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First published online March 16, 2007; 10.1104/pp.107.097154 Plant Physiology 144:793-805 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Cowpea Chloroplastic ATP Synthase Is the Source of Multiple Plant Defense Elicitors during Insect Herbivory1,2,[W],[OA]Center of Medical, Agricultural, and Veterinary Entomology, United States Department of Agriculture, Agricultural Research Service, Chemistry Research Unit, Gainesville, Florida 32608
In cowpea (Vigna unguiculata), fall armyworm (Spodoptera frugiperda) herbivory and oral secretions (OS) elicit phytohormone production and volatile emission due to inceptin [Vu-In; +ICDINGVCVDA–], a peptide derived from chloroplastic ATP synthase  -subunit (cATPC) proteins. Elicitor-induced plant volatiles can function as attractants for natural enemies of insect herbivores. We hypothesized that inceptins are gut proteolysis products and that larval OS should contain a mixture of related peptides. In this study, we identified three additional cATPC fragments, namely Vu-GE+In [+GEICDINGVCVDA–], Vu-E+In [+EICDINGVCVDA–], and Vu-In–A [+ICDINGVCVD–]. Leaf bioassays for induced ethylene (E) production demonstrated similar effective concentration50 values of 68, 45, and 87 fmol leaf–1 for Vu-In, Vu-E+In, and Vu-GE+In, respectively; however, Vu-In–A proved inactive. Shortly following ingestion of recombinant proteins harboring cATPC sequences, larval OS revealed similar concentrations of the three elicitors with 80% of the potential inceptin-related peptides recovered. Rapidly shifting peptide ratios over time were consistent with continued proteolysis and preferential stability of inceptin. Likewise, larvae ingesting host plants with inceptin precursors containing an internal trypsin cleavage site rapidly lost OS-based elicitor activity. OS containing inceptin elicited a rapid and sequential induction of defense-related phytohormones jasmonic acid, E, and salicylic acid at 30, 120, and 240 min, respectively, and also the volatile (E)-4,8-dimethyl-1,3,7-nonatriene. Similar to established peptide signals such as systemin and flg22, amino acid substitutions of Vu-In demonstrate an essential role for aspartic acid residues and an unaltered C terminus. In cowpea, insect gut proteolysis following herbivory generates inappropriate fragments of an essential metabolic enzyme enabling plant non-self-recognition.
Plant responses to biotic and abiotic perturbation often reduce the severity of future damage through complex changes, including increased cell wall lignification, hypersensitive responses that limit pathogen mobility, production of direct defenses toxic to the offending organism, or volatile emissions that act indirectly in the attraction of natural enemies of the pests (Hiraga et al., 2001  ; Kessler and Baldwin, 2002; Greenberg and Yao, 2004). The phytohormones ethylene (E) and jasmonic acid (JA) are known to be important regulators in plant responses to mechanical damage, insect herbivory, and pathogens (Howe, 2004; Zhao et al., 2005). Phytohormone-mediated plant responses are initiated by complex interactions of exogenous and endogenous signals, many of which have been identified as peptides (Boller, 2005; Matsubayashi and Sakagami, 2006). For example, in tomato species (Solanum lycopersicum and Solanum peruvianum), damage-induced E and JA-mediated plant responses are amplified by the action of an endogenous 18-amino acid peptide termed systemin (Pearce et al., 1991; Felix and Boller, 1995; Howe et al., 1996). Cellular damage initiates proteolysis or release of the 200-amino acid prosystemin protein, systemin production, ligand binding to the membrane-bound Leu-rich repeat (LRR) receptor kinase (RK) SR160, mitogen-activated protein kinase cascades, and ultimately increased JA biosynthesis leading to the production of defensive proteins (Pearce et al., 1991; Howe et al., 1996; Stratmann and Ryan, 1997; Dombrowski et al., 1999; Scheer and Ryan, 2002). Endogenous wound-related Hyp-rich peptides serving similar functions are also liberated from cell wall proteins in tobacco (Nicotiana tabacum) and tomato (Pearce et al., 2001; Pearce and Ryan, 2003). These functionally defined systemins (Ryan and Pearce, 2003) promote plant defenses in response to insect herbivory, yet are not known to enable specific discrimination between biotic and abiotic damage.
Mechanistic knowledge detailing how plants specifically recognize biotic attack comes primarily from pathology research and includes the established role of small peptides, derived from both plants and pathogens, as critical signals. In Arabidopsis (Arabidopsis thaliana), the isolated 23-amino acid peptide AtPep1 is produced from the 92-amino acid PROPEP1 protein and is both up-regulated by JA/E and requires functional JA/E signaling for defense gene (PDF1.2) expression (Huffaker et al., 2006
Despite advances in our understanding of pathogen-derived and endogenous peptide signals, the production of bioactive peptides specifically during insect herbivory has only recently been demonstrated. We utilized an induced E production bioassay in cowpea (Vigna unguiculata) leaves to guide the biochemical fractionation of elicitors from oral secretions (OS) of fall armyworm (Spodoptera frugiperda) larvae and isolated an 11-amino acid protein fragment. This peptide, termed inceptin, is derived from the proteolytic cleavage of chloroplastic ATP synthase To better understand how legumes recognize insect herbivores via peptide signals derived from plant metabolic proteins, we further examined components of armyworm larval OS and their role in eliciting cowpea responses. In this study, we investigated: (1) the range of inceptin-related peptides present in larval OS and their comparative biological activity; (2) the progressive pattern of peptide abundance and activity in larval OS following ingestion of proteins containing cATPC sequences; (3) the importance of leaf-derived OS containing inceptin-related peptides in triggering the rapid and dynamic production of phytohormone and volatile metabolites; and (4) structural features and amino acid residues of inceptin required for biological activity in cowpea, with a focus on induced E, SA, and DMNT volatile production. Consistent with the predicted proteolytic complexity of insect guts, we identify three additional inceptin-related peptides present in larval OS. We then demonstrate that peptides with additional N-terminal amino acids retain full elicitor activity, while C-terminal deletions abolish activity. Additionally, we show that inceptin-related peptides are rapidly generated in OS upon larval consumption of cATPC proteins and, consistent with gut proteolysis, exhibit preferential losses of larger peptide elicitors over time. Furthermore, we show that 1 µL of shoot-derived larval OS containing inceptin-related peptides initiates a rapid, sequential, and maximal induction of JA, E, and SA/DMNT in cowpea leaves at 30, 120, and 240 min, respectively. Comparatively, wounding plus cowpea root-derived OS lacking inceptins promotes only moderate JA increases. Finally, we demonstrate that the activity of modified inceptins in cowpea can vary slightly depending upon the induced biochemical marker selected but overall is highly sensitive to select modifications near the C terminus and relatively tolerant to numerous changes at the N terminus.
cATPC Is the Source of Multiple Active Elicitors
Lepidoptera larvae contain a diverse array of digestive proteases capable of converting plant proteins into complex peptide mixtures (Bown et al., 1997
The Inceptin C Terminus Is Required for Elicitor Activity To address how the differential proteolysis of inceptin-related peptides influences activity, we compared the Vu-In-induced E responses of cowpea leaves to the peptides containing additional N-terminal (Vu-E+In, Vu-GE+In) and cleaved C-terminal amino acids (Vu-In–A). Peptides sharing the same C terminus, namely Vu-In, Vu-E+In, and Vu-GE+In, displayed similar effective concentration (EC)50 values for induced E production in cowpea leaves of 68, 45, and 87 fmol leaf–1, respectively (Fig. 3 ). In contrast, Vu-In–A proved inactive at all concentrations tested (Fig. 3). These results demonstrate the occurrence of at least three active peptide elicitors in the OS of armyworm and also identify proteolytic sites resulting in peptide inactivation.
Time-Dependent Changes in the Composition of Active Elicitors Cowpea-derived armyworm OS contain a mixture of active and inactive inceptin-related peptides, yet it was unknown how the levels of these peptides vary over time following larval ingestion of cATPC. To quantify the production and dynamics of inceptin-related peptides, we generated an Escherichia coli-expressed recombinant protein derived from eight repeated sequences of the chloroplast-specific region of soybean (Glycine max) atpC (termed glutathione S-transferase [GST]-GmIn8H), fed this protein to larvae, and collected OS either 2 or 6 h after the initiation of feeding. In the 2-h sample, the final NP-HPLC purification step resulted in two distinct regions of strong E-inducing activity eluting between 20 and 24 min (Fig. 4A ). In contrast, the 6-h sample produced only a single fraction with predominant E-inducing activity (Fig. 4C). In both samples, early (20.5 min) and late (22–24 min) eluting activity closely corresponded with the relative amounts of [M + H]+ m/z 1,105.5 (Gm-In; +ICDVNGVCVDA–), 1,234.5 (Gm-E+In), and 1,291.5 (Gm-GE+In; Fig. 4, B and D). The relatively poor elution behavior and fmol activity level of the inceptin-related peptides harboring an additional acidic group (E+In and GE+In) resulted in chromatographic tailing and a broad range of HPLC fractions promoting E induction (Figs. 1C and 4, A and C). These results indicate that levels and ratios of inceptin-related peptides in OS change over time following ingestion of cATPC.
OS Levels of Inceptin Display Preferential Stability over Related Peptides To better understand the dynamics and relative contribution of inceptin-related peptides to OS elicitor activity after ingestion of cATPC, we quantitatively analyzed a time course of selected peptides present in armyworm OS. Within 15 min of complete GST-GmIn8H protein consumption by larvae, defined here as time zero, Gm-In, Gm-E+In, and Gm-GE+In constituted a nearly equal mixture representing 36%, 38%, and 26% of the total active peptides, respectively (Fig. 5 ). Two hours after consumption of the GST-GmIn8H protein, the relative abundance of active peptides Gm-In, Gm-E+In, and Gm-GE+In shifted dramatically to 80%, 17%, and 3%, respectively. Rapid loss of the larger peptides Gm-GE+In and Gm-E+In follows predictions of continued gut proteolysis over time. Despite trace abundance, levels of Gm-In–A increased 3.6-fold from 3.1 ± 0.6 to 11.3 ± 1.8 pmol larvae–1 between 0 and 2 h, then declined back to 3.7 ± 1.1 pmol larvae–1 at 6 h. While Gm-In–A levels do not quantitatively account for the loss of active elicitors, this pattern is consistent with catabolism concordant with the dramatic loss of Gm-GE+In and Gm-E+In. Of the 492 pmol of inceptin-related peptides contained within the GST-GmIn8H protein consumed by each larva, 80.8% ± 1.9%, 42.2% ± 3.0%, 24.5% ± 5.5%, 11.1% ± 1.4%, and 4.9% ± 1.3% was recovered in the OS at times designated as 0, 2, 4, 6, and 8 h, respectively. These results demonstrate: (1) the rapid and efficient proteolysis of cATPC proteins into inceptin-related peptides; (2) the preferential stability of inceptin; and (3) the gradual loss of all inceptin-related peptides over time.
A Trypsin Cleavage Site within Inceptin Promotes the Loss of OS Elicitor Activity
The composition of armyworm OS during feeding is a rich combination of both plant and insect biochemicals and their reciprocal postingestive enzymatic products (Paulillo et al., 2000
Inceptins in Larvae OS Induce Sequential Increases in JA, E, SA, and Volatiles
We previously demonstrated that repeated applications of synthetic Vu-In promote increases in E, JA, SA, and volatile pools of DMNT in cowpea leaves at a single sampling time point; however, the temporal coordination of these events was unknown. To address the phytohormone dynamics elicited by a natural mixture of inceptin-related peptides, we analyzed and bioassayed OS collected from larvae actively feeding on either cowpea roots or shoots. Root-derived OS lacked inceptins (<10 fmol µL–1), while shoot-derived OS contained Vu-In, Vu-E+In, and Vu-GE+In at concentrations of 616 ± 59, 170 ± 17, and 150 ± 15 fmol µL–1, respectively (n = 4, ±SEM). Wounding plus root-derived OS-induced increases in JA levels above unwounded control leaves at 30 min (Fig. 7A
); however, shoot-derived OS induced the highest JA accumulations at 30, 60, and 90 min. Root-derived OS treatments failed to result in significant increases in E, SA, or DMNT (Fig. 7, B–D), while shoot-derived OS induced a sequential maximal production of JA, E, and SA at 30, 120, and 240 min, respectively (Fig. 7, A–C). A similar yet delayed sequential activation of phytohormones has been described during Xanthomonas pathogen infection of tomato (O'Donnell et al., 2003
Ala Substitution of Inceptin Reveals Roles for Asp, Cys, and the C Terminus Of all the potential peptides present in the OS of actively feeding larvae, inceptin-related fragments are hypothesized to possess a unique sequence that promotes activity. Here, we consider the cyclic structure, the role of individual Vu-In residues via Ala substitutions, and flexibility (via additions and deletions) of both termini in the elicitation of E, SA, and DMNT as different markers for activity. Peptides and their abbreviations considered in this experiment are detailed in Figure 8 . In the E bioassay, replacement of the penultimate C-terminal Asp (Vu-In-A10) completely abolished activity (Fig. 8A). Surprisingly, replacement of the Cys proximal to the N terminus (Vu-In-A2) resulted in statistically insignificant reduction of activity, suggesting a nonessential role of the disulfide bridge and cyclized peptide. Significant reductions in E-inducing activity were detected in Vu-In-A3, Vu-In-A8, and Vu-In-A10, demonstrating important functions for both Asp residues and the Cys proximal to the C terminus. Both removal (Vu-In–A) and addition (Vu-In+A) of Ala to the C terminus significantly reduced activity (Fig. 8A), whereas additions (Vu-E+In; Fig. 3) and truncations (Vu-I–In; Fig. 8A) at the N terminus had no significant effect on E production. The only substitutions, additions, or truncations that abolished induced E activity were within the last four amino acids of the C terminus.
Similar to the E responses, SA production was significantly lower in response to modifications at the C terminus, including Vu-In-A10, Vu-In–A, and Vu-In+A (Fig. 8B). However, amino acid sequence modifications proximal to the N terminus (Vu-In-A3) and substitution of either Cys residue (Vu-In-A2 and Vu-In-A8) failed to significantly reduce SA accumulation compared to Vu-In (Fig. 8B). This contrasts the pattern established for E production and suggests that the SA responses may occur at either lower elicitor concentrations or have different ligand-binding requirements. Accumulation of tissue levels of DMNT more closely mirrored elicitor-induced changes in E production. Substitution of both Asp residues (Vu-In-A3 and Vu-In-A10) significantly reduced DMNT accumulation compared to Vu-In (Fig. 8C). Likewise, removal or addition of Ala to the C terminus in peptides Vu-In–A and Vu-In+A also suppressed DMNT levels. Substitution of either Cys (Vu-In-A2 and Vu-In-A8) produced weak intermediate DMNT responses not statistically different from numerous active and inactive peptides. JA levels were also measured at 4 h but, as expected, did not display strong patterns, in part due to the relaxation of the response at this time point (Fig. 7A; E.A. Schmelz, unpublished data). These activity assays demonstrate a significant role for Asp residues, relative flexibility of additions or truncations at the N but not at the C terminus, and a nonessential role of the Vu-In disulfide bridge for phytohormone activity.
As a model, we propose that herbivory by armyworm larvae on cowpea leaves generates three active peptide elicitors, namely Vu-In, Vu-E+In, and Vu-GE+In, during gut proteolysis of ingested cATPC proteins (Fig. 9 ). During herbivory, larval OS contact the feeding sites (Truitt and Pare, 2004 ) and promote inceptin-mediated responses (Schmelz et al., 2006). Larvae that have previously fed on nonphotosynthetic tissue or have moved between alternate host plants may initially lack these elicitors. Inceptin triggers the sequential induction of defense-related phytohormones JA, E, and SA in addition to the induction of volatile biochemical defenses, including DMNT, (E)- -ocimene, MeSA, indole, (E)--farnesene, (E,E)- -farnesene, and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (Schmelz et al., 2006). These elicitor-induced volatiles have established roles in the attraction of predators and parasitoids to arthropod herbivores (Kessler and Baldwin, 2002; Degenhardt et al., 2003; De Boer and Dicke, 2004; Kappers et al., 2005). Inceptin-related peptides are active in the low femtomole range, exhibit essential Asp residues, and require a conserved C terminus consistent with systemin and flg22 receptor-ligand models (Pearce et al., 1993; Felix et al., 1999). Proteolytic cATPC fragments represent the first identified class of small peptide elicitors that are specifically generated by and mediate the interaction between insects and plants.
Peptides regulate numerous processes, including rapid responses to biotic and abiotic stress. Biochemically characterized endogenous peptides include systemins, AtPep1, phytosulfokine, S-locus Cys-rich protein, and the rapid alkalinization factor, which are involved in wound-induced defenses, Pythium resistance, somatic embryogenesis, self-incompatibility, and root growth inhibition, respectively (Huffaker et al., 2006 ; Matsubayashi and Sakagami, 2006). Many of these peptides are active in the femtomolar to picomolar range and are derived from single or multiple homologous genes encoding precursor proteins. Bacterial peptides flg22 and elf18 (derived from elongation factor Tu) exhibit similar activity ranges and elicit resistance in Arabidopsis to Pseudomonas syringae and A. tumefaciens (Zipfel et al., 2004, 2006). In general, these PAMPs are derived from essential proteins that display infrequent mutations, whereas endogenous plant peptides are more variable between plant families.
In contrast to previously described plant peptide signals, prior to proteolysis, inceptin functions in planta as a critical dithiol region and redox switch that controls ATPase activity (Samra et al., 2006
Unlike pathogens, receptor-ligand pairs mediating specific nematode and insect resistance in plants remain unknown. Over a dozen nematode and insect R genes have been either cloned or mapped in close association with NBS-LRR gene clusters, yet no corresponding ligands have been identified (Rossi et al., 1998
For elicitors derived from variable length polypeptides, understanding the exact size of predominant native ligands is often not trivial. For example, despite considerable research, the actual contribution to elicitation in planta caused by prosystemin (23 kD) and flagellin (33 kD) relative to their respective active core peptides remains unclear. Comparatively, both intact proteins display 100-fold lower EC50s for alkalinization responses than the corresponding core peptides, yet still retain potent activity in the picomolar to nanomolar range (Dombrowski et al., 1999
To assess the production of bioactive peptides, we fed larvae recombinant GST-GmIn8H proteins derived from the soybean cATPC gene. This sequence encodes inceptin-related peptides identical to maize, established to have activity in cowpea equivalent to Vu-In (Schmelz et al., 2006
Peptide signals are known to mediate complex reorganizations in transcription and metabolite production, in part through phytohormone signaling pathways (Navarro et al., 2004
Inceptin activity depends on a limited number of conserved amino acid residues. As with systemin and flg22 deletion studies (Pearce et al., 1993
Through the overexpression of precursor proteins, plant-derived peptide signals have been used to elevate the expression of plant defenses and resistance to biotic attack (McGurl et al., 1994
Plant and Insect Material
Armyworm (Spodoptera frugiperda) eggs were obtained from Dr. R. Meagher (U.S. Department of Agriculture-Agricultural Research Service, Gainesville, FL) and reared on a pinto bean (Phaseolus vulgaris) diet (Schmelz et al., 2003a
All experiments used 2- to 3-week-old plants containing two fully expanded pairs of trifoliate leaves. For all induction assays, the adaxial sides of new fully expanded leaves were superficially scratched with a razor in three areas, removing approximately 5% of the total waxy cuticle. The damage sites (2 cm2 each) included the central leaf tip spanning both sides of the midrib and two midbasal sections on opposite sides of the midrib. Test solutions in 5 µL water were immediately applied and dispersed over the damage sites. Leaves remained on the intact plants for specified lengths of time prior to E and leaf metabolite sampling. Large-scale collection of OS from the sixth instar larvae was as previously described (Turlings et al., 1993
As previously reported, a 100-mL sample of cowpea-derived armyworm OS was initially fractionated using RP-C18 and strong anion exchange Discovery solid phase extraction columns (Supelco; Schmelz et al., 2006
Edmund chemical N-terminal sequencing was performed as previously described (Schmelz et al., 2006
Synthetic Vu-GE+In, Vu-E+In, Vu-In, and Vu-In–A peptides were HPLC purified, dissolved into water, and applied (n = 4) at 1.3, 4, 12, 45, 130, 430, 1,280, 4,300, and 12,800 fmol leaf–1 to a total of 144 cowpea leaves. Immediately prior to the construction of these dilutions, peptide concentrations in stock solutions were analyzed by LC/MS and adjusted. Compared to Vu-In, natural and synthetic Vu-GE+In and Vu-E+In consistently displayed greater losses of these peptides in solution suggesting lower aqueous solubility.
Primers CTCGAGATGGCTGATGATGTTTTCTCAC and GCGGCCGCATAGATCTATCCCTCTCCACAGTCAAC, which contain XhoI and NotI sites, respectively, were used to amplify cDNA from soybean leaves with the resulting 253-bp PCR product cloned into a TOPO pCRII vector (Invitrogen). The soybean chloroplastic atpC partial sequence generated was deposited in GenBank (EF185283). This fragment contains a BamHI site 95 bp downstream of the XhoI site and a BglII site 34 bp upstream of the NotI site. The 230-bp XhoI-BglII fragment and the 145-bp BamHI-NotI fragment were ligated together with pBluescript SK+ cut with XhoI and NotI to create a construct with two inceptin fragments. This SK+ GmIn2 construct was cut with BglII + NotI and BamHI + NotI, and then the gel-purified 3.2-kb and 145-bp fragments were ligated to create SK+ GmIn4. This process was repeated once more to generate the SK+ GmIn8 construct. To introduce a polyhistidine tag, primers Bgl2HIS-F (GATCACCACCACCACCACCAC CACCAC) and Blg2HIS-R (GATCGTGGTGGTGGTGGTGGTGGTGGT) were phosphorylated with T4 polynucleotide kinase (Invitrogen), annealed with one another to create BglII-compatible overhangs, and ligated with BglII-cut SK+ GmIn8. The 1.2-kb XhoI-NotI GmIn8H fragment was then ligated to SalI-NotI cut pET41b(+) (Novagen). This construct was transformed into BL21DE3pLysS Escherichia coli (Novagen) and was used to express GST-GmIn8H upon the addition of 1 mM isopropylthio- To estimate the amount of GST-GmIn8H fed to armyworm larvae, a dilution series containing the equivalent of 0.1, 0.3, 1.0, and 3.0 µL of protein was subject to SDS-PAGE next to known concentrations of pure His-tagged GST. Duplicate gels were either stained with Coomassie Blue or transferred to polyvinylidene difluoride membrane for western blot with mouse monoclonal anti-6x His antibody (Sigma) followed by peroxidase conjugated antimouse (Sigma). Reactive proteins were visualized by chemiluminescent staining followed by exposure to x-ray film. Both films and stained gels were photographed with the Bio-Rad Gel-Doc system and quantified with Quantity One software (Bio-Rad). The slope of the line for the GST standard was used to calculate the concentration of GST-GmIn8H in each lane and average molecules/microliter was determined based on the molecular mass of GST and GST-GmIn8H.
To examine inceptin-related peptide production in armyworm OS, we allowed 24 individual larva to consume 10-mm2 pieces of cellulose filter paper saturated with 20 µL of 20% Suc solution containing 9.6 µg GST-GmIn8H for either 2 or 6 h. Amounts of filter paper consumed varied greatly, and larval OS were pooled into single 280-µL samples at each time point. The 2- and 6-h samples were purified using a combination of RP-C18/strong anion exchange SPE and RP-C18 HPLC with all active fractions combined for the final NP-HPLC separation. To aid in reproducibility of diet consumption by larvae and quantitatively explore the dynamics of inceptin production, isolated sixth instar armyworm were deprived of artificial diet for 12 h, then allowed to consume a 5-mm2 piece of cellulose filter paper saturated with 10 µL of 20% Suc solution containing 4.8 µg GST-GmIn8H. All larvae completed consumption of the protein between 45 and 60 min after initial access to the filter paper diet. Within 15 min of completed diet consumption (
Quantification of inceptin-related peptides was achieved using ubiquitously labeled 13C and 15N Val-N-FMOC (V*; Cambridge Isotope Laboratories) incorporated into the synthetic peptide [Vu-I; +ICDING-V*-CVDA–]. Aliquots of crude OS (typically 50–100 µL) were sequentially spiked with 50 ng of the internal standard peptide, 5 µL HCOOH, vortexed, and centrifuged at 12,000g for 5 min. The aqueous phase was mixed with an equal volume of ethanol, stored at –70°C for 30 min, and centrifuged 12,000g for 2 min. Samples were diluted to 5% ethanol, loaded on 100-mg RP-C18 SPE columns, washed with 2 mL of water, and eluted with 9:1 CH3CN:water. Samples were then concentrated to dryness under vacuum, brought up in 50 µL 5:95 CH3CN:water containing 10 mM HCO2NH4 and 10 µL analyzed by LC-MS as described. Quantification was based on peak retention times (9.7–10.2 min) and monitoring of [M + H]+ ions with a m/z of 1,125.5 (ISTD), 1,034.5 (Gm-In–A), 1,105.5 (Gm-In), 1,234.5 (Gm-E+In), 1,291.5 (Gm-GE+In), 1,048.5 (Vu-In–A), 1,119.5 (Vu-In), 1,248.5 (Vu-E+In), and 1,305.5 (Vu-GE+In). The identity of each peptide was confirmed with MS2 daughter ion spectra.
Gas chromatography-based quantification of elicitor-induced E production followed from Schmelz et al. (2006)
To consider amino acid residues important for inceptin signaling, peptides with additional and deleted amino acids at both the N and C termini were examined along with an Ala substitution series of Vu-In. These peptides were synthesized by Genosys (Sigma-Aldrich) and subsequently HPLC purified using RP-C18 to >90%. Significant solubility differences in these peptides necessitated estimation of actual aqueous concentrations using LC/MS and selected [M + H]+ m/z ion monitoring to determine peak areas. Analysis of dilutions resulted in a final concentration of 4 pmol leaf–1 for all peptides assayed. Based on the timing of the dynamics of induced phytohormone changes (Fig. 7, A–D), we focused on E production between 60 and 120 min and SA/DMNT pools at 240 min. In this experiment, the cowpea leaf tip (n = 4) was excised at 1 h for E analysis while the treated basal leaf section remained attached and was harvested at 4 h for metabolite analysis. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number EF185283.
The following materials are available in the online version of this article.
We thank R.L. Meagher, N. Lowman, and C. Dillard for supplying armyworm larvae, A.Y. Chung for peptide synthesis, G. Pearce and G. Munske for amino acid sequencing, T. Davis and J. Meredith for experimental assistance, H.J. Klee and A.R. Zangerl for useful editorial feedback, and two anonymous reviewers for improving the manuscript. Received January 31, 2007; accepted March 5, 2007; published March 16, 2007.
1 This work was supported by the U.S. Department of Agriculture, Agricultural Research Service (base funds directed to project no. 6615–22430–002–00D). 
2 The use of trade, firm, or corporation names in this publication (or page) is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Eric A. Schmelz (eschmelz{at}gainesville.usda.ufl.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.097154 * Corresponding author; e-mail eschmelz{at}gainesville.usda.ufl.edu; fax 352–374–5707.
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ABSTRACT
RESULTS
= 200 Å) are overlaid on an arbitrary scale. Active fractions (*) were sequentially collected, desalted, evaporated, and resolubilized in water for bioassays. C, Final purification resulted in fractions (*) used for MS, Edmund N-terminal sequencing, and confirmation by comparison with synthetic peptides.
-diols, termed bruchins, present in pea (Pisum sativum) weevil oviposition fluid and the fatty acid amino acid conjugates (FACs) from lepidopteran OS (Doss et al., 2000
1 h from the start of feeding) larval OS were collected from the first group, defined as time zero, and pooled from three individual larva (n = 4). Similar larvae were sampled 2, 4, 6, and 8 h later. The peptides Gm-GE+In, Gm-E+In, Gm-In, and Gm-In–A were quantified in these samples using RP-C18 LC/MS analysis. To examine OS activity from larvae on different host plants, sixth instar armyworm were allowed to feed on either cowpea or spinach for 48 h prior to removal. A total of 96 larvae were used to generate 32 independent samples, with the OS of three larvae pooled (n = 4) at 0, 1, 2, and 4 h after isolation from host plants and used for induced E bioassays in cowpea leaves. 
