Plants are exposed to two completely different environments: the aboveground and the belowground. Aboveground and belowground plant parts thus develop their own endogenous rhythms [10]. Clock components in the shoot and root oscillate in a similar way under light/dark cycles but not under constant light. The roots express approximately four-fold fewer oscillating genes than do shoots in Arabidopsis [10]. However, this does not mean that the endogenous clock is less important for root physiology. Important physiological processes such as root bending and lateral root formation occur every 6 h and are controlled by the internal clock [11]. In addition, clock-regulated water contents in roots are reduced during the day and increase during the night [12]. Nicotiana attenuata is a native tobacco plant growing in the Great Basin Desert of southwestern USA. It germinates in post-fire ecosystems and shows diverse intra-specific and inter-specific interactions. We have studied N. attenuata growing in its ecological niche in natural habitats for more than two decades and observed several diurnal rhythms in its physiology. N. attenuata interacts with different pollinators in a light-dependant manner [13,14]. It produces two kinds of flowers. Night-opening flowers (NoFs) that open their corollas during the night and close during the day. Morning-opening flowers (MoFs) open their corollas during the early morning. NoFs emit benzyl acetone only during the night to attract nocturnal hawkmoth pollinators (Manduca sexta and M. quinquemaculata). Early morning flower visitors such as humming birds mainly nectar at MoFs and transfer pollen. Plant-herbivore interactions are also regulated in a day/night cycle. The accumulation of two lipoxygenase trancripts (NaLOX2 and NaLOX3) involved in the biosynthesis of green leaf volatiles and jasmonic acid (JA) show diurnal rhythms in the leaf [15]. In addition, the generalist predator, Geocoris spp. feeds on the eggs and neonates of the specialist herbivore, M. sexta, usually during the day. Metabolites produced in leaves and roots are essential elements determining the outcome of plants’ aboveground and below- ground interaction with other organisms. If most of the organisms on earth are governed by their endogenous clocks, the rhythms of the metabolites they produce should help us understand plant- plant, plant-animal interactions. Even though many genes involved in metabolism show diurnal expressions, large-scale screenings of tissue-specific oscillating metabolites and their regulation by herbivory remain largely unknown. Here, we examined tissue-specific diurnal rhythms of metabolites in leaves and roots of N. attenuata for two days. To find interconnections among oscillating metabolites and herbivore-induced plant defenses, we treated mechanical wounds in the leaves of N. attenuata with oral secretions (OS) from M. sexta larvae to mimic herbivore-induced changes [16] and precisely time the onset of elicitations to analyze the changes in oscillating metabolite levels with the oscillations of the transcripts of their associated genes. Results and Discussion Experimental Design N. attenuata plants were grown in 16 h light/8 h dark cycle for 5 weeks, and source/sink leaves and roots were collected every 4 h for two days without any treatment to identify oscillating metabolites (Figure 1A). Diluted OS (1:5 with distilled water) from larvae of the specialist herbivore, M. sexta, were applied to puncture wounds in leaves created with a pattern wheel (W+OS) to mimic herbivory at 1 pm on a second day (Figure 1A). We also treated wounds with water (W+W) to distinguish OS-specific responses from wound-induced responses. Two source leaves (at nodes +2, +1) and one transition leaf (at node 0) were treated with Diurnal Rhythms in Plant Defense Metabolites water or OS, and collected to examine local response in oscillating metabolites and transcripts. We also collected two sink leaves (at nodes 21, 22) and roots to examine systemic response in untreated tissues (Figure 1B). The enormous diversity of metabolites among different plant taxa and their diverse chemical properties means that metabo- lomic analysis must be optimized for each plant species. In previous studies, we developed an efficient method to extract defense-related metabolites in N. attenuata [17,18] and used this 40% methanol-based extraction method (Figure 1C) and separat- ed the metabolites by rapid separation liquid chromatography (RSLC). The separated metabolites were ionized by electrospray in both negative and positive modes and the exact mass to charge ratio of ions was measured with time of flight (TOF)-mass spectrometry. We also used a cDNA library and a microarray system of N. attenuata to explore the overall molecular mechanism in plant-herbivore interactions. The three-dimensional data (retention time, mass, intensity) of the mass spectrometry analysis was processed using the peak picking freeware XCMS [19], and diurnally oscillating metabolites were extracted by a model-based peak picking algorithm of the HAYSTACK [20] program. To reduce the information redundancy in the dataset, isotope peaks were clustered and annotated using the pseudo-spectrum decon- volution freeware CAMERA and removed from the analysis. Fragment and adduct ions detected in negative mode were included as there is no certain way to date of selecting only mother ions in large scale experiments, and negative ionization produces fewer daughter ions and adducts compared to positive ionization mode. In the negative ionization analysis, a total of 2209 and 1463 ions were detected from leaves and roots, respectively. With these platforms, we identified diurnally oscillating metabolites and their related transcripts accumulation (Figure 1C). Leaves and roots have distinctive diurnal patterns Pattern analysis revealed that 8% of total leaf metabolites and 12% of total root metabolites detected in negative mode had diurnal rhythms (Figure 2, Table S1 and File S1). Oscillating metabolites separated roughly into two groups by hierarchical clustering (Figure 2A and 2C). One group of metabolites was highly induced during the day, and the other group peaked at dusk or night. In leaf extracts, 72% of oscillating metabolites peaked during the day (Figure 2A), whereas 81% of root oscillating metabolites peaked at dusk or night (Figure 2C). The number of ions that show highest accumulations at each time clearly showed distinctive patterns in the two different tissues (Figure 2B and 2D). Moreover, only 10 ions (among 182 in leaves and 179 in roots) had diurnal accumulations in both tissues (Figure 2E) and among them, only one ion had the same diurnal rhythm. After the pattern analysis, we identified the fragment and adduct ions among the oscillating ions by CAMERA and by Pearson correlations dependent upon time and treatments [17] and found that a still smaller number of ions (8 ions among 122 and 132 ions in leaves and roots, respectively) were diurnal-regulated in both tissues (Figure 2F). The results show that oscillating metabolites in N. attenuata accumulated in a tissue-specific manner and a few metabolites were commonly oscillating in both leaves and roots (Figure 2). According to the transcriptome analyses performed in root and shoot of Arabidopsis, the root has fewer oscillating genes than does the shoot under constant light condition [10]. However, our analysis of oscillating metabolites, which are the final products of gene regulations, demonstrates that roots also have strong diurnal rhythms which are distinct from those found in the leaves of N. attenuata. PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26214