Diurnal Rhythms in Plant Defense Metabolites Figure 1. Experimental procedures used to identify oscillating and herbivore-induced metabolites and their associated genes in different tissues of Nicotiana attenuata. (A) Wild type (WT) N. attenuata plants were harvested every 4 h for two days during the initiation of stem elongation. To mimic herbivory, oral secretions (OS) of the larvae of the specialist herbivore, M. sexta, were immediately applied to puncture wounds made in leaves at 1 pm. Water treatment of puncture wounds on separate plants was used to distinguish OS-specific from wound-induced changes in metabolites and transcripts. (B) Metabolites from three different tissues, source leaves, sink leaves, and roots of N. attenuata were isolated. The leaf at node 0 had completed the sink to source transition and the leaf at node +1 was older by one leaf position than the leaf at node 0 and so forth. Source leaves (at nodes +2, +1, 0) were wounded with a fabric pattern wheel and treated with 20 ml of M. sexta OS, which was diluted 1:5 with water. Untreated leaves (at nodes 21, 22) and roots were harvested to monitor systemic responses. (C) After sample preparation from six biological replicates, a 40% methanol extraction method optimized for the defense metabolites of N. attenuata was used and the metabolites separated with a rapid separation liquid chromatography (RSLC) on a C18 column and detected by ESI-TOF-MS (electrospray ionization time-of-flight mass spectrometer) for parents and their daughter ions. Peak picking and alignments were performed with the XCMS package [19]. Diurnal oscillating metabolites were extracted by the pattern matching algorithms of HAYSTACK tool [20]. In-house and public databases were used to identify oscillating metabolites and a 44K Agilent microarray designed for N. attenuata was used to examine the expression of metabolite-related genes. doi:10.1371/journal.pone.0026214.g001 Next, we annotated the oscillating ions using in-house and public databases, and analyzed their accumulation after W+W and W+OS treatments. Here, we describe the stories of 7 suites of oscillating metabolites, tales that speak of interesting interactions among the diurnal rhythms of metabolites and plant defense responses against herbivory. A disaccharide and its related genes We first focused our attention on two signals corresponding to the m/z 341.11 and its dimer, m/z 683.23, at 90 s both with the same strong diurnal rhythms that peaked at dusk only in roots (Figure 3A and S1). While, the same signals were detected in treated and systemic leaves they did not pass our selection filter in these tissues (Figure 3A). We calculated the elemental formulas (m/ 2 2 z 341.11, C12H21O11 ; m/z 683.23, C24H43O22 ) using Smart- Formula. Molecular mass and standard compound injections verified that m/z 341.11 at 90 s is a disaccharide and m/z 683.23 at 90 s is a dimer of the disaccharide. Changes in primary metabolism strongly influence plant- herbivore interactions [21]. We therefore examined the regulation of disaccharide levels with their related genes (Figure 3B) after W+W and W+OS treatments. The result showed that only OS- elicitation rapidly reduced disaccharide levels in roots (Figure 3A, green line) within 1 h (P,0.05, one-way ANOVA followed by Bonferroni post hoc test). In untreated systemic leaves, disaccharide levels (Figure 3A, blue line) were significantly reduced by W+OS treatment after 13 h and W+W treatment after 17 h (P,0.05, one-way ANOVA followed by Bonferroni post hoc test). However, disaccharide accumulations in treated leaves did not change within 21 h after treatments (Figure 3A, red line), except for a small reduction at 17 h after W+W treatment (P = 0.0134, one-way ANOVA followed by Bonferroni post hoc test). Most of the genes involved in sugar metabolism have diurnal rhythms [9,22]. We blasted the Arabidopsis and other plant species homologues of these genes (Figure 3B) against our cDNA database of N. attenuata and identified the transcripts with high similarity (Table S2). We observed two distinct diurnal patterns of transcript accumulation for these genes using the HAYSTACK algorithm (Figure 3C and File S1). The first group peaked in the middle of the day and was low during the night (Figure 3C, left). The second group peaked at dawn and remained at low levels for the rest of the day (Figure 3C, right). We analyzed the regulation of these genes after OS-elicitation. Interestingly, the genes involved in sucrose transport (suc transporter and sugar exporter, SWEET [23]) were significantly up-regulated and several other genes (b-amylase, suc synthase, suc phosphatase and sugar exporter) were down-regulated by W+OS treatment in treated leaves (Figure 3D; P,0.05, Student’s t-test), while disaccharide levels in treated leaves (Figure 3A) were not changed. The first group of genes peaked in the day was usually down-regulated in treated PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26214