0.1% (v/v) acetonitrile (Baker, HPLC grade) and 0.05% (v/v) formic acid. Solvent B consisted of acetonitrile and 0.05% (v/v) formic acid. The following gradient conditions were used for the chromatography: 0–0.5 min 10% B, 0.5–6.5 min linear gradient 80% B, 6.5–10 min 80% B, and re-equilibration at 10% B for 3 min. The flow rate was 300 mL/min. An ESI-TOF mass spectrometer (Bruker Daltonic, Bremen, Germany) was used to determine the molecular mass of ionized molecular fragments and the amounts of the eluted analytes. The capillary voltage was 4500 V, and dry gas (200uC) flow rate was 8 L/min. Detected ion range was from m/z 200 to 1400 at a repetition rate of 1 Hz. The mass calibration was achieved using a sodium formate solution (10 mM sodium hydroxide and 0.2% formic acid in isopropanol/water 1:1, v/v). Raw data processing Raw data files from Bruker software (Data Analysis v4.0) were exported as netCDF format, and processed using the XCMS R package [19] (http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics/ Peak_Alignment/xcms/). Peak detection was performed using the centWave algorithm with the following parameter settings: ppm = 20, snthresh = 10, peakwidth = c(5,18). Retention time correction was accomplished using the XCMS retcor function with the following parameter settings: mzwid = 0.01, minfrac = 0.5, bw = 3. Missing peak data were filled using the fillPeaks function. The CAMERA package was used to annotate isotope and adduct ions (http://bioconductor. org/packages/devel/bioc/html/CAMERA.html). After 75 percentile normalization and log2 transformation, XCMS output files were processed using Microsoft Excel and the Statview software for statistical test, and TIGR’s Multiexperiment Viewer software for visualization and clustering. Diurnal pattern analysis and metabolites annotation The model-based HAYSTACK [20] algorithm was used to identify diurnal-regulated metabolites (http://haystack.cgrb. oregonstate.edu/). Isotope ions detected using CAMERA were removed before processing using pattern matching algorithm of HAYSTACK. With the models from HAYSTACK we selected oscillating metabolites, typically with the following values in the selection filters: correlation cutoff 0.8, fold cutoff 1.5, P-value cutoff 0.05. Molecular formulas were generated using the SmartFormular algorithm in Data Analysis v4.0 software (Bruker). The following maximum elemental composition CaHbNcOdNaeKf and restrictions were used: 1#b/a#3; e = 0 or 1; f = 0 or 1; a, b, c and d not limited. Rings plus double bonds values from 20.5 to 40, the nitrogen rule and ions of even electron configuration were considered. The structural annotation based on tandem MS measurements of the precursor ions of selected diterpene glucosides and feruloyl putrescine has been published by our group in Gaquerel et al. [17]. Tandem MS measurements for these metabolites are available in the supplemental online material associated with this article. Lyciumoside I has initially been characterized in N. attenuata by means of NMR in Heiling et al. [31]. Phenylalanine, tyrosine, 12-oxo-phytodienoic acid and jasmonic acid were identified after comparison with authentic standard material. Manual annotation using metabolite informa- tion from the literature was performed in the case of tyramine conjugates. Feruloyl tyramine and coumaroyl tyramine are well described in tobacco cells’ metabolites whose biosynthesis highly increases by wounding [59,60]. Besides elemental formula calculation, the annotation of these metabolites was facilitated by typical ion signatures corresponding to coumaroyl- (m/z 147.04) and feruloyl- (m/z 177.04) residues released after the break of the Diurnal Rhythms in Plant Defense Metabolites ester bound linking them to a core tyramine molecule during in- source fragmentation. Microarray data and analysis Three biological replicates among the six replicates harvested at each harvest time used for metabolites analysis were also used for RNA isolation (six harvest time for control and W+OS treatments, three harvest time for W+W treatments). Total RNA was isolated with TRIZOL reagent and labeled cRNA with the Quick Amp labeling kit (Agilent). Each sample was hybridized on Agilent single color technology arrays (4644K 60-mer oligonu- cleotide microarray designed for N. attenuata transcriptome analysis, http://www.agilent.com, GEO accession number GPL13527). Agilent microarray scanner (G2565BA) and Scan Control software were used to obtain intensity of the spots. All microarray data with each probe name were deposited in the NCBI GEO database (accession number GSE30287). We confirm that all details are MIAME compliant. The resulting gene expression profiles were analyzed using GeneSpring GX software (Silicon Genetics, Redwood City, CA). Raw intensities were normalized using the 75th percentile value and log2 and baseline transformed prior statistical analysis. Probes were filtered based on their Quality control Metrics. The HAYSTACK algorism was also used to examine the diurnal rhythm of genes after we selected genes of interest involved in biosynthesis or signaling of oscillating metabolites. With the models from HAYSTACK we examine the diurnal rhythm of gene of interest, typically with the following values in the selection filters: correlation cutoff 0.8, fold cutoff 2, P-value cutoff 0.05. Supporting Information Figure S1 Accumulation of m/z = 683.23 at retention time, 90 s, in roots. Mean (6SE) levels of normalized intensity of m/z = 683.23 at 90 s in roots. Calculated molecular formula 2 (C24H43O22 ) and retention time (90 s) indicated that it is a disaccharide dimer. After W+W (dashed lines with colors) or W+OS (solid lines with colors) treatments, compound levels were examined in roots (green). Gray boxes depict the dark period. Asterisks indicate significant differences among the treatments at the indicated time point (* = P,0.05, one-way ANOVA with Bonferroni post hoc test). (TIF) Figure S2 Accumulation of Phe in leaves, roots and open flowers. Mean (6SE) levels of normalized intensity of Phe in different tissues. Phe accumulation was quantified in leaves (red), roots (green), and flowers (black). To calculate relative accumu- lation, 75th percentile normalized intensity at each harvest time was divided by average value over all time points. We collected open flowers from 7 week-old plants and extracted metabolites with a 40% methanol extraction method. Gray box depicts the dark period. (TIF) Figure S3 Transcript abundance of NaADT1/2, Na- PAL1/2, and NaC4H1/2 in roots. Mean (6SE) levels of normalized intensity of NaADTs, NaPALs and NaC4Hs in roots. ADT, arogenate dehydratase; PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase. Gray boxes depict the dark period. Asterisks indicate significant differences between control plants and M. sexta oral secretions-treated plants (W+OS) at indicated time points (* = P,0.05, as determined by Student’s t-test). (TIF) PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e26214