Diurnal Rhythms in Plant Defense Metabolites Figure 4. Diurnal rhythms and OS elicitation of glucose-containing secondary metabolites in different tissues. Mean (6SE) levels of normalized intensity of lyciumoside I (m/z 629.35 at 339 s, C32H53O122) in treated (A), and untreated systemic leaves (C) at each harvest time for two days (gray dotted lines) in control plants. Lyciumoside I was not detected in roots. After W+W (dashed lines with colors) or W+OS (solid lines with colors) treatments, lyciumoside I levels were examined in treated leaves (A) and untreated systemic leaves (C). Gray boxes depict the dark period. Asterisks indicate significant differences among the treatments at the indicated time points (* = P,0.05, one-way ANOVA with Bonferroni post hoc test). (B), (D) Effects of W+W and W+OS on relative transcript abundance (6SE) of NaGGPPS (N. attenuata geranylgeranyl diphosphate synthase), a gene involved in producing the diterpenoid precursor, geranylgeranyl diphosphate [31]. Different letters (a, b and c) reflect significant differences among the treatments at the indicated time points (P,0.05, one-way ANOVA with Bonferroni post hoc test). doi:10.1371/journal.pone.0026214.g004 Phenylalanine/tyrosine and their related genes The aromatic amino acids, phenylalanine (Phe) and tyrosine (Tyr) are well-known primary metabolites with diurnal rhythms [9]. Phe and Tyr, which are synthesized via the shikimate pathway, are used in the production of lignin, anthocyanins, alkaloids, floral scents and defensive metabolites [33]. These compounds were also identified by our method for identifying oscillating metabolites (Figure 5C and 5G). The genes involved in their biosynthesis or catabolism showed similar diurnal patterns as well (Figure 5A, 5B and Table S3). Most of these genes increased during the day and remained at low levels at dusk and night (Figure 5B). Interestingly, Phe accumulation in different tissues peaked at different times: 2 or 6 pm in leaves and 10 pm in roots (Figure 5C and S2). While Phe levels in roots peaked later than in leaves, we did not find a diurnal pattern of the Phe biosynthetic gene transcripts, arogenate dehydratases (NaADTs) in roots, which are thought to convert arogenate to Phe, (Figure S3A and Table S3). The diurnal rhythm of Phe levels in the roots may be linked to the diurnal accumulation of NaADT1/2 transcripts in leaves (Figure 5B and 5D) and the translocation of Phe from leaves to roots through the phloem [34–36]. Phe levels in flowers are particularly interesting because it is a precursor of the nocturnally emitted pollinator attractant, benzyl acetone [37,38]. Phe levels in flowers were elevated during the night (Figure S2). Three different tissues (leaf, root and flower) have their own diurnal rhythms of Phe, which would be connected to specific roles of Phe in different tissues. Tyr levels were constitutively high in roots, exceeding the levels found in leaves even during peak accumulations (Figure 5G). W+W and W+OS treatments induced similar increases of Phe levels in treated leaves (Figure 5C, red line; P,0.05, one-way ANOVA followed by Bonferroni post hoc test), but OS-elicitation resulted in much stronger systemic responses (Figure 5C, blue line; P,0.05, one-way ANOVA followed by Bonferroni post hoc test). Phe accumulation was also increased in roots, but the OS-elicited increase was less than in treated/systemic leaves (Figure 5C, green line). Transcripts of two NaADTs were significantly increased within 1 h of elicitation followed by dramatic decreases in treated leaves (Figure 5D, red line; P,0.05, Student’s t-test), which may explain Phe’s peak 5 h after treatment. Transcripts of the same genes (Figure 5D, blue line) were also increased in systemic leaves (P,0.05, Student’s t-test) but with a 4 h delay. Phe accumulation in systemic leaves peaked twice at 5 and 13 h after OS-treatment but only once at 5 h after W+W treatment. It is possible that a part of the induced Phe accumulation in younger leaves originated from Phe produced in treated leaves. Phenylalanine ammonia lyase (PAL) is the first enzyme in phenylpropanoid biosynthesis (Figure 5A). NaPAL1/2 transcript accumulations followed a similar pattern to that of NaADT1 (Figure 5E) with OS-elicited increases within 1 h in treated leaves and within 5 h in systemic leaves (P,0.05, Student’s t-test). The other phenylpropanoid pathway gene, C4H (cinnamate-4-hydrozylase) showed a similar pattern of increase observed in NaPALs transcript levels (Figure 5F; P,0.05, Student’s t-test). The accumulation of NaADTs, NaPALs and NaC4Hs transcripts in roots was less affected by W+W and W+OS treatments than in leaves (Figure S3). W+W and W+OS treatments increased the amount of Tyr in treated and systemic leaves (Figure 5G; P,0.05, one-way ANOVA followed by Bonferroni post hoc test), while OS-treatment increased its level with an 8 h delay in systemic compared to treated leaves (Figure 5G). Only W+W treatment increased Tyr accumulation in roots at 9 and 13 h (Figure 5G; P,0.05, one-way ANOVA followed by Bonferroni post hoc test). We identified two arogenate dehydrogenase (TyrA) genes, which are thought to be involved in Tyr biosynthesis (Figure 5H), with OS-elicited increases in treated and systemic leaves (P,0.05, Student’s t-test). NaTyrA1/2 transcript levels increased more in treated leaves compared to systemic leaves (Figure 5H), but induced Tyr levels in both tissues were similar (Figure 5G). The induced Tyr PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26214