Temporal and ontogenetic aspects of protein induction in foliage of the tomato, Lycopersicon esculentum

Damage to foliage of the tomato, Lycopersicon esculentum, causes the induction of proteinase inhibitors and of the oxidative enzymes polyphenol oxidase, peroxidase, and lipoxygenase. The time courses of induction of these proteins by feeding of two caterpillar species (Manduca sexta and Helicoverpa zea) were studied in a series of experiments. In another series of experiments, the effects of plant age on the inducibility of these proteins were studied. In the time course experiments, induction of proteinase inhibitors and oxidative enzymes in the damaged leaflet was rapid, with higher protein activities evident in damaged leaflets within 12–24 h of damage, depending on the enzyme and the species of insect used to damage the plant. Systemic induction of proteinase inhibitors was also rapid, but systemic induction of polyphenol oxidase was delayed relative to systemic induction of proteinase inhibitors, possibly because high constitutive polyphenol oxidase activities obscured expression of systemic induction at earlier time points. Lipoxygenase and peroxidase were not induced systemically. Induction of all proteins persisted for at least 21 days. In the phenology experiments, inducibility of all proteins decreased in magnitude and was less consistent as plants aged. The results of these experiments exemplify the numerous constraints on induction in tomato plants. Knowledge of these physiological constraints is important to an understanding of the ecological role and causal basis of induced resistance.     

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.     

Characterization of induced resistance in tomato plants

We have characterized, using several types of bioassays, the resistance induced in young tomato plants by feeding of the corn earworm, Helicoverpa zea. Beet armyworm larvae, Spodoptera exigua, and leafminers, Liriomyza trifolii, were used to assay the induced resistance. In whole-plant experiments, damage localized to a single leaflet of fourleaf tomato plants induced a systemic increase in resistance such that beet armyworm larvae confined to previously damaged (induced) plants grew at a rate about half that of larvae raised on control plants and consumed less leaf tissue from induced plants than from control plants. In experiments using excised leaves, beet armyworm larvae suffered increased mortality when reared on leaves from induced plants. The strength of this induced resistance varied spatially relative to the damaged position; moreover, the spatial distribution of induced resistance changed over a three-week period following damage. Other experiments demonstrated that the mechanisms of induced resistance in tomato foliage involves both a decrease in larval preference for and a decrease in the nutritional value of induced foliage. Induction also retarded the oviposition and/or early development of leafminers. Thus, induced resistance has relatively severe effects on the biology of subsequent herbivores. These data should allow us to begin to elucidate cause-effect relationships between induced resistance and induced chemistry in tomato plants.     

Nitrogen and water affect direct and indirect plant systemic induced defense in cotton

Plants have direct and indirect constitutively produced and inducible defenses against herbivores and pathogens, which can substantially aid in their ability to defend themselves. However, very little is known about the influence of agronomic factors on such defenses. Here, we tested the effects of nitrogen levels and water availability on the ability of cotton plants to deter feeding by Spodoptera exigua through induction of anti-feedants, and to attract Microplitis croceipes through systemic induction of volatile emission. Cotton plants were grown with various nitrogen levels and were either exposed to water stress or normal water before being exposed to S. exigua for 48 h for induction of defenses. Dual choices of various nitrogen and water treatments were provided to M. croceipes in flight tunnel bioassays. Dual choices of leaf tissue from the various nitrogen and water treatments were provided to S. exigua larvae. Both water stress and nitrogen levels under and over the recommended levels increased leaf tissue consumption and decreased attraction of M. croceipes to the plants. Analyses of induced volatiles released from herbivore damaged plants indicate that their concentrations differ among the nitrogen levels tested with plants receiving no nitrogen or twice the recommended dose having amounts much lower than plants receiving the recommended dose. Because both direct and indirect plant defense mechanisms are negatively affected by improper nitrogen and insufficient water, we argue that these factors should be considered for a better natural control of pests in cotton and most probably in other crops.     

Variation in damage to cotton affecting larval feeding preference of Spodoptera littoralis

Feeding experiments with larvae of Spodoptera littoralis were performed with leaves from cotton plants subjected to damage and from undamaged plants. In the experiments, four different time intervals (1, 3, 7, and 14 days) after damage induction and two different levels (high and low) of herbivore damage were tested. Seven days after damage induction larvae fed less on the young top leaves from damaged plants for both levels of damage. At the high damage level, the larvae fed less on leaves from the damaged plants after just three days, and this effect still remained 14 days after damage infliction. When mature leaves from the middle of the plant were compared, no difference between treatments was observed.Two plant sizes were tested, small plants with 4–5 true leaves and large plants with 8–10 true leaves. In small plants the induced changes affecting larval feeding were found mainly in the youngest leaf at the top of the plant, while in large plants the induced effects were found in both the youngest and the second youngest leaves.In plants subjected to artificial damage, larvae fed less on top leaves of the damaged plants when compared to leaves from undamaged plants. When leaves from plants that had been artificially damaged were directly compared with leaves from plants damaged by herbivores, larvae fed more on the youngest leaves from artificially damaged plants when the plants were large. In small plants no significant difference was found when comparing artificial and herbivore damage.     

Induction of defense responses in tomato against Pseudomonas syringae pv. tomato by regulating the stress ethylene level with Methylobacterium oryzae CBMB20 containing 1-aminocyclopropane-1-carboxylate deaminase

This study aimed to examine the induction of defense responses in tomato elicited by Methylobacterium oryzae CBMB20 as a consequence of reduced stress ethylene level possibly through its ACC deaminase activity. Significantly increased activities of pathogenesis-related (PR) proteins and defense enzymes such as β-1,3-glucanase, phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase were noted in M. oryzae CBMB20 pretreated and challenged with Pseudomonas syringae pv. tomato (Pst) compared to either control or M. oryzae-treated tomato plants in both growth chamber and greenhouse conditions. Increased PR proteins and defense enzyme activities were correlated with the reduction of stress ethylene level. M. oryzae CBMB20 reduced the stress ethylene level about 27% and 55% when challenged with Pst, in growth chamber and greenhouse on day 7 respectively and the effect was comparable to that of the chemical ethylene biosynthesis inhibitor AVG, L-α-(2-aminoethoxyvinyl)-glycine hydrochloride. As a consequence of reduced stress ethylene level and its effect on defense response in crop plants, the disease severity was reduced 26% in M. oryzae CBMB20-treated plants challenged with pathogen. Therefore, inoculation of M. oryzae CBMB20 would induce the defense enzymes and contribute to the enhanced resistance of tomato plants against the pathogen Pst.     

Fitness costs of jasmonic acid-induced defense in tomato, Lycopersicon esculentum

The resource allocation hypothesis is based on the assumption that defenses are costly, but relatively few studies have quantified the reproductive price of induced defenses, which represent the best means of measuring such costs in isolation from the genotypic costs that confound research involving constitutive defenses. Jasmonic acid (JA) is a plant signal molecule involved in the defensive responses of plants. It induces many of the same chemicals that are associated with herbivore damage, and thus offers a means of inducing plants without the removal of leaf area, which incurs its own costs. In tomato plants, JA induced resistance to Manduca sexta and increased levels of two defensive enzymes, polyphenol oxidase and peroxidase. We measured the impact of JA-induced defenses in tomato, Lycopersicon esculentum (Solanaceae), on several variables associated with reproductive success: fruit number, fruit weight, ripening time, time of fruit-set, number of seeds per fruit, total seeds per plant, the relationship between fruit weight and seed number, and germination success. Plants were grown in a pest-free greenhouse and treated biweekly with solvent or with JA at either of two concentrations: 10 mM or 1 mM. The high concentration of JA led to fewer but larger fruits, longer ripening time, delayed fruit-set, fewer seeds per plant, and fewer seeds per unit of fruit weight. The reproductive impact of induction was reduced at the lower dose, but still significant; 1 mM JA resulted in delayed fruit-set and fewer seeds per unit of fruit weight, compared to control plants. Our research indicates that JA-induced defenses impose significant costs on tomato plants.     

Timing of induced resistance in a clonal plant network

After local herbivory, plants can activate defense traits both at the damaged site and in undamaged plant parts such as in connected ramets of clonal plants. Since defense induction has costs, a mismatch in time and space between defense activation and herbivore feeding might result in negative consequences for plant fitness. A short time lag between attack and defense activation is important to ensure efficient protection of the plant. Additionally, the duration of induced defense production once the attack has stopped is also relevant in assessing the cost–benefit balance of inducible defenses, which will depend on the absence or presence of subsequent attacks. In this study we quantified the timing of induced responses in ramet networks of the stoloniferous herb Trifolium repens after local damage by Mamestra brassicae larvae. We studied the activation time of systemic defense induction in undamaged ramets and the decay time of the response after local attack. Undamaged ramets became defense‐induced 38–51 h after the initial attack. Defense induction was measured as a reduction in leaf palatability. Defense induction lasted at least 28 days, and there was strong genotypic variation in the duration of this response. Ramets formed after the initial attack were also defense‐induced, implying that induced defense can extend to new ramet generations, thereby contributing to protection of plant tissue that is both very vulnerable to herbivores and most valuable in terms of future plant growth and fitness.     

Accumulation of gentisic acid as associated with systemic infections but not with the hypersensitive response in plant-pathogen interactions

In the present work we have studied the accumulation of gentisic acid (2,5-dihydroxybenzoic acid, a metabolic derivative of salicylic acid, SA) in the plant-pathogen systems, Cucumis sativus and Gynura aurantiaca, infected with either prunus necrotic ringspot virus (PNRSV) or the exocortis viroid (CEVd), respectively. Both pathogens produced systemic infections and accumulated large amounts of the intermediary signal molecule gentisic acid as ascertained by electrospray ionization mass spectrometry (ESI-MS) coupled on line with high performance liquid chromatography (HPLC). The compound was found mostly in a conjugated (β-glucoside) form. Gentisic acid has also been found to accumulate (although at lower levels) in cucumber inoculated with low doses of Pseudomonas syringae pv. tomato, producing a nonnecrotic reaction. In contrast, when cucumber was inoculated with high doses of this pathogen, a hypersensitive reaction occurred, but no gentisic-acid signal was induced. This is consistent with our results supporting the idea that gentisic-acid signaling may be restricted to nonnecrotizing reactions of the host plant (Bellés et al. in Mol Plant-Microbe Interact 12:227–235, 1999). In cucumber and Gynura plants, the activity of gentisic acid as inducing signal was different to that of SA, thus confirming the data found for tomato. Exogenously supplied gentisic acid was able to induce peroxidase activity in both Gynura and cucumber plants in a similar way as SA or pathogens. However, gentisic-acid treatments strongly induced polyphenol oxidase activity in cucumber, whereas pathogen infection or SA treatment resulted in a lower induction of this enzyme. Nevertheless, gentisic acid did not induce other defensive proteins which are induced by SA in these plants. This indicates that gentisic acid could act as an additional signal to SA for the activation of plant defenses in cucumber and Gynura plants.     

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.