidase and proteinase inhibitors, are regulated by an octadecanoid-based signalling pathway which includes the wound hormone, jasmonic acid. It is not known whether other responses to wounding are also regulated by this same signalling pathway. In this paper, we show that application of jasmonates (jasmonic acid or its volatile derivative, methyl jasmonate) in low concentrations to foliage of young tomato plants induced, in a dose-dependent manner, the same protein responses-polyphenol oxidase, proteinase inhibitors, lipoxygenase, and peroxidase-as doesHelicoverpa zea Boddie feeding. Application of jasmonic acid to a single leaflet of four-leaf tomato plants induced these four proteins in a spatial pattern nearly identical to that produced by localized feeding ofH. zea. Exogenous jasmonic acid also decreased suitability of foliage for the beet armyworm,Spodoptera exigua Hubner in the laboratory. Based on these results, we conducted an experiment to measure the effects of jasmonic acid spray under field conditions. We provide the first evidence that jasmonic acid spray on field plants induces production of chemical defenses above the levels found in unsprayed controls. Exogenous jasmonic acid sprayed on plants in agricultural plots increased levels of polyphenol oxidase and proteinase inhibitors. Because application of jasmonic acid induces these defensive compounds at low concentrations in a manner similar to natural wounding, it may prove to be a useful tool for stimulating plant resistance to insects in the field.

Antinutritive and toxic components of plant defense against insects

Chemical defense of the tomato plant against noctuid larvae is argued to result from suites of interactive chemical traits that simultaneously impair the acquisition of nutrients and toxify the insect. Defense results from tomatine, catecholic phenolics and phenol oxidases, proteinase inhibitors, and lipoxygenase. The catalytic role of plant oxidative enzymes in activating a variety of defense mechanisms is discussed. It is argued that the terms “digestibility reducer,” “toxin,” and “nutrient” signify ecological outcomes, not properties of molecules. Current views on the roles and the modes of activity of plant natural products against herbivorous insects are challenged. It is proposed that chemical context and mixture are critical determinants of biological activity, and that viewing natural products as isolated defensive factors is often misleading.

Tomatine and parasitic wasps: potential incompatibility of plant antibiosis with biological control.

α-Tomatine, an alkaloid in tomato plants, is toxic to an endoparasite of a major lepidopterous pest of tomatoes. The parasite acquires the alkaloid from its host after the host ingests the alkaloid. This form of interaction creates a potential dilemma to controlling herbivorous pests through chemical antibiosis in plants.

Trade-Offs in Plant Defense Against Pathogens and Herbivores: A Field Demonstration of Chemical Elicitors of Induced Resistance

Two signaling pathways, one involving salicylic acid and another involving jasmonic acid, participate in the expression of plant resistance to pathogens and insect herbivores. In this study, we report that stimulation of systemic acquired resistance in field-grown tomato plants with the salicylate mimic, benzothiadiazole: (1) attenuates the jasmonate-induced expression of the antiherbivore defense-related enzyme polyphenol oxidase, and (2) compromises host-plant resistance to larvae of the beet armyworm, Spodoptera exigua. Conversely, treatment of plants with jasmonic acid at concentrations that induce resistance to insects reduces pathogenesis-related protein gene expression induced by benzothiadiazole, and partially reverses the protective effect of benzothiadiazole against bacterial speck disease caused by Pseudomonas syringae pv. tomato. We conclude that effective utilization of induced plant resistance to the multiple pests typically encountered in agriculture will require understanding potential signaling conflicts in plant defense responses.

Impact of oxidized plant phenolics on the nutritional quality of dietar protein to a noctuid herbivore, Spodoptera exigua

Abstract Treatment of selected dietary proteins (i.e. casein, soy protein, gluten, zein and tomato foliar protein) with chlorogenic acid and polyphenol oxidase significantly reduced protein quality to Spodoptera exigua larvae as measured by larval growth rate. The reduction in growth was negatively correlatable with the content of lysine, histidine, cystein and methionine for each protein. These amino acids contain nucleophilic centres (e.g. -SH, -NH2) that are susceptible to alkylation by quinones formed from the enzymatic oxidation of chlorogenic acid. The treatment of the proteins with chlorogenic acid and polyphenol oxidase caused significant losses in alkylatable amino acids (i.e. cysteine, histidine, methionine and lysine). The amount of chlorogenic acid bound to each protein was significantly increased by polyphenol oxidase activity and was dependent upon the content of the alkylatable amino acids. In the absence of polyphenol oxidase, the influence of alkylatable amino acids content was different. The toxicity of chlorogenic acid was directly proportional to the content of alkylatable amines in a model protein, casein. Also, the ability of chlorogenic acid to bind to protein was inversely proportional to alkylatable amine content. The importance of phenolics and phenolic oxidizing enzymes in determining protein quality to insect herbivores is discussed.

Jasmonate-mediated induced plant resistance affects a community of herbivores

1. The negative effect of induced plant resistance on the preference and performance of herbivores is a well‐documented ecological phenomenon that is thought to be important for both plants and herbivores. This study links the well‐developed mechanistic understanding of the biochemistry of induced plant resistance in the tomato system with an examination of how these mechanisms affect the community of herbivores in the field.

Specificity of induced resistance in the tomato, Lycopersicon esculentum

Abstract Specificity in the induced responses of tomato foliage to arthropod herbivores was investigated. We distinguished between two aspects of specificity: specificity of effect (the range of organisms affected by a given induced response), and specificity of elicitation (ability of the plant to generate distinct chemical responses to different damage types). Specificity of effect was investigated by examining the effect of restricted feeding by Helicoverpa zea on the resistance of tomato plants to an aphid species (Macrosiphum euphorbiae), a mite species (Tetranychus urticae), a noctuid species (Spodoptera exigua), and to a phytopathogen, Pseudomonas syringae pv. tomato. Prior H. zea feeding was found to increase the resistance of tomato plants to all four organisms. Specificity in elicitation was investigated by examining the effect of aphid feeding on the activities of four defense-related proteins and on the suitability of foliage for S. exigua. Aphid feeding was found to induce peroxidase and lipoxygenase activities but not polyphenol oxidase and proteinase inhibitor activities; this response is distinct from the response to H. zea feeding, which induces polyphenol oxidase and proteinase inhibitors but not peroxidase. Leaflets which had been fed upon by aphids were better sources of food for S. exigua than were leaflets which had not been fed upon by aphids. Studies of both these aspects of specificity are needed to understand the way in which plants coordinate and integrate induced responses against insects with other physiological processes.

Plant Proteinase inhibitors: A defense against herbivorous insects?

Using the tomato plant, Lycopersicon esculentum L., and the beet armyworm, Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae), we have demonstrated that insect herbivory induces a rapid decline in plant quality. This decline in plant quality manifests itself by a highly significant reduction in rate of larval growth on a medium containing foliage from insect‐damaged as opposed to undamaged tomato plants. The induction of tomato Proteinase inhibitors, as a result of larval feeding, is invoked as a factor that systemically reduces leaf quality.