Michael J. Stout

COSTS OF INDUCED RESPONSES AND TOLERANCE TO HERBIVORY IN MALE AND FEMALE FITNESS COMPONENTS OF WILD RADISH

Theory predicts that plant defensive traits are costly due to trade‐offs between allocation to defense and growth and reproduction. Most previous studies of costs of plant defense focused on female fitness costs of constitutively expressed defenses. Consideration of alternative plant strategies, such as induced defenses and tolerance to herbivory, and multiple types of costs, including allocation to male reproductive function, may increase our ability to detect costs of plant defense against herbivores. In this study we measured male and female reproductive costs associated with induced responses and tolerance to herbivory in annual wild radish plants (Raphanus raphanistrum). We induced resistance in the plants by subjecting them to herbivory by Pieris rapae caterpillars. We also induced resistance in plants without leaf tissue removal using a natural chemical elicitor, jasmonic acid; in addition, we removed leaf tissue without inducing plant responses using manual clipping. Induced responses included increased concentrations of indole glucosinolates, which are putative defense compounds. Induced responses, in the absence of leaf tissue removal, reduced plant fitness when five fitness components were considered together; costs of induction were individually detected for time to first flower and number of pollen grains produced per flower. In this system, induced responses appear to impose a cost, although this cost may not have been detected had we only quantified the traditionally measured fitness components, growth and seed production. In the absence of induced responses, 50% leaf tissue removal, reduced plant fitness in three out of the five fitness components measured. Induced responses to herbivory and leaf tissue removal had additive effects on plant fitness. Although plant sibships varied greatly (49–136%) in their level of tolerance to herbivory, costs of tolerance were not detected, as we did not find a negative association between the ability to compensate for damage and plant fitness in the absence of damage. We suggest that consideration of alternative plant defense strategies and multiple costs will result in a broader understanding of the evolutionary ecology of plant defense.

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.

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.

Differential induction of tomato foliar proteins by arthropod herbivores

The effects of mechanical and chemical damage and three types of biotic damage on the activities of four foliar proteins of the tomato plant (Lycopersicon esculentum Mill var. Castlemart) were assayed. Proteinase inhibitor, polyphenol oxidase, peroxidase, and lipoxygenase activities were assayed in damaged leaflets and compared with activities in undamaged leaflets. These proteins are putative plant defenses in tomato. Differential induction of these proteins by the various damage-treatments was demonstrated, such that different subsets of the four proteins were induced by different types of damage. This study clearly demonstrates the ability of plants to respond differentially to different types of damage. Possible mechanisms for this differential induction and the implications of differential induction for plant defense are discussed.

Effect of Nitrogen Availability on Expression of Constitutive and Inducible Chemical Defenses in Tomato, Lycopersicon esculentum

Young tomato plants (Lycopersicon esculentum) grown in sand in a greenhouse and subjected to different fertilization regimes were used to test the effects of nitrogen availability on constitutive levels of phenolics and on constitutive and inducible activities of polyphenol oxidase and proteinase inhibitors. Theories that emphasize physiological constraints on the expression of phytochemicals predict an increase in levels of carbon-based allelochemicals under moderate nitrogen stress but predict, under the same conditions, an attenuation of chemical responses involving nitrogen-containing compounds such as proteinase inhibitors and polyphenol oxidase. We found that nitrogen availability had a strong effect on constitutive levels of phenolics; weaker effects on constitutive polyphenol oxidase activity, constitutive proteinase inhibitor activity, and inducible polyphenol oxidase activity; and no effect on inducible proteinase inhibitor activity. These results point to a need for the integration of theories that emphasize physiological influences on secondary metabolism with those that emphasize ecological influences on secondary metabolism and suggest that current theories of plant defense do not adequately account for enzymatic and proteinaceous defenses against arthropods.

Drought stress in tomatoes : changes in plant chemistry and potential nonlinear consequences for insect herbivores

Insect herbivores respond variably to drought-stressed plants; in some cases herbivore performance (e.g. fecundity, survival) is enhanced, in others it is negatively affected. We used tomato plants to examine the consequences of drought stress intensity on plant chemistry potentially important to insect herbivores in order to gain insights into resolving these conflicting results. In a series of experiments conducted out-of-doors, we measured the concentration of five chemicals previously shown to act as growth reducers against herbivores (2 oxidative enzymes, 2 phenolics, proteinase inhibitor) in control plants and drought-stressed plants over a period of about 14 d. Three of the five defenses increased in response to increasing drought (polyphenol oxidase, rutin, chlorogenic acid), one showed no response (peroxidase) and one was inconsistent in its response (proteinase inhibitor). Simultaneously, we measured three chemicals thought to be important nutrients for insect herbivores (water content, total nitrogen, and soluble carbohydrates). Water content declined with increasing drought stress intensity, total nitrogen increased at severe stress in one experiment and did not respond in a second, and soluble carbohydrates showed a convex response to drought stress. In one experiment we also fed leaf tissue, incorporated into non-nutritive agar, from control and drought-stressed plants to neonate Spodoptera exigua larvae. Growth of these larvae over a 7-d period was negatively affected by drought stress while survival was not significantly affected. This study represents one of the first to assess both chemical defenses and nutrients in plants subjected to drought as it develops from slight stress to a very severe condition. Depending on the direction and shape of response curves for nutrients and defenses, many possible effects on herbivores are predicted, some of them complex and nonlinear. By studying the particular kinetics of defenses and nutrients in plants we may be in a better position to understand why insect herbivores show such variable responses to drought-stressed plants.

Identity, spatial distribution, and variability of induced chemical responses in tomato plants

Using four‐leaf tomato plants (Lycopersicon esculentum Mill) as a model system, we examined the spatial distribution of damage‐induced changes in foliar protein activities. Terminal leaflets of third leaves of tomato plants were subjected to one of four types of damage, and the activities of four putative defenses – polyphenol oxidase, peroxidase, lipoxygenase, and proteinase inhibitors – were determined at four leaflet positions relative to the damaged leaflet. Multiple proteins were differentially induced by the different damage types. For a given damage type, the spatial pattern of induction was different for different proteins. More exhaustive spatial mapping of the polyphenol oxidase response to feeding by Helicoverpa zea Boddie revealed that damaged plants were more variable, both within and between plants, in the activity of this enzyme than undamaged plants. The spatial patterns of induction of these four putative defenses throughout the plant suggest that the induced plant is chemically heterogeneous and that different mechanisms of defense operate in different regions of the plant. These data are critical to an elucidation of cause‐effect relationships between induced chemicals and induced resistance in tomato foliage. In addition, these data suggest that induction functions, in part, to increase chemical variation in tomato plants; the potential role of phytochemical variation in plant defense is discussed.

Stimulation and attenuation of induced resistance by elicitors and inhibitors of chemical induction in tomato (Lycopersicon esculentum) foliage

Elicitors and inhibitors of chemical induction were used to manipulate the activities of several putative defense‐related proteins in leaves of the tomato, Lycopersicon esculentum Mill. The four presumptive defenses manipulated were proteinase inhibitors, polyphenol oxidase, peroxidase, and lipoxygenase. The elicitors used were jasmonic acid, methyl jasmonate, ultraviolet light, and feeding by larvae of the noctuid, Helicoverpa zea Boddie; the inhibitors used were salicylic acid and acetylsalicylic acid. These chemical manipulations were combined with short‐term growth assays using larvae of the generalist noctuid, Spodoptera exigua Hubner, in order to assess the relative roles of the proteins in induced resistance to S. exigua. When activities of proteinase inhibitors and/or polyphenol oxidase in leaf tissue were high (e.g., in damaged or elicited plants), growth rates of larvae of S. exigua were low; when activities of polyphenol oxidase and proteinase inhibitors were low (e.g., in undamaged or damaged, inhibited plants), growth rates of larvae were high. In contrast, high activities of peroxidase and lipoxygenase were not associated with decreases in suitability of leaf tissue for S. exigua. The association of high levels of proteinase inhibitors and polyphenol oxidase with resistance to S. exigua– irrespective of the presence or absence of damage – strongly implicates these proteins as causal agents in induced resistance to S. exigua.