Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphigidae) and Its Natural Host Nicotiana attenuata. VIII. An Unbiased GCxGC-ToFMS Analysis of the Plant's Elicited Volatile Emissions

摘要

Treating wounds in Nicotiana attenuata leaves with Manduca sexta oral secretions (W+OS) mimics most changes elicited by M. sexta herbivory, but an unbiased analysis of the effect of the different OS constituents on volatile emissions is lacking. We used two-dimensional gas chromatography/time-of-flight (GCxGC-ToF) mass spectrometry combined with multivariate statistics to parse volatiles into regulatory patterns. Volatiles released by wounding alone and by the alkalinity of OS were assessed by applying a buffer known to mimic the pH-mediated changes of OS elicitation (pectin methyl esterase activation and methanol release). The activities of fatty acid amino acid conjugates, well-known elicitors of antiherbivore defenses, and of 2-hydroxyoctadecatrienoic acid, a newly discovered signal in OS, were determined. Approximately 400 analytes were detected after deconvolution and alignment of GCxGC data; 35 volatiles were significantly regulated upon W+OS. Two-thirds of these were specifically regulated by OS, being either amplified (most terpenoids and certain hexenylesters) or strongly repressed (many short-chain alcohols and some aromatic and hexenylester derivatives). Fatty acid amino acid conjugates played a central role in this pattern of regulation, since they induced the emission of half of OS-elicited volatiles and inhibited the production of almost all OS-repressed volatiles; 2-hydroxyoctadecatrienoic acid influenced emission of trans--bergamotene, while other unknown OS constituents amplified hexenylester production. We conclude that the complex bouquet of herbivory-elicited volatiles results from the complex modulations of the wound response by diverse cues found in OS. This work also underscores the value of ultra-high-resolution GCxGC-ToF analysis combined with the nontargeted mining of the resulting data. nnnn--------------------------------------------------------------------------------nPlants employ a complex arsenal of defense mechanisms to protect themselves from insect herbivores. In addition to constitutive defenses such as trichomes or thick secondary walls, plants also use induced defenses, which are deployed specifically when herbivores start to chew on a leaf (Karban and Baldwin, 1997). These conditional responses allow plants to forgo the costs of defense production (Zavala et al., 2004) and optimize their ability to compete with neighboring plants or to tolerate the damage of resistant herbivores (Stowe et al., 2000; Schwachtje et al., 2006). Induced defenses function either directly via the production of amino acid-catabolizing enzymes, antidigestive proteinase inhibitors, or toxic and repelling chemicals that render plant tissues less suitable as food for herbivores (Duffey and Stout, 1996) or indirectly through traits that increase the apparency of infested plants to the natural enemies of their herbivores (Karban and Baldwin, 1997; Pare and Tumlinson, 1999). nAn intensively studied example of an inducible indirect defense is the production and emission of volatile organic compounds (VOCs) by plants attacked by insects (for review, see Turlings and Wäckers, 2004). The first complete evidence that plants use odors to attract natural enemies of their predators was obtained from studies on plant-mite interactions (Sabelis and van de Baan, 1983; Dicke et al., 1988; Dicke et al., 1990a, 1990b). These first studies and many since, performed under optimized laboratory conditions (Dicke, 1994; Mattiacci et al., 1995 Alborn et al., 1997; Turlings et al., 1990, 1991a, 1991b, 1998) or under field conditions (Shimoda et al., 1997; De Moraes et al., 1998; Bernasconi Ockroy et al., 2001; Kessler and Baldwin, 2001), have demonstrated that parasitoids and predators make effective use of herbivory-induced plant VOCs (HIPV) to locate their prey. Certain of these volatiles have notably been shown to decrease oviposition rates and increase egg predation on the emitting plant (Feeny et al., 1989; Kessler and Baldwin, 2001; Colazza et al., 2004; Johne et al., 2006). Moreover, some of these plant odors can also serve as direct repellents against insect herbivores (Quiroz et al., 1997; Bernasconi et al., 1998; Landolt et al., 1999). In addition, the volatiles can be perceived by neighboring plants and increase or potentiate their defensive abilities (Arimura et al., 2000a, 2000b; Baldwin et al., 2006; Kessler et al., 2006; Kost and Heil, 2006). On the other hand, volatile signaling is also used by some plant species to overcome the vascular constraints on systemic signaling to elicit defense responses in adjacent leaves with little or no vascular connection with the attacked leaves (Frost et al., 2007). nnDespite their common feature of being volatile at ambient temperatures, these major players of plant signaling are chemically highly diverse (Holopainen, 2004). HIPV bursts have been partially characterized in several plant species (for overview, see van Poecke and Dicke, 2004; Van Dam and Poppy, 2008), including, for instance, maize (Zea mays), cotton (Gossypium hirsutum), lima bean (Phaseolus lunatus), and native tobacco (Nicotiana attenuata). The major constituents of these VOC bouquets consist of products of the shikimic acid pathway, terpenes and fatty acid (FA) derivatives. The best characterized metabolites from this latter subpopulation are the green leaf volatiles (GLVs), originating from the degradation of C18 FAs (linolenic and linoleic acids) when hydroperoxidated by a lipoxygenase and cleaved into C12 and C6 units by a hydroperoxide lyase (Halitschke et al., 2004; Matsui, 2006). nnFatty acid amino acid conjugates (FACs), produced in the insect gut by conjugation of host-derived FAs to amino acids (Spiteller et al., 2000) and contained in the oral secretions (OS) of many caterpillar species, were the first nonenzymatic elicitors of HIPV characterized (Alborn et al., 1997; Halitschke et al., 2001). More generally, recent "omic" approaches have demonstrated that FACs are responsible for eliciting a large portion of the hundreds of genes regulated during the plant-herbivore interaction (Halitschke et al., 2003; Roda et al., 2004) as well as the reconfiguration of the proteome (Giri et al., 2006). However, such untargeted analyses have not been conducted for HIPV, and until now the regulation of only a few components of plants' volatile blends (e.g. trans--bergamotene in N. attenuata [Halitschke et al., 2001] and hexenylacetate or trans-β-farnesene in maize [Alborn et al., 1997]) has been associated with FAC signaling. nnNotably, FACs are not always active: in lima bean and cowpea (Vigna unguiculata), for instance, FAC treatment of wounds does not elicit VOCs (Spiteller et al., 2001). Rather, Mithöfer et al. (2005) demonstrated that a continuous wounding treatment that resembled insect feeding was sufficient for HIPV emission. In cowpea, inceptin, an elicitor produced from the postingestive digestion of host plant ATP synthase, triggers changes in phytohormone and VOC production similar to those detected during herbivory (Schmelz et al., 2006; Carroll et al., 2008). In N. attenuata, the large methanol burst, which is many times larger than the GLV burst elicited by Manduca sexta herbivory, originates from the demethylation of pectin cell walls and in turn is elicited by the alkalinity of OS (von Dahl et al., 2006). β-Glucosidase hydrolytic enzymes from Pieris brassicae OS also trigger HIPV emissions when applied to cabbage (Brassica capitata) plants (Mattiacci et al., 1995). 2-Hydroxyoctadecatrienoic acid (2-HOT) is a newly identified component of M. sexta OS (E. Gaquerel, A. Steppuhn, and I.T. Baldwin, unpublished data). This oxylipin is produced from linolenic acid through the action of the -dioxygenase (-DOX; Hamberg et al., 2002, 2003). In N. attenuata, the production of 2-HOT during herbivory and its accumulation in OS allow the plant to monitor the progression of the insect's attack and to sustain its production of jasmonic acid, the central hormone that coordinates antiherbivore defenses. The striking postingestive stability and high activity of N. attenuata's -DOX1 proteins in the alkaline and proteolytic milieu of the M. sexta midgut mediates 2-HOT's particular signaling role (E. Gaquerel, A. Steppuhn, and I.T. Baldwin, unpublished data). nnGiven the rapid advances in characterizing the active elicitors of M. sexta OS, a full unbiased analysis of N. attenuata's HIPV blend, an analysis that has not been conducted for any plant, is overdue. To examine the extent to which FACs, 2-HOT, and alkalinity contribute to the OS-elicited HIPV bouquet, we used comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GCxGC-ToFMS) in combination with multivariate statistics. This nontargeted approach was applied to a suite of elicitation treatments in which the amount of mechanical wounding was held constant.