Alkamide-Regulated Transcriptional Networks Figure 1. Overview of N-isobutyl decanamide responsive genes in Arabidopsis seedlings. Number of genes (vertical axis) Up-regulated (red) and Down-regulated (blue) by N-isobutyl-decanamide treatment at 1, 3, 7, and 14 d.a.t. (A). Edwards-Venn diagrams showing common or distinct responsive genes identified at every time evaluated (B). The number of genes up- or down-regulated in a single condition is shown in bold letters. The number of genes regulated at all sampled-times are shown in bold italic font. Agglomerative hierarchical clustering of differentially expressed genes at every sampled times (C). Clustering was performed using the Smooth correlation and average linkage clustering in GeneSpring GX 7.3.1 software (Agilent TechnologiesH). Blue color indicates Down-regulated, red Up-regulated and white unchanged values, as shown on the color scale at the right side of the figure. doi:10.1371/journal.pone.0027251.g001 developmental and defense process. Among the N-isobutyl decanamide differentially expressed genes, at least 70 belonging to the functional group ‘‘oxygen and radical detoxification’’ were regulated by alkamide treatment (Table S2), having their highest expression level at days 3 and 7 after transfer. Up-regulated genes included ten peroxidases (At4g08780, At5g06730, At4g08770, At5g06720, At5g58390, At5g05340, At2g18150, At3g49960, At3g49110 and At2g18140), two thioredoxin H-type TH8 and TH7 (At1g69880, At1g59730) and a glutaredoxin (At5g40370), two glutathione peroxidases ATGPX4 and ATGPX6 (At2g48150, At4g11600), five FAD-binding oxidoreductases (At1g26410, At1g26380, At1g26390, At1g26400 and At1g26420), the catalase CAT3 (At1g20620) and HYDROPEROXIDE LYASE1 (At4g15440) (Table S1 & S2). Given this overrepresentation, we decided to explore whether ROS accumulation coincided with the increase in transcript level of the group of oxygen and radical detoxification genes. We detected hydrogen peroxide (H2O2) production in situ in Arabidopsis seedlings that were transferred for 7 d from MS 0.26 medium to a medium containing N-isobutyl decanamide. At this stage the seedlings were treated with 3,3-diaminobenzidine (DAB), which in the presence of peroxidases polymerizes as soon as it comes into contact with H2O2, forming a brown precipitate. Leaves from N-isobutyl decanamide-treated seedlings clearly showed an increase in H2O2 (Figure 3C) and NO production (Figure 3D) when compared to solvent-treated seedlings. Overall, these results suggest that general defense-associated responses elicited by N-isobutyl decanamide appear to be related to both hormonal and oxidative stress response. Endogenous levels of JA and their corresponding transcripts are induced by N-isobutyl decanamide Virtually all genes encoding for biosynthetic enzymes for JA production were regulated by N-isobutyl decanamide (Figure 4A). Canonical JA-dependent inducible genes involved in JA signaling and response pathways showed predominantly induction profiles (Figure 4C). Among them CORONATINE-INDUCED3 (CORI3, At4g23600), NAC DOMAIN-CONTAINING PROTEIN81 (ATAF2/ ANAC081, At5g08790), JASMONATE-ZIM-DOMAIN (JAS1/ JAZ10, At5g13210), ETHYLENE RESPONSE FACTOR2 (ERF2, PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27251