Jurgen Engelberth, Department of Biology, University of Texas at San Antonio, San Antonio, Texas, USA

September, 2006

The response of plants to insect herbivore damage provides an excellent example for the physiological complexity and diversity of induced defenses. Mechanical damage, recognition of insect elicitors, induction of direct defenses, and "the cry for help"—the recruitment of parasites and predators of the attacking herbivore through the release of so-called volatile organic compounds (VOC)—are some of the major events associated with this response. A central role in these processes is played by jasmonic acid (JA), a fatty-acid-derived signaling compound common to most, if not all, plants. JA is produced through the octadecanoid signaling pathway starting with a-linolenic acid (18:3 fatty acid) after the insertion of oxygen and the formation of an instable allene oxide, the allene-oxide cyclase form 12-oxo phytodienoic acid (OPDA). The cyclopentenone moiety is then reduced to a cyclopentanone by 12-oxo phytodienoic acid reductase and, after three cycles of ß-oxidation, JA is released. While the first steps in the biosynthesis are located in the chloroplast, reduction of the cyclopentenone and ß-oxidation are performed in the peroxisomes. JA is best described in the context of the wound response, but it is also now an established signal in herbivore-related defense responses. It has been shown in numerous publications that JA does induce direct defenses like the production of defensive proteins (e.g., proteinase inhibitors), but is also important in the induction of secondary metabolites, which can either serve as a direct defense due to their toxicity, or act—when released as VOC—as attractants for natural enemies of the attacking herbivore. This phenomenon, often referred to as tritrophic interaction, has been a subject of intensive research for many years and has led to the establishment of Chemical Ecology as an integrative research area, combining entomology, plant biology, and chemistry in an attempt to better understand the complexity of interactions in nature, mediated by secondary metabolites.

Herbivore-induced VOC, a mixture of volatile secondary metabolites from various pathways like terpenes, shikimic acid-derived products [salicylic acid-methyl ester (MeSA), anthranilic acid-methyl ester, and indole], and fatty-acid-derived products [jasmonic acid-methyl ester (MeJA), cis jasmone, and green leafy volatiles (GLV) like hexenal, hexenol, and hexenyl acetate], can serve as signals, not only to attract predators and parasites of attacking herbivores but they can also be recognized by neighboring plants, resulting in the activation of defense-related signaling events and gene expression. The first reports on VOC serving as signals in interplant communication were published more then 20 years ago by Rhoades (1983) and Baldwin and Schultz (1983). Since then, scientists have struggled with the phenomena, and for each publication claiming to present evidence for this type of communication, there was one declaring that this cannot be—especially when considering the complex situation in nature. And so it did not come as a surprise that the next series of publications demonstrating the effect of VOC on receiver plants resulted from experiments performed in the controlled environment of a laboratory. In 2000, Arimura et al. demonstrated convincingly the capability of complex blends of volatiles derived from herbivore-infested plants on the gene expression of uninfested receiver plants. In addition, they were able to attribute this activity to certain terpenes like ocimene and the homoterpenes, MeSA, MeJA and to GLV, as well. GLV are released within seconds at the site of wounding as an immediate wound response, but can also be released systemically—like other VOC—several hours after insect herbivory. Like JA, GLV are also fatty-acid-derived (mainly, 18:2 and 18:3 fatty acids) compounds. After the insertion of oxygen by the activity of a lipoxygenase in position 13, the resulting fatty acid hydroperoxides are rapidly cleaved by a hydroperoxide lyase, resulting in a C₆ aldehyde, which is often further processed to the corresponding alcohol and esters. The remaining nonvolatile C₁₂ part is transformed into traumatin, a classical wound hormone, which is made responsible for wound healing and callus formation. The effects of GLV on plant defense responses had been shown earlier by several labs, but neither the mechanisms nor the physiological consequences of this treatment were described. Only Bate and Rothstein (1998) showed that exposure to certain GLV did induce the induction of some genes in Arabidopsis, some of which were defense-related. The effects of those exposures to either herbivore-induced VOC or pure chemicals, however, were always weaker or incomplete when compared to actual herbivore damage or exogenous application of JA as the major regulator in these processes, questioning the effectiveness of such a form of defense response. This dilemma was partially solved when in 2004 Engelberth et al. not only demonstrated the direct effect of GLV-exposure on intact plants, but also treated the same plants with insect-derived elicitors after a certain period of exposure in an attempt to mimic insect herbivory. The results from that study showed for the first time that previous exposure to GLV caused the plant (corn seedlings, Zea mays) to produce more JA as the major signaling compound and more VOC as a major defense measure, when induced by insect-derived elicitors. Additionally, it was shown that this effect only concerned defense-related responses, for the wound-induced production of JA was not affected. This was the first report on priming of plants against insect herbivore attack, and GLV seem to play an important part in this process.

But What Does Defense Priming Mean?

Defense "priming" describes a process in which responses to a challenge (i.e., pathogen infection or herbivore infestation) are accelerated, enhanced, or potentiated by prior stimulation. Besides being well-established in the animal system, analogous priming phenomena have also been characterized in plants (see Conrath et al. 2002, and references therein for a review) in the context of plant-pathogen interactions. Priming can be hastened by asymptomatic prior exposure to pathogenic microbes, nonpathogenic microbes, microbial elicitors, salicylate, or salicylate analogs and is often also referred to as systemic acquired resistance (SAR). For example, treating parsley cell cultures with SAR inducers like salicylic acid, isonicotinic acid or benzothiodiazole does not induce cellular defense responses but does sensitize the cells so that they respond subsequently to noninducing concentrations of elicitors from Phytophthora sojae. Primed responses can comprise an enhanced oxidative burst and increased production of secondary metabolites. Also, Thulke and Conrath (1998) found that some defense-related genes and enzymes (e.g., anionic peroxidases) were activated by low, priming concentrations of SA, while others were potentiated but not activated. For example, more than 500 µm of SA is needed to directly increase phenylalanine ammonia-lyase (PAL) activity, while only 10 µm of SA greatly increases PAL′s responsiveness to subsequent elicitation. Numerous examples for primed responses can be found in the literature, all requiring that the plants be previously challenged by either nonpathogenic strains of potential pathogens or treated with chemicals or analogs of those chemicals that are usually involved in defense-response signaling. The nonprotein amino acid, ß-aminobutyric acid (BABA), has long been known to prime defense responses in many plants. Unlike SA, JA or their analogs, BABA does not elicit any responses directly. Its only effect is to enhance and/or accelerate responses to subsequent challenges (Conrath et al. 2002). Among the challenges primed by BABA are not only those concerning defense responses but also drought stress, as an example for abiotic stresses that can be primed. However, among the signaling compounds involved in these processes, only the salicylic MeSA and MeJA represent volatile signals with the potential to serve in interplant communication. And, although MeSA is induced to a certain extent by insect herbivores, it is rather unlikely that it has a priming function in this context. MeSA has often been described as an antagonist of JA-related signaling processes, and inhibiting the JA signaling pathway would be rather counterproductive. MeJA, on the other hand, seems to have a priming function. Kessler et al. (2006) described the priming of plant defense responses in nature by volatile signals in the interaction between Artemisia tridentate and Nicotiana attenuata. Artemisia releases a significant amount of MeJA when mechanically damaged, and the authors described not only the direct effects of this interaction but also the priming-related processes, in great detail. However, in their study, MeJA was not the only active volatile compound. Certain GLV—like hexenyl acetate—were also abundant in significant amounts among the VOC released after mechanical damage and they have the same effect as MeJA.

There is now an increasing number of publications describing the priming effects of typical insect herbivore-induced VOC, and while certain individual components like species-specific terpenes have been shown to affect gene expression in receiver plants, the common feature of all these studies is the activity of GLV as a volatile priming agent. And, while all plants investigated so far did release GLV as part of their volatile bouquet, MeJA is only produced and released by certain plant species; for example, it cannot be found among the VOC released by corn seedlings upon insect herbivore damage. Of course, this does not mean that GLV are the only active compounds in the priming process. Ethylene, a well-known volatile plant hormone, often acts in a synergistic fashion with other signaling components and is also massively produced upon pathogen infection or insect herbivore infestation. Ethylene has been shown to potentiate GLV-, insect elicitor-, and insect herbivore-induced VOC production, and a synergistic effect on the priming response cannot be excluded (Ruther and Kleier, 2005; Schmelz et al. 2003). Contrary to the priming by GLV, the effects of ethylene could not be attributed to increased JA production.

Priming of plant defense responses by volatiles is currently one of the hot topics in the area of plant insect interactions, and while the phenomenon was already described more then 20 years ago, we are just at the beginning of our understanding on how priming against impending insect herbivory is regulated on the molecular level. It appears that priming by GLV works through the octadecanoid-signaling pathway. The result is unique insofar as specifically defense-related processes seem to be affected, while other functions of the same pathway remain untouched. The advantages of this kind of advanced preparedness lie in the relatively low investments in defense, and therefore, they do not affect the general physiology of the plant, as the full induction of all possible countermeasures would do. Improvements in methodology, access to large data bases, and the identification of defensive measures affected by priming—which will vary among different plant species—will lead to an increasing knowledge about the physiology of priming plant defense responses with all its potential implications for the development of environmentally sound pest-management strategies.

References

Arimura, G., Ozawa, R., Shimoda, T., Nishioka, T., Boland, W., and Takabayashi, J. (2000) Herbivory-induced volatiles elicit defence genes in lima bean leaves. Nature 406: 512–515.

Baldwin, I. T., and Schultz, J. C. (1983) Rapid changes in tree leaf chemistry induced by damage: Evidence for communication between plants. Science 221: 277–279.

Bate, N. J., and Rothstein, S. J. (1998) C-6-volatiles derived from the lipoxygenase pathway induce a subset of defense-related genes. Plant J. 16(5): 561–569.

Conrath, U., Pieterse, C. M. J., and Mauch-Mani, B. (2002) Priming in plant-pathogen interactions. Trends Plant Sci. 7(5): 210–216.

Engelberth, J., Alborn, H. T., Schmelz, E. A., and Tumlinson, J. H. (2004) Airborne signals prime plants against herbivore attack. Proc. Natl. Acad. Sci. USA 101: 1781–1785.

Kessler, A., Halitschke, R., Diezel, C., and Baldwin, I. T. (2006) Priming of plant defense responses in nature by airborne signaling between Artemisia tridentata and Nicotiana attenuata. Oecologia 148: 280–292.

Rhoades, D. F. (1983) Responses of alder and willow to attack by tent caterpillars and webworms: Evidence for pheromonal sensitivity of willows. In P. A. Hedin, Editor, American Chemical Society Symposium Series 208, Washington, D.C., USA, 55–68.

Ruther, J., and Kleier, S. (2005) Plant-plant signaling: Ethylene synergizes volatile emission in Zea mays induced by exposure to (Z)-3-Hexen-1-ol. J. Chem. Ecol. 31(9): 2217–2222.

Schmelz, E. A., Alborn, H. T., and Tumlinson, J. H. (2003) Synergistic interactions between volicitin, jasmonic acid and ethylene mediate insect-induced volatile emission in Zea mays. Physiol. Plantarum 117(3): 403–412.

Thulke, O., and Conrath, U. (1998) Salicylic acid has a dual role in the activation of defence-related genes in parsley. Plant J. 14(1): 35–42.