in vitro growth of some plant microbial pathogens, including bacteria and fungi [35]. To explore the structure-activity relationships of alkamides, we previously evaluated the root developmental responses of Arabidopsis seedlings to application of a group of affinin-derived natural and/or synthetic fatty acid amides with similar chain length [42]. We found that N-isobutyl decanamide, a C:10 saturated alkamide that is naturally produced in Acmella radicans [43] and Cissampelos glaberrima [44], was the most active compound in inhibiting primary root growth and stimulating lateral root formation. Interestingly, root developmen- tal alterations induced by N-isobutyl decanamide were related to a sustained increase in nitric oxide (NO) production and required the activity of the DRR1 protein [45]. To further understand the molecular responses to fatty acid amides, in this work we performed whole-genome transcriptional profiling of Arabidopsis thaliana seedlings in response to N-isobutyl decanamide. Our results show the activation of defense-related gene expression, concomitant to an increase in JA accumulation and in the expression of JA-responsive and senescence-associated genes. Moreover, N-isobutyl decanamide application to mature Arabidopsis leaves conferred resistance against fungal necrotizing pathogen Botrytis cinerea in a process involving JA-dependent signaling. Results Transcriptomic profiling of Arabidopsis in response to N-isobutyl decanamide To characterize at the transcriptional level the molecular responses of Arabidopsis to N-isobutyl decanamide, Col-0 WT seedlings were germinated and grown for 6 d on 0.26 MS medium and then transferred to fresh medium supplied with or without 60 mM of N-isobutyl decanamide to directly compare their effect on whole-genome transcriptional profile after 1, 3, 7 and 14 d of treatment (Figure S1) employing a two-channel long- oligonucleotide microarray platform (see Methods). According to a stringency level of FDR 0.05 (fold change $2), a total of 1,281 genes showed differential expression in at least one of the four sampled time points. The complete list of differentially expressed genes is provided in Table S1. Among differentially expressed genes, 727 were found to be up-regulated and 554 down-regulated by N-isobutyl decanamide (Figure 1A). Only 22 from the 727 induced genes and 33 down-regulated genes were common to all time points evaluated (Figure 1B). Of these overlapping genes, highest expression values were reached on the seventh day of N-isobutyl decanamide treatment (Figure 1C). Analysis of expression patterns by agglomerative hierarchical clustering showed that the number of differentially regulated genes increased from day 1 to day 7 after treatment and then decreased at day 14 (Figure 1C). In addition to the statistical methods described (see Materials and Methods), validation of microarray data was achieved by real- time quantitative PCR (qRT-PCR) of 15 randomly chosen genes, including up- and down-regulated genes. These experiments were carried out using RNA extracted from an independent batch of control and treated plants than those used for microarray analysis experiments. qRT-PCR gene expression profiles obtained for the analyzed loci were quite consistent with those generated by the microarray analysis (Figure S2). Functional categories of genes up-regulated by N-isobutyl decanamide Differentially expressed genes were classified into functional categories according to the Munich Information Center for Alkamide-Regulated Transcriptional Networks Protein Sequences classification (MIPS) using the FunCat database [46]. The categories ‘‘Metabolism’’ (290 genes), ‘‘Storage protein’’ (16 genes), ‘‘Cellular transport, transport facilities and transport routes’’ (161 genes), ‘‘Cell rescue, defense and virulence’’ (189 genes), ‘‘Interaction with the environment’’ (165 genes), ‘‘Systemic interaction with environment’’ (92 genes), ‘‘Cell fate’’ (31 genes) and ‘‘Biogenesis of cellular components’’ (90 genes) were identified as significantly over-represented MIPS categories among N- isobutyl decanamide responsive genes (Table S2). Most of these genes belong to defense- and stress-related categories, including subcategories belonging to the ‘‘Metabolism’’ set such as, ‘Metabolism of glutamate, polyamines, nitrogen and related groups, chitin and others polysaccharides, and secondary metab- olism’ (Table S2). When we performed functional categorization per day of treatment, we found that the highest percentage of genes in every category was represented at the seventh day (Figure 2A). Two remarkable over-represented categories identified were ‘‘Cell rescue, defense and virulence’’ and ‘‘Systemic interaction with environment’’ (Figure 2A). Detailed analyses of these two categories showed significant overrepresentation of the ‘stress response’, ‘disease, virulence and defense’, ‘detoxification’, ‘plant/ fungal specific systemic sensing and response’ and ‘animal systemic sensing and response’ subcategories (Figure 2B). Within these subcategories, we found 70 genes involved in oxygen and radical detoxification, 75 genes involved in hormone-related responses (auxin, ethylene, cytokinin and abscisic acid), and particularly, genes encoding enzymes involved in JA synthesis and associated responses (Table S2; Figure 3). Additional differentially regulated genes encoded proteins related to biotic stress, including different secreted pathogenesis-related proteins (PR) such as chitinases and glucanases (At4g07820, At2g19990, At2g14610, At3g57260, At3g04720, At1g75040, At2g19970, At2g14580, At4g33720 and At2g14580) (Table S1). Overrepresentation of biotic stress-related categories can be appreciated more clearly in the functional categorization of N-isobutyl decanamide-induced genes (Figure 2C). These results suggest that alkamides are likely involved in triggering defense-associated responses in Arabidopsis. General defense responses but not salicylic acid biosynthesis are activated by N-isobutyl decanamide Because N-isobutyl decanamide increased the transcript level of a wide class of PR genes, we examined its effect on the production of salicylic acid (SA) and signaling molecules related to local and systemic responses in defense processes. SA is a phenolic hormone whose activity is required to successfully respond against several different invading pathogens [47] and their biosynthesis succeeds in association with changes in redox homeostasis producing reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H2O2) [48]. In turn, SA and H2O2 release is accompanied by another reactive signalling molecule, nitric oxide (NO). Whole-transcriptional profiling regulated by N-isobutyl decanamide showed that PATHOGENESIS-RELATED1 (PR1, At2g14610), a marker for SA signaling, and overall defense responses [49,50,51] increased its transcript level by 7.5-fold at day 7 (Table S1). However, none of the genes encoding enzymes related to SA biosynthesis were significantly up-regulated. Moreover, N-isobutyl decanamide did not appear to significantly affect the overall SA content despite an observed induction of the PR1:GUS reporter-gene expression (Figure 3A &B), suggesting that N-isobutyl decanamide-mediated gene expression of PR1 occurred independently of SA accumulation. It is well documented that some stress-associated molecules, such as ROS, play signaling roles as second messengers in PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27251