Wilhelm Boland

Herbivory-induced volatiles elicit defence genes in lima bean leaves

In response to herbivore damage, several plant species emit volatiles that attract natural predators of the attacking herbivores. Using spider mites (Tetranychus urticae) and predatory mites (Phytoseiulus persimilis), it has been shown that not only the attacked plant but also neighbouring plants are affected, becoming more attractive to predatory mites and less susceptible to spider mites. The mechanism involved in such interactions, however, remains elusive. Here we show that uninfested lima bean leaves activate five separate defence genes when exposed to volatiles from conspecific leaves infested with T. urticae, but not when exposed to volatiles from artificially wounded leaves. The expression pattern of these genes is similar to that produced by exposure to jasmonic acid. At least three terpenoids in the volatiles are responsible for this gene activation; they are released in response to herbivory but not artificial wounding. Expression of these genes requires calcium influx and protein phosphorylation/dephosphorylation.

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves. II. Continuous Mechanical Wounding Resembling Insect Feeding Is Sufficient to Elicit Herbivory-Related Volatile Emission

Herbivore feeding elicits defense responses in infested plants, including the emission of volatile organic compounds that can serve as indirect defense signals. Until now, the contribution of plant tissue wounding during the feeding process in the elicitation of defense responses has not been clear. For example, in lima bean (Phaseolus lunatus), the composition of the volatiles induced by both the insect caterpillar Spodoptera littoralis and the snail Cepaea hortensis is very similar. Thus, a mechanical caterpillar, MecWorm, has been designed and used in this study, which very closely resembles the herbivore-caused tissue damage in terms of similar physical appearance and long-lasting wounding period on defined leaf areas. This mode of treatment was sufficient to induce the emission of a volatile organic compound blend qualitatively similar to that as known from real herbivore feeding, although there were significant quantitative differences for a number of compounds. Moreover, both the duration and the area that has been mechanically damaged contribute to the induction of the whole volatile response. Based on those two parameters, time and area, which can replace each other to some extent, a damage level can be defined. That damage level exhibits a close linear relationship with the accumulation of fatty acid-derived volatiles and monoterpenes, while other terpenoid volatiles and methyl salicylate respond in a nonlinear manner. The results strongly suggest that the impact of mechanical wounding on the induction of defense responses during herbivore feeding was until now underestimated. Controlled and reproducible mechanical damage that strongly resembles the insects feeding process represents a valuable tool for analyzing the role of the various signals involved in the induction of plant defense reactions against herbivory.

Biotic and heavy metal stress response in plants: evidence for common signals

In higher plants, biotic stress (e.g., herbivore or pathogen attack) as well as abiotic stress (in particular heavy metals) often induce the synthesis and accumulation of the same defense‐related secondary metabolites. This well‐known finding still awaits an explanation regarding the common features of both stress types. In this study, a mechanism is proposed that links reactive oxygen species (ROS) generation with lipid oxidation processes, ultimately resulting in the formation of similar, highly active signalling compounds. The generation of ROS is a common event in both heavy metal treatment and biotic stress although it can depend on quite different, enzymatic and non‐enzymatic reactions. Regardless, ROS are involved in the oxidation of unsaturated fatty acids which initiate the formation of oxylipins, a highly variable class of lipid‐derived compounds in plants. Oxylipins represent new endogenous signals involved in biotic‐ and abiotic‐induced stress responses.

Whiteflies interfere with indirect plant defense against spider mites in Lima bean.

Plants under herbivore attack are able to initiate indirect defense by synthesizing and releasing complex blends of volatiles that attract natural enemies of the herbivore. However, little is known about how plants respond to infestation by multiple herbivores, particularly if these belong to different feeding guilds. Here, we report the interference by a phloem-feeding insect, the whitefly Bemisia tabaci, with indirect plant defenses induced by spider mites (Tetranychus urticae) in Lima bean (Phaseolus lunatus) plants. Additional whitefly infestation of spider-mite infested plants resulted in a reduced attraction of predatory mites (Phytoseiulus persimilis) compared to attraction to plants infested by spider mites only. This interference is shown to result from the reduction in (E)-β-ocimene emission from plants infested by both spider mites and whiteflies. When using exogenous salicylic acid (SA) application to mimic B. tabaci infestation, we observed similar results in behavioral and chemical analyses. Phytohormone and gene-expression analyses revealed that B. tabaci infestation, as well as SA application, inhibited spider mite-induced jasmonic acid (JA) production and reduced the expression of two JA-regulated genes, one of which encodes for the P. lunatus enzyme β-ocimene synthase that catalyzes the synthesis of (E)-β-ocimene. Remarkably, B. tabaci infestation concurrently inhibited SA production induced by spider mites. We therefore conclude that in dual-infested Lima bean plants the suppression of the JA signaling pathway by whitefly feeding is not due to enhanced SA levels.

Herbivore-induced volatiles: The emission of acyclic homoterpenes from leaves of Phaseolus lunatus and Zea mays can be triggered by a β-glucosidase and jasmonic acid

The treatment of healthy, undamaged plants of the Lima bean Phaseolus lunatus with solutions of a β‐glucosidase from bitter almonds (at 5 U·ml−1) through the petiole results in an enhanced emission of volatiles to the environment. The compounds are identical with those emitted in response to infestation with the red spotted spider mite Tetranychus urticae. Dominant products are the two acyclic homoterpenes 4,8‐dimethyl‐1,3E,7‐dimethylnonatriene (homoterpene I) and 4,8,12‐trimethyl‐1,3E,7E,11‐tridecatetraene (homoterpene II) which are of sesquiterpenoid and diterpenoid origin. Therefore, a β‐glucosidase of the herbivore may be considered as the true elicitor for the odor induction. Homoterpene I and most other of the herbivore‐induced volatiles can also be triggered by treatment of the plant with solutions of jasmonic acid (JA) at 100 nmol·ml−1 to 10 μmol·ml−1. The C16 homoterpene II is not significantly induced by JA. The time—course of the enzymatic‐ and the JA‐triggered induction of the volatiles is identical. The dose—response to JA parallels previous reports on alkaloid induction in cell cultures. In corn plants (Zea mays) JA triggers the emission of all volatiles which are known to be emitted in response to the damage by the beet army worm Spodoptora exigua. In summary, the emission of volatiles after damage by a herbivore resembles the production of phytoalexins in response to an attacking microorganism and uses similar elicitors and internal transduction pathways.

Recognition of herbivory-associated molecular patterns.

During their long, approximately 350 million-year period of coexistence, plants, insects, and other arthropods evolved a variety of different interactions (Gatehouse, 2002). Some interactions can be beneficial for the plant, as in the case of insect-mediated pollination or seed dispersion, and others are deleterious, as in the case of attack by herbivorous insects (Fig. 1). To successfully combat aggressors, plants must be equipped with a sophisticated sensory system to perceive signals fast and efficiently from their environment and thereby detect potential enemies and subsequently translate and integrate such signals into appropriate biochemical and physiological responses. Thus, upon attack, a number of reactions are detectable in plant cells, including changes in ion flux and protein phosphorylation, formation of reactive oxygen species and oxylipins, as well as initiation of various defense reactions in the host plant (Kessler and Baldwin, 2002; Maffei et al., 2007b). Intriguing questions arising from these observations are how plants recognize the particular herbivores, what kinds of signals are involved, how such signals are perceived, and how they are converted into downstream signaling pathways involved in plant defense activation. Signal perception in the plant cell may rely on the presence of specific receptors for chemical signals or on general recognition processes based on localized tissue injuries. In principle, the feeding process combines two sites of the same coin: mechanical wounding of the infested tissue and introduction of oral secretions that are delivered from the feeding organism into the wounded tissue (i.e. the attacked plant is challenged by both a mechanical as well as a chemical stimulus). This Update introduces herbivore-derived metabolites, which represent serious candidates for signaling compounds; we will also discuss advances in herbivore recognition, namely, the perception of insect-derived signals by specific binding proteins. The properties of these binding proteins suggest their involvement in signal perception.

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves. III. Membrane Depolarization and Involvement of Hydrogen Peroxide

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H2O2) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H2O2 was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H2O2 was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H2O2 in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H2O2 prompted a transient transmembrane potential (Vm) depolarization, with a Vm depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca2+-sensing aequorin system, increasing amounts of added H2O2 correlated with a higher cytosolic calcium ([Ca2+]cyt) concentration. In MD and HW leaves, H2O2 also triggered the increase of [Ca2+]cyt, but MD-elicited [Ca2+]cyt increase was more pronounced when compared to HW leaves after addition of exogenous H2O2. The results clearly indicate that Vm depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves : I. Membrane Potentials, Intracellular Calcium Variations, Oral Secretions, and Regurgitate Components

Membrane potentials (Vm) and intracellular calcium variations were studied in Lima bean (Phaseolus lunatus) leaves when the Mediterranean climbing cutworm (Spodoptera littoralis) was attacking the plants. In addition to the effect of the feeding insect the impact of several N-acyl Glns (volicitin, N-palmitoyl-Gln, N-linolenoyl-Gln) from the larval oral secretion was studied. The results showed that the early events upon herbivore attack were: a) a strong Vm depolarization at the bite zone and an isotropic wave of Vm depolarization spreading throughout the entire attacked leaf; b) a Vm depolarization observed for the regurgitant but not with volicitin {N-(17-hydroxy-linolenoyl)-Gln} alone; c) an enhanced influx of Ca2+ at the very edge of the bite, which is halved, if the Ca2+ channel blocker Verapamil is used. Furthermore, the dose-dependence effects of N-acyl Gln conjugates-triggered influx of Ca2+ studied in transgenic aequorin-expressing soybean (Glycine max) cells, showed: a) a concentration-dependent influx of Ca2+; b) a configuration-independent effect concerning the stereochemistry of the amino acid moiety; c) a slightly reduced influx of Ca2+ after modification of the fatty acid backbone by functionalization with oxygen and; d) a comparable effect with the detergent SDS. Finally, the herbivore wounding causes a response in the plant cells that cannot be mimicked by mechanical wounding. The involvement of Ca2+ in signaling after herbivore wounding is discussed.

Herbivory, induced resistance, and interplant signal transfer in Alnus glutinosa

Abstract Field experiments with manually defoliated black alders ( Alnus glutinosa ) showed that defoliation affected herbivory by the major alder antagonist, the leaf beetle Agelastica alni . Herbivore damage increased with increasing distance to the defoliated tree, suggesting induced resistance not only on the damaged tree, but also on the neighbouring trees. The beetles also avoided leaves from the nearest neighbours for both feeding and oviposition in a laboratory assay, so the alders showed interplant resistance transfer. Natural enemies did not appear to shape this pattern, because the number of entomophagous arthropods and predator–prey ratios even increased with increasing distance to the defoliated tree. The numbers of all specialist, but not the generalist, herbivore species paralleled the increase in the attack of the specialist A. alni , supporting the view that specialists are more affected by plant resistance than generalists. Mechanisms causing this pattern, found in the field, were studied in more detail using biochemical analyses and further bioassays. Responses of alder leaves to herbivory of A. alni were shown to include ethylene emission and the release of a blend of volatiles with mono-, sesqui- and homoterpenes. Changes in leaf chemistry after herbivory included increases in the activity of oxidative enzymes (polyphenoloxidase, PPO, lipoxygenase, LOX, and peroxidase, POD) and proteinase inhibitors (PIs), and an increase in the phenolic contents of the leaves. Quantification of the endogenous jasmonic acid (JA) showed the activation of the octadecanoid pathway following herbivory. The active components in mediating a possible interplant signal transfer via airborne volatiles may have included ethylene, β -ocimene, 4,8-dimethylnona-1,3,7-triene (DMNT), and 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). The incubation with volatiles resulted in an increase in the activity of catalase (CAT) and PIs (after MeJA application) and in an increase in the content of phenolics and PI activity (after ethylene application). Further evidence that airborne interplant communication may be important in the response of alder trees to beetle attack came from container experiments. In airtight chambers, unattacked leaves significantly increased the activity of proteinase inhibitors when they were associated with leaves previously attacked by beetle larvae. In conclusion, field experiments, bioassays in the laboratory as well as biochemical analyses suggest the existence of interplant resistance transfer in A. glutinosa , with airborne volatiles as a possible mechanism. However, the relative importance of airborne and possible soil-borne signals as well as unknown effects of intensified nutrient absorption of defoliated trees, possibly reducing foliage quality of undamaged neighbours, remains to be shown.

Involvement of siderophores in the reduction of metal-induced inhibition of auxin synthesis in Streptomyces spp

Unlike synthetic metal chelators, microbe-assisted phytoremediation provides plants with natural metal-solubilizing chelators which do not constitute a potential source of environmental pollution. Concurrently with microbial chelators, plant growth promotion can be enhanced through bacterially-produced phytohormones. In this work, the simultaneous production of siderophores and auxins by Streptomyces was studied to gain insight for future application in plant growth and phytoremediation in a metal-contaminated soil. Standard auxin and siderophore detection assays indicated that all of the investigated Streptomyces strains can produce these metabolites simultaneously. However, Al(3+), Cd(2+), Cu(2+), Fe(3+) and Ni(2+), or a combination of Fe(3+) and Cd(2+), and Fe(3+) and Ni(2+) affected auxin production negatively, as revealed by spectrophotometry and gas chromatography-mass spectrometry. This effect was more dramatic in a siderophore-deficient mutant. In contrast, except for Fe, all the metals stimulated siderophore production. Mass spectrometry showed that siderophore and auxin-containing supernatants from a representative Streptomyces species contain three different hydroxamate siderophores, revealing the individual binding responses of these siderophores to Cd(2+) and Ni(2+), and thus, showing their auxin-stimulating effects. We conclude that siderophores promote auxin synthesis in the presence of Al(3+), Cd(2+), Cu(2+) and Ni(2+) by chelating these metals. Chelation makes the metals less able to inhibit the synthesis of auxins, and potentially increases the plant growth-promoting effects of auxins, which in turn enhances the phytoremediation potential of plants.