Figure 3. Effects of N-isobutyl decanamide on defense-related metabolite production and PR1 expression. Arabidopsis seedlings were grown for 6 days on N-isobutyl decanamide-free medium and transferred to control plates with or without N-isobutyl decanamide for 7 additional days. Salicylic acid (SA) accumulation was determined by measuring free and conjugated SA by GC-MS (A). Benzoic acid was used as internal standard, data are means of three independent experiments 6 SD. Transgenic Arabidopsis line carrying PR1:GUS was stained for GUS expression (B). Detection of hydrogen peroxide (H2O2) was made by staining leaves from control and treated seedlings with DAB (C). Images were captured with a Nomarski microscope. Nitric oxide (NO) from leaves of control and treated seedlings was detected by analyzing fluorescent signal of DAF-2DA with a confocal microscope (D). All photographs are representative individuals from at least 9 seedlings analyzed. doi:10.1371/journal.pone.0027251.g003 Alkamide-Regulated Transcriptional Networks etry (GC-MS), JA accumulation in seedlings treated with the solvent or 60 mM N-isobutyl decanamide . A nearly two-fold increase in JA level was observed in response to alkamide treatment (Figure 5A), indicating that the effects of this alkamide on gene expression might be mediated, at least in part, by increasing JA production in the plant. To gain further insight into the transcriptional activation of JA-dependent responses to N- isobutyl decanamide, transgenic seedlings containing a chimeric gene in which the LOX2 promoter is fused to the GUS reporter gene (LOX2:GUS) were treated with 30 and 60 mM N-isobutyl decanamide, and GUS histochemical analysis performed in 7 d- old seedlings. Interestingly, increased expression of this marker was observed throughout the shoot in alkamide-treated seedlings in a dose-dependent manner (Figure 5B). Because a concentration of 30 mM N-isobutyl decanamide was able to induce LOX2 expression, we evaluate the expression levels of JA-responsive transcripts by qRT-PCR in seedlings treated for 7 days with this alkamide concentration. Among the biosynthetic genes, we focused on genes encoding the LOX2 and LOX3 lipoxigenases, ALLENE OXIDE SYNTHASE (AOS) and ALLENE OXIDE CYCLASE2 (AOC2) enzymes, and OPDA REDUCTASE3 (OPR3). We also examined the expression levels of the JA- inducible genes JAZ8, VSP2 and ERF2. All tested genes were induced by N-isobutyl decanamide application more strongly than JA itself (Figure 5C). It has been reported that JA-responsive gene expression occurs in a short time after JA perception [52]. For this reason, the JA insensitive mutant coronatine insensitive1 (coi1-1) was employed as negative control to determine if an intact JA signaling pathway was required for the long-term (7 days) response. As shown in Figure 5C, most tested genes either did not respond or were repressed in response to JA or to N-isobutyl decanamide treatment in the coi1-1 mutant. Additionally, we evaluated local activity of N-isobutyl decana- mide to induce transcriptional activation of defense-related genes in fully developed leaves, which were excised and incubated for 24 h in media supplemented with 30 mM N-isobutyl decanamide. Alkamide-treated leaves showed increased expression of the LOX2:GUS reporter gene in a dose-dependent manner as compared to untreated controls (Figure S3A). Moreover, the relative expression level of OPR3, VSP2 and PAD3 genes was at least two-fold higher in treated wild-type leaves than in the controls (Figure S3B). These results show that at least some transcriptional networks modulated by N-isobutyl decanamide are active even in detached tissue. N-isobutyl-decanamide confers resistance to fungal necrotizing pathogen Botrytis cinerea JA accumulation and JA-responsive gene expression analyses suggest that N-isobutyl decanamide may function as a potential defense-inducing factor. To determine whether N-isobutyl decan- amide could effectively activate defense mechanisms that lead to pathogen resistance, we tested the responses of leaves from 20 d- old Arabidopsis plants to the necrotrophic pathogen Botrytis cinerea. In these experiments, again, fully developed leaves were transferred 24 h to agar plates supplied with 30 mM N-isobutyl decanamide or with the solvent as control. A 10 ml droplet of B. cinerea spores was inoculated on the leaf surface and disease symptoms evaluated 3, 4 and 5 days after inoculation (d.a.i.). In leaves transferred to control medium and inoculated for 3 days, the fungus induced necrotic lesions in over 90% of inoculated leaves (Figure 6A), whereas in N-isobutyl decanamide leaves treated only 10% presented necrotic lesions (Figure 6A). Four d.a.i., it was found that 100% of the control leaves showed necrotic lesions, whereas in N-isobutyl decanamide-treated leaves, around PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27251