( A ) EFN secretion [mg soluble solids per gram leaf fresh mass and hour], ( B ) time course in endogenous jasmonic acid (JA) levels [ng per gram leaf fresh mass], ( C ) endogenous JA level 60 min after different treatments. Identical colors in different panels indicate identical treatments, values shown are means ± SE (of n = 7 biological replicates in panel A and n = 5 in panels B and C). Bars or lines marked with different letters indicate treatments that were significantly different (P<0.05) according to posthoc analysis with least significant difference tests. Abbreviations: Con = control, E = leaf extract, EFN = extrafloral nectar, F = flame wounding, Fru = fructose, Glu = glucose, Sor = sorbitol, Suc = sucrose, W = mechanical wounding.

Wild-type plants of P. lunatus were subjected to flame wounding, or to mechanical wounding with subsequent application of either water or 1 mM aqueous solutions of glucose, fructose, sucrose, ATP, or JA (positive control). EFN secretion quantified 2 h after treatment differed significantly among treatments (univariate ANOVA: F = 8.340, P<0.001, n = 7). Wounding alone increased EFN secretion over control levels but the response was significantly stronger after application of ATP, sucrose, leaf extract, or flame wounding. Glucose and fructose caused no further increase as compared to wounding alone ( Fig. 1A ). Sucrose is the major photosynthetic product and the dominant sugar in the phloem [24] and its extracellular concentration will inevitably increase upon tissue disruption, whereas neither glucose nor fructose is an abundant sugar in the leaf tissue. A sudden increase in the extracellular concentration of sucrose, but less so of the monosaccharides, would therefore appear to be a suitable indicator of severe tissue disruption. ATP is released from cells by secretion or by wounding and it has been suggested that ATP plays a role in plant defense signaling [25] . Flame wounding physically destroys entire cells, releasing their contents into the extracellular space. Flame wounding as applied in our study can induce proteinase inhibitor genes, which are classical markers of wound-induced genes whose expression depends on JA signaling [26] , [27] , and has been related to Ca 2+ signaling [28] , which is a well-known early step in the perception of herbivore-feeding by plants [29] , [30] . Finally, leaf extract contains all the molecules that are released when cells become disrupted. Patterns in EFN secretion thus support the prediction that common plant molecules or their fragments are monitored in the extracellular space for damaged-self recognition in lima bean.

Endogenous JA levels were quantified (ng per gram leaf fresh mass) at different times after the treatment in ( A ) Arabidopsis, ( B ) sesame, ( C ) tomato, ( D ) strawberry, and ( E ) maize. Lines marked with different letters indicate statistically significant differences among treatments (least significant difference post hoc tests, n = 5 biological replicates per mean).

We applied the above treatments and quantified endogenous JA using gas chromatography coupled with single-ion mass spectrometry (GC-SIM-MS). Wounding induced endogenous JA, but leaf extract, sucrose and flame wounding had significantly greater effects ( Fig. 1B , Table 1 ). The effect of wounding alone was dependent on the severity of the damage, and glucose, sorbitol or salt (NaCl) at 1 mM had no significant effect on the induction of endogenous JA compared with the levels of JA induced by wounding alone ( Fig. 1C ), which excludes osmotic shock as the reason for the JA response. Similar effects were observed in other plant species. Mechanical wounding of leaves of Arabidopsis thaliana (Brassicaceae), tomato (Solanum lycopersicum, Solanaceae), strawberry (Fragaria×ananassa, Rosaceae), sesame (Sesamum indicum, Pedaliaceae) and maize (Zea mays, Poaceae) caused a significant induction of endogenous JA in all species compared with the level of endogenous JA in intact leaves ( Fig. 2 , Table 1 ). The application of sucrose induced the greatest increase in endogenous JA levels in maize, whereas in sesame and tomato the application of leaf extract induced a greater increase in JA concentration than that induced by mechanical damage ( Fig. 2 ), confirming earlier reports of an induction of JA-dependent plant defenses after the application of leaf extract [1] , [31] , [32] , [33] . Therefore, both the amount of damage ( Fig. 1C ) and the species ( Fig. 2 ) can affect whether any of the damaged-self signals boost the JA response above the levels that are seen after mere mechanical damage. We conclude that all the plants investigated here can respond to mechanical damage to some degree, whereas the specific signals perceived and the degree of the response varies among species. Damaged-self recognition represents a taxonomically common mechanism that is subject to physiological and evolutionary flexibility.

Transcriptional changes after damaged-self recognition

The application of leaf extract or sucrose induced a greater increase in endogenous JA than that induced by mere wounding in lima bean, sesame, tomato and maize, and mere wounding increased endogenous JA over control levels in all species investigated here (Figs. 1 and 2). In lima bean, an increase in the severity of the wounding led to an increase in the level of endogenous JA that was dependent on the severity of the damage, whereas the application of leaf extract or sucrose elicited a strong JA response that was independent of the damage level applied before (Fig. 1C). Finally, several different damaged-self signals were able to induce EFN secretion by lima bean (Fig. 1A). All these observations appear contradictory to reports that HAMPs are required for a full resistance response [5], [9], [10], [12], [14], [15], [16], [18], [33], [34], [35]. To resolve this apparent contradiction, we must consider the quantitative aspect: studies aimed at elucidating the particular role of damaged-self recognition in the overall response to herbivore feeding could compare endogenous JA concentrations after different damage levels (with and without the application of leaf extract) to the levels observed after damage caused by different types of herbivores, and they should consider the effects downstream of JA. Our preliminary results indicate that the relative importance of damaged-self recognition is likely to vary among species.

How similar are transcription patterns after damaged-self recognition to the full set of JA-dependent transcriptional responses in lima bean leaves? We used 454 pyrosequencing [36] of cDNA libraries to investigate transcriptomic changes (in relation to the transcriptome of untreated control plants) after JA treatment, flame wounding, mechanical wounding, and mechanical wounding with subsequent application of leaf extract or sucrose. We extracted mRNA, produced cDNA, tagged the resulting libraries and then subjected them to two sequencing runs [37]. During 454 sequencing, the number of sequencing reads representing the same gene can be taken as a measure of transcript abundance (see Text S1).

The overall patterns of up- and down-regulated genes (see Table S1 for the list of contigs, accession numbers, annotation results and expression levels of differentially expressed genes) as compared to the control were surprisingly similar among lima bean leaves that had been treated with JA and leaves that had been subjected to fire or to mechanical wounding with subsequent application of leaf extract (Fig. 3). By contrast, mechanical wounding alone and mechanical wounding with subsequent application of sucrose solution caused clearly distinct patterns (Fig. 3).

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 3. Transcriptomic response of lima bean leaves to damaged-self signals and jasmonic acid (JA). (A) Transcriptomic patterns (genes up- and downregulated in comparison to untreated controls) are presented for JA treatment (JA) in comparison to flame wounding (F), mechanical wounding (W), and mechanical wounding with subsequent application of leaf extract (E) or 1 mM solution of sucrose (S). (B) Scatter plot of Hj (diversity) versus δj (specialization) for each treatment tested. https://doi.org/10.1371/journal.pone.0030537.g003

Leaf extract application and particularly flame wounding elicited very similar transcriptomic patterns to those elicited by JA (Fig. 3A). Forty-three genes were induced and 71 were repressed by all three treatments. The transcriptome elicited by JA overlapped with a further 48 genes (29 up, 19 down) in the wounding-induced transcriptome and with 36 genes (33 up, 3 down) in the extract-induced transcriptome (Table 2). According to a Gene Ontology classification using the BioMaps tool from the VirtualPlant webpage (http://virtualplant.bio.nyu.edu/cgi-bin/vpweb/) [38] most of the upregulated genes represent categories such as defense and virulence, interaction with the environment and responses to wounding or stress (Table S2). Several of the highly upregulated genes (by factors >10) are involved in JA synthesis (e.g. LOX, lipoxygenase; AOS, allene oxide synthase; and AOC, allene oxide cyclase) [3] or in the regulation of JA-isoleucine-responsive genes downstream of JA (JAZ1, jasmonate ZIM-domain) [39], [40], [41]. Genes related to photosynthesis or primary metabolism appeared downregulated, which is in line with the repressing effect of JA on photosynthesis and other growth-related processes [42]. Only a few of the induced genes were related to osmotic or salt stress (Table S3), which further supports our view that our treatments caused a specific response rather than general osmotic stress.

Because the metabolic pathways that we obtained from the P. lunatus unigene set yielded an almost complete coverage of a global metabolic map according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) classification (Fig. 4), we are confident that our transcriptome is sufficiently complete as to allow conclusions concerning global changes in gene expression. To quantify the similarities among these transcriptomes, we compared their diversity (H j ) and specialization (δ j ), defining H j as the Shannon entropy of the frequency distribution of a transcriptome and δ j as the average specificity of the genes expressed under each condition [43]. Plotting the transcriptomes in a two-dimensional space defined by H j and δ j as well as calculating Euclidean distances among the distributions of the transcriptomes confirmed that the transcriptomes obtained after JA treatment, leaf extract application and flame wounding clustered closely together (Fig. 3B, Table 3). Apparently, the global changes in gene expression after the application of leaf extract after mechanical wounding or after damaging the tissue via flame wounding were mediated via jasmonate signaling.

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 4. Metabolic pathways represented in the Phaseolus lunatus unigene set. Global metabolism map constructed combining existing pathway maps and corresponding genes referenced in the KEGG database for Arabidopsis, Populus, Vitis, Oryza and Sorghum (green lines). (B) Global metabolism map represented by the P. lunatus unigene set (magenta lines). (C) Overlap comparison of the KEGG metabolic global map of flowering plants (Arabidopsis, Populus, Vitis, Oryza and Sorghum) with the metabolic map represented by the P. lunatus unigene set. https://doi.org/10.1371/journal.pone.0030537.g004

Mere mechanical wounding overall elicited similar transcriptomic patterns but was clearly distinguishable from the transcriptomic patterns elicited by JA, flame wounding, or leaf extract application after mechanical wounding. We observed a set of genes which were either induced only after wounding or that were repressed by wounding but induced by JA, or when wounding was caused by fire or complemented by the application of leaf extract (see lower parts in Fig. 3A). Several of the genes that were repressed by mechanical wounding alone, but not repressed by JA or flame wounding or when leaf extract was applied to the wounded leaves, appeared to be related to photosynthesis (Table S1). Genes that were induced at least fourfold by mechanical wounding, but not induced by leaf extract application to wounded leaves, or by JA application or flame wounding, appeared, among others, to have ATPase and other ATP binding functions (Table S1).

Mechanical damage as applied in our study is likely to release some damaged-self signals. However, the transcriptomic patterns elicited by our mechanical wounding treatment differed in some parts from the patterns that resulted when the entire contents of many cells were applied to the wounded tissue. Sucrose caused gene expression alterations that overlapped partly, but in general differed from the gene expression induced by the other treatments (Fig. 3). Although sucrose induced both EFN secretion and endogenous JA-levels in lima bean (Fig. 1), sucrose-signaling is also involved in multiple independent physiological responses [24], [44]. It therefore appears to be likely that an exogenous application of sucrose induced a mixed response whose components can be related to both damaged-self recognition and other physiological processes.

The expression patterns of 14 genes were confirmed with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR, see Table S4 for primer sequences). We found a good correlation between expression levels obtained by 454 sequencing and those obtained by qRT-PCR (Figs. 5 and 6). Interestingly, leaf extract, sucrose and flame wounding treatments induced the expression of AOS, AOC, LOX, OPR and JAZ1 more strongly than JA itself (Fig. 5). At the phenotypic level, the same treatments induced high levels of endogenous JA in lima bean, with flame wounding eliciting the highest levels, a result that is fully congruent with the strong induction of EFN secretion that occurred following this treatment (Fig. 1). NaCl and sorbitol induced gene expression patterns that were the opposite of those induced by the damaged-self signals. The level of induction of most of the up-regulated genes was much lower following glucose application than for the other treatments, and MYB73, a transcription factor that was induced by all damaged-self signals, including sucrose, was repressed by glucose (Figure 5C). Although the signals that trigger the defense response represent common plant molecules (or changes in their extracellular concentration), the specificity that we observed in the transcriptomic changes appears to be high enough to allow a fine-tuning of the response.