Increasing insight into induced plant defense mechanisms using elicitors and inhibitors

One of the strategies that plants employ to defend themselves against herbivore attack is the induced production of carnivore-attracting volatiles. Using elicitors and inhibitors of different steps of the signal-transduction pathways can improve our understanding of the mechanisms underlying induced plant defenses. For instance, we recently showed that application of jasmonic acid, a key hormone in the octadecanoid pathway involved in herbivore-induced defense, to Brassica oleracea affects gene expression, hormone levels, and volatile emission, as well as oviposition by herbivores and host location behavior by parasitoids. Such defense responses vary with the dose of the elicitor and with time since application. This addendum describes how the use of inhibitors, in addition to the use of elicitors like jasmonic acid, can be applied in bio-assays to investigate the role of signal-transduction pathways involved in induced plant defense. We show how inhibition of different steps of the octadecanoid pathway affects host location behavior by parasitoids.Key words: Brassica oleracea, Cotesia glomerata, elicitor, herbivore-induced plant defense, inhibitor, jasmonic acid, octadecanoid pathway, phenidone, propyl gallate, diethyldithiocarbamic acid (DIECA)Chemical information plays an important role in the interactions between plants and insects. When a plant is damaged, it can respond with the production of specific volatiles and toxins.¹ Insects associated with these plants can use the resulting chemical information to find their host plants and to determine the suitability of a plant for feeding or oviposition. Application of chemicals acting as elicitors can be used to mimic such plant responses, while knowledge of the signal transduction pathways involved can be used to select potential inhibitors of induced plant response. Compared to exposing plants to herbivores, the application of elicitors and inhibitors allows for manipulation of defined steps in signal-transduction pathways, as well as to induce plants in a dose-controlled manner.² However, also with elicitors and inhibitors it is often difficult to link the applied dose to the strength of the induction of the plant, as the plant may use alternatives routes to express certain traits and the manipulation can result in unwanted effects on other processes in the plant, such as flowering or senescence.^3,4 Hence, experiments using elicitors or inhibitors should preferably use rather short incubation times (hours to days), to avoid developmental differences due to treatment.⁵JA is a key hormone in the octadecanoid pathway, involved in direct as well as indirect plant defenses against herbivores. Application of this phytohormone is known to mediate induction of volatile emission, increase toxin levels and to upregulate defense gene expression. In turn, the changes in these plant traits affect members of the insect community associated with the plant and may result in higher parasitism rates of herbivores, attraction of predators, and reduced oviposition and development of herbivores.^6–12JA is often used to mimic herbivory in studies on induced plant responses. However, recent studies on JA-application to e.g., Brassica oleracea var. gemmifera L. (Brussels sprouts) also indicate that the JA-induced volatile emission differs from volatile emission induced by herbivores.¹² More nectar was secreted by flowers of herbivore-infested Brassica nigra L. (black mustard) than by flowers from JA-induced plants.⁶ The intensity of the behavioral responses of herbivores and parasitoids differs between JA- and herbivore-induced plants, but compared to non-induced plants, both treatments are favored by parasitoids on both Brussels sprouts and black mustard plants,^6,12 while Pieris butterflies avoid oviposition on induced Brussels sprouts plants.¹¹ The results indicate that JA-mediated responses do play an important role in plant defense against herbivorous insects, and can be used to induce defense responses in many plant species. However, cross-talk with other phytohormones, as well as visual cues will also affect plant defense responses.While JA application induces the octadecanoid pathway, inhibitors of steps in this pathway are also available (Fig. 1). This approach allows including visual cues of feeding damage while eliminating or reducing chemical cues. Three inhibitors of different steps of the octadecanoid pathway are phenidone (1-phenyl-pyrazolidinone), DIECA (diethyldithiocarbamic acid) and n-propyl gallate (3,4,5-trihydroxybenzoic acid propyl ester; all obtained from Sigma-Aldrich, St. Louis, MO; Fig. 1). The redox-active compound phenidone is known to inhibit the activity of lipoxygenases (LOXs),^13–15 by reducing the active form of LOX to an inactive form. Therefore, phenidone is an effective inhibitor of an early step in the octadecanoid pathway, and thus of the plant’s induced defense system.^16,17 DIECA reduces 13-hydroperoxylinolenic acid to its corresponding alcohol 13-hydroxylinolenic acid, which is not a signaling intermediate and cannot be converted into JA.^18–20 Propyl gallate is a less specific inhibitor inhibiting both LOX and allene oxide cyclase (AOC), an enzyme catalyzing the step to 12-oxo-phytodienoic acid (OPDA) in the octadecanoid pathway.^14,21,22 We investigated the effects of these three inhibitors on herbivore-induced parasitoid attraction. For all three inhibitors 2 mM aqueous solutions with 0.1% Tween were applied to the plants.Open in a separate windowFigure 1Representation of the octadecanoid pathway with indication of which step of the signal-transduction pathway is affected by the different elicitors (+) and inhibitors (−).The response of the parasitoid Cotesia glomerata was tested to Pieris brassicae-infested plants (15 2^nd instar larvae) treated with inhibitor solution, Pieris brassicae-infested plants treated with a solution without inhibitor or intact plants sprayed with inhibitor solution. Recently, Bruinsma et al.¹⁷ showed that Pieris brassicae- infested plants treated with phenidone were less attractive to C. glomerata than infested plants treated with control solution (binomial test, N = 42, p = 0.008, Fig. 2). However, infested plants treated with phenidone were still more attractive than intact plants sprayed with phenidone (binomial test, N = 39, p < 0.001, Fig. 2). Thus, phenidone did reduce the induction of parasitoid attractants, but did not eliminate the induction completely. Here, we present additional experiments with the inhibitors DIECA and propyl gallate. DIECA application shows similar results as phenidone application; infested plants treated with DIECA are less attractive to C. glomerata than infested plants treated with control solution, but are more attractive than uninfested plants treated with DIECA (binomial test, N = 46, p = 0.026 and N = 26, p < 0.001, respectively, Fig. 2). Treatment with propyl gallate resulted in lower attractiveness of infested inhibitor-treated plants compared to infested control plants, but not significantly so (binomial test, N = 45, p = 0.072, Fig. 2), and propyl gallate-treated infested plants were more attractive than propyl gallate-treated intact plants (binomial test, N = 28, p < 0.001; Fig. 2). Summarizing, phenidone and DIECA treatment of Brussels sprouts plants resulted in a reduced attractiveness of caterpillar-infested B. oleracea plants to C. glomerata. Although propyl gallate-treated plants also attracted fewer parasitoids, this difference was marginally insignificant. Of the three inhibitors, the LOX inhibitor phenidone had the largest effect on the attraction of the parasitoid C. glomerata.Open in a separate windowFigure 2Attraction of Cotesia glomerata to plants sprayed with the inhibitors phenidone, DIECA, or propyl gallate, or sprayed with a control solution, with or without infestation with Pieris brassicae. Numbers to the left of the bars indicate the total number of parasitoids tested, numbers between brackets the number of parasitoids that landed on a plant (binomial test, ***p < 0.001, **p < 0.01, *p < 0.05).Our data show that both elicitors and inhibitors can be used in bio-assays to demonstrate the importance of certain steps in defense pathways.^5,23 Although application of the inhibitors to herbivore-infested plants did not abolish the response of the plants and the parasitoids still preferred them over non-induced plants, the inhibition of the octadecanoid pathway did reduce the attractiveness of the plants to the parasitoids. This implies that the octadecanoid pathway is involved in attracting parasitoids, but it is not the only factor determining parasitoid host location. This shows that use of inhibitors can provide interesting opportunities to comparatively investigate ecological interactions of genetically identical plants that differ in the degree of defense expression. Integrating knowledge on mechanisms with studies on ecological interactions and applying this to studies of increasingly complex interactions will further promote the understanding of induced defense in a community ecology context.^24,25