Figure 2. Accumulation of oscillating metabolites in N. attenuata show tissue-specific rhythms. Oscillating metabolites in source leaves (A) and roots (C) were roughly divided into two groups, one peaking during the day, and the other peaking at night. The heat map displays all of the Z-transformed oscillating metabolites levels in a false-color scale where green indicates low and red indicates high values. Each metabolite is represented by a single row with the average linkage hierarchical clustering tree obtained using Euclidean distances as metric. We counted the number of ions (y-axis) that peak at a particular harvest times (x-axis) in source leaves (B) and roots (D). Gray boxes depict the dark period. (E) Venn diagram of the oscillating metabolites selected across source leaves (black solid line) and roots (gray dashed line). (F) Venn diagram of the oscillating metabolites after removing adduct and daughter ions. CAMERA package and Pearson correlation [17] were used to select adduct and daughter ions detected from extracts of source leaves (black solid line) and roots (gray dashed line). doi:10.1371/journal.pone.0026214.g002 Diurnal Rhythms in Plant Defense Metabolites leaves by OS-elicitation but the regulation of the second group by OS-elicitation was highly variable (File S1). This pattern of regulation might be affected by the timing of induction (1 pm in this experiment). It will be interesting to evaluate in future studies how elicitations at night influence these patterns. In systemic leaves and roots, these genes were less regulated (Figure 3D), whereas suc levels reduced quickly after treatments (Figure 3A). However, root SWEET and suc invertase transcript accumulations were rapidly increased within 5 h of OS-treatment (Figure 3D; P,0.05, Student’s t-test). The increase in suc invertase transcript levels is consistent with previous results of increased invertase activity in roots after OS-treatment [24]. Sucrose is synthesized from photosynthesis during the day and loaded into the phloem for transport to sink leaves and roots [25]. Starch is synthesized from sugars during the day and degraded into maltose to provide energy for the metabolic requirements of the dark phase [26]. A plant’s clock anticipates the end of the night and regulates the rate of starch degradation to prevent energy limitations at night [27]. The sugar content in plants is known to have a diurnal rhythm [28]. A weak diurnal rhythm of disaccharide in source leaves was observed in early-elongated stage of N. attenuata but did not pass our selection filter (Figure 3A). The fact that our analytical procedures do not distinguish sucrose from maltose, which has a rhythm opposite to that of sucrose, may be the explanation, because sucrose accumulations are known to peak at dusk, while maltose peaks at night [27]. Testing this possibility will require calculating the ratio of sucrose and maltose in N. attenuata and examining separately their regulation during herbivory. The other reason may be the relatively strong selection filter that we employed. Consistent with this, the kinetic of this disaccharide in source leaves but not in sink leaves was detected as diurnally rhythmic when the stringency was lowered (data not shown). OS-elicitation significantly altered many sugar-metabolic and transporter genes in treated leaves within 5 h (Figure 3D) but the disaccharide levels in treated leaves was not changed significantly within 21 h (Figure 3A). This indicates that the rapid turnover of disaccharide levels may maintain sugar levels in treated leaves, which is used to produce secondary metabolites for direct defense responses and supply carbon for allocation from shoot to root that would increase a plant’s tolerance of herbivory [24,29,30]. In contrast, disaccharide levels in sink tissues, systemic leaves and roots were significantly reduced (Figure 3A). The OS-elicited decrease in disaccharide levels in roots during a day may be mainly due to a reduction of sucrose because maltose levels remain low during the day and increase during the night [26,28]. The reduced disaccharide levels in roots may have increased the gradient force driving sucrose translocation from shoot to root [24]. However, it remains an open question whether the decrease in disaccharide levels in sink leaves is linked to a reduction of sucrose or maltose levels or whether it results from increased sucrose transport from source leaves to sink leaves. A sugar-containing diterpene glycoside and its related genes The analysis identified a sugar-containing defense metabolite with strong diurnal patterns of accumulation. We found that the 2 accumulation of lyciumoside I (m/z 629.35 at 339 s, C32H53O12 ), a precursor of various diterpene glycosides (DTGs) in N. attenuata [31], peaked at dusk in treated and systemic leaves but this metabolite was not detected in roots (Figure 4A and 4C). Sink leaves contained more lyciumoside I than did source leaves (Figure 4A and 4C). OS-elicitation did not alter the accumulation of lyciumoside I in treated leaves within 21 h, whereas W+W PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26214