Diurnal Rhythms in Plant Defense Metabolites Figure 3. Accumulation of disaccharides and sugar related genes in three different tissues. (A) Mean (6SE) levels of normalized intensity of disaccharides (m/z 341.11 at 90 s, C12H21O112) in source leaves, sink leaves and roots at each harvest time for two days (gray dashed lines) in control (Con) plants. After wounding and treating puncture wounds with either water (W+W, dashed lines with colors) or M. sexta OS (W+OS, solid lines with colors), disaccharides levels were examined in treated leaves (red), untreated systemic leaves (blue) and roots (green). Gray boxes depict the dark period. Asterisks indicate significant differences among the treatments at the indicated harvest time (* = P,0.05, one-way ANOVA with Bonferroni post hoc test). (B) Schematic overview of sucrose (suc) metabolism. (C) Two diurnal patterns of sugar metabolism-related genes (Table S2) accumulation in source leaves. Gray boxes depict the dark period. Sweet: sugar exporter [23]. Ratio to average: Ratio of transcript abundance at the time point shown, to the mean abundance of the same transcript across all time points. (D) Volcano plot analysis of the transcript levels of sugar- related genes in treated leaves (red dot), systemic untreated leaves (blue dot) and roots (green dot) 1 and 5 h after M. sexta OS-elicitation. The log2 ratio of mean intensities (OS-elicited/Con, with microarray expression data) plotted against the negative log10-transformed P value derived from Student’s t tests. The horizontal dashed line indicates the threshold for statistically significant expression at P = 0.05 and the vertical dashed line, the threshold for two-fold changes in gene expression. doi:10.1371/journal.pone.0026214.g003 treatment (Figure 4A, red line) increased its levels in treated leaves at 5, 13 and 17 h (P,0.05, one-way ANOVA followed by Bonferroni post hoc test). Consistent with these results was the observation that NaGGPPS (N. attenuata geranylgeranyl diphosphate synthase) transcripts (Figure 4B), which are involved in the biosynthesis of lyciumoside I [31], significantly increased only after W+W treatment (P,0.05, one-way ANOVA followed by Bonferroni post hoc test). Systemic signaling also elicited the accumulation of lyciumoside I (Figure 4C) and NaGGPPS transcript (Figure 4D) in systemic leaves (P,0.05, one-way ANOVA followed by Bonferroni post hoc test). W+W and W+OS treatments resulted in similar increases in lyciumoside I, whereas OS-elicitation resulted in larger increases in NaGGPPS transcripts in systemic leaves after wounding (Figure 4D). OS-elicitation reduced the wound-induced genes (type I) or amplified the regulation of genes by wounding (type II) [32]. Interestingly, NaGGPPS transcript showed a type I expression pattern in treated leaves and a type II expression pattern in systemic leaves. In flowering stage plants, several DTGs accumulate mainly in young leaves and reproductive tissues [31]. Induction of Lyciumoside I by OS-elicitation also followed this pattern which might enhance plant fitness by protecting these young fitness-enhancing tissues first, as predicted by Optimal Defense Theory. PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26214