Diurnal Rhythms in Plant Defense Metabolites (dashed lines with colors) or W+OS (solid lines with colors) treatments, Phe (C) and Tyr (G) levels were quantified in treated leaves (red), untreated systemic leaves (blue) and roots (green). Effects of W+W and W+OS treatments on relative transcript abundance (6SE) of genes related Phe (D–F) and Tyr (H–J) metabolism. Gray box depicts the dark period. Different symbols (* and #) indicate significant differences among the treatments at the indicated time point (P,0.05, one-way ANOVA with Bonferroni post hoc test). doi:10.1371/journal.pone.0026214.g005 accumulation in treated leaves may also be translocated into systemic leaves. Tyramine N-hydroxycinnamoyltransferase (THT) is an enzyme that conjugates cinnamoyl-CoA, caffeoyl-CoA, or feruloyl-CoA to tyramine [39]. Interestingly, two NaTHTs transcript levels (Figure 5I) were increased after OS-elicitation only in treated but not in systemic leaves (P,0.05, Student’s t-test). NaTHT1 (Figure 5J) and NaTHT2 (Figure S4) transcripts accumulated to high levels in roots and only NaTHT1 transcript displayed diurnal accumulation only in roots. OS-elicitation increased NaTHT1 (Figure 5J) and decreased NaTHT2 (Figure S4) transcript levels in roots (P,0.05, Student’s t-test). Phenylpropanoid-polyamine conjugates Coumaroyl tyramine (m/z 284.10 at 165 s, C17H18NO3+) peaked during the day (Figure 6A) with an OS-elicited increase within 1 h in treated leaves (P,0.05, one-way ANOVA followed by Bonferroni post hoc test). The levels of two feruloylamine conjugates [17] were also increased by W+W or W+OS treatments (Figure 6B and 6C). The accumulation of feruloyl putrescine (m/z 265.152 at 212 s, C14H21N2O3+) increased in treated and systemic leaves after OS-elicitation but not after W+W treatment (Figure 6B; P,0.05, one-way ANOVA followed by Bonferroni post hoc test). N-feruloyl tyramine accumulated (m/z 314.140 at 319 s, C18H20NO4+) markedly after W+W and W+OS treatments, but only in treated leaves (Figure 6C; P,0.05, one-way ANOVA followed by Bonferroni post hoc test). Systemic leaves did not accumulate N-feruloyl tyramine after any treatments was consis- tent with the low accumulation of NaTHT1/2 transcripts in systemic leaves (Figure 5I and 6C). However, no change in N- feruloyl tyramine levels was observed in roots (Figure S5) despite the significant increases in NaTHT1 transcripts in these tissues (Figure 6J). THT enzyme activity of barley and wheat is known to be stronger in roots than in shoots [40]. N-feruloyl tyramine levels in roots (Figure S5) were higher than in leaves (Figure 6C) before or after W+OS treatment, perhaps a reflection of these NaTHTs transcript levels, but did not show diurnal rhythms (Figure S5). Identification of oscillating tyramine conjugates in roots will be helpful to understand plant defenses against root herbivores or pathogens that have their own diurnal activity rhythms [41,42]. OPDA and JA in roots and their related genes We found two jasmonates with root-specific diurnal patterns of accumulation. OPDA (12-oxophytodienoic acid, m/z 291.20 at 479 s, C18H27O32) and JA (m/z 209.12 at 368 s, C12H17O32) levels peaked at night only in roots (Figure 7A and 7B). OPDA is a precursor of JA, which is an important phytohormone known to activate defense responses against herbivore attack [43]. JA accumulation is well known to increase after W+W and to be amplified by the OS-elicitation (Figure 7B, red line). Without OS- elicitation, OPDA and JA were detected at higher levels in roots than in leaves (Figure 7A and 7B). W+W and W+OS treatments did not dramatically alter the accumulation of OPDA or JA in roots as they did in treated leaves (Figure 7A and 7B; P,0.05, one- way ANOVA followed by Bonferroni post hoc test). Only OS- treatment slightly increased the accumulation of OPDA after 17 h (Figure 7A; P = 0.0064, one-way ANOVA followed by Bonferroni post hoc test). We identified several oscillating transcripts involved in JA signaling (Figure 7C and Table S4).While the levels of JA in leaves did not show diurnal patterns, the JA biosynthetic genes, Figure 6. OS-elicitation affects secondary metabolites in the phenylpropanoid pathway. Mean (6SE) levels of normalized intensity of coumaroyl tyramine (A; m/z 284.10 at 165 s, C17H18NO3+), feruloyl putrescine (B; m/z 265.15 at 212 s, C14H21N2O3+) and N-feruloyl tyramine (C; m/z 314.14 at 319 s, C18H20NO4+) in treated and systemic leaves at each harvest time for two days (gray dotted lines) in control plants. Feruloyl putrescine and N-feruloyl tyramine were not detected in roots. After W+W (dashed lines) or W+OS (solid lines) treatments their levels were examined in treated leaves (red) and untreated systemic leaves (blue). 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). doi:10.1371/journal.pone.0026214.g006 PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26214