Fatty acid-amino acid conjugates (FACs) in M. sexta OS induce herbivore-specific defense responses in N. attenuata [16,44]. The phytohormone, JA plays an important role in FACs-induced plant defense signaling against herbivore attack [43]. JA biosynthesis occurs in two organelles, the chloroplast and the peroxisome. a- linolenic acid released from membrane is converted into OPDA by LOX, AOS, and AOC (allene oxide cyclase), all enzymes of the chloroplast. The OPDA is then imported into the peroxisome and converted into JA [43]. Even though root cells have no chloroplasts, transcripts encoding LOX, AOS and AOC proteins are expressed in roots of several plant species, likely in the leucoplasts of roots, and jasmonates are detected in their roots [45–48]. To our knowledge, our study represents the first report of a diurnal rhythm of OPDA and JA in roots of plants. Basal levels of JA in leaves did not show a diurnal rhythm, although JA biosynthesis genes, LOX3 [15] and AOS (Figure 7C) transcripts in N. attenuata’s leaves did (Figure 7C). It is still unclear whether the oscillation in JA levels in roots results from JA synthesis in leaves and transport into roots or their de novo synthesis in the roots. Wang et al. treated wounded N. attenuata’s leaves with isotope- labeled (13C6) Ile and detected JA-13C6-Ile in treated leaves of N. attenuata [49]. However, labeled JA-Ile was not detected in systemic leaves and roots. These data suggest that N. attenuata’s roots have the ability to de novo synthesize jasmonates [47,49]. Stress-induced biosynthesis of JA initiates direct and indirect defense responses in plants [29]. The diurnal rhythm of JA levels in roots may be linked to the daily occurrence of these stresses in roots. One of the possible stresses is elicited by pathogen attack. The circadian clock component, CCA1 has been recently shown to be critical for plants in the anticipation of leaf pathogen attack and in turn regulate the expression of defense related genes against the pathogen [4]. Arabidopsis AOS gene has one CCA1 binding site (AAAAAATCT) and one evening element (AAAATATCT) in its promoter region [5,50,51]. LOX3 in Arabidopsis have also one evening element [50,51]. Homologous genes in N. attenuata showed diurnal accumulations (Figure 7C) either in leaves or in roots [15]. It will therefore be extremely interesting to analyze the promoter sequences of JA-related genes in N. attenuata. Leaf pathogens usually attack at dawn when temperature and moisture conditions are conducive for pathogen infection. However, roots in the soil are subjected a completely different microclimate. Diurnal rhythms of JA accumulation in roots therefore may play other roles. Oscillating JA levels may facilitate root penetration into hard soil. The soil of N. attenuata’s native habitat, the Great Basin Desert in Utah is usually dry and hard. We used sand-grown roots, rather than hydroponic culture, to mimic as close as possible these natural conditions. Comparisons of the JA contents of hydropon- ically-grown and sand-grown roots are not simple, because these different culture conditions produce roots with different patterns of growth and morphology. Nonetheless, when we compared the JA contents in the same mass of roots, we usually observed higher JA levels in sand-grown roots than in hydroponically-grown roots (data not shown). The pattern of JA accumulation that peaked at night may be associated with the growth rhythm of roots and the production of secondary metabolites to protect root cells from infection and attack during growth-associated wounding. Even though JA levels in leaves did not show a diurnal rhythm, JA signaling components, COI1 and several JAZs transcript levels in N. attenuata were diurnal-regulated (Figure 7C). Far-red light treatment increases AOC and JAZ1 transcript accumulation of Arabidopsis and phytochrome A elicits the degradation of JAZ1 proteins [52], which suppress COI1-dependent JA signaling. Extrafloral nectar secretion in lima bean is regulated by JA levels Diurnal Rhythms in Plant Defense Metabolites but also in a light dependent manner [53]. These results suggest that diurnal changes in light quality in nature may influence JA signaling pathway in a time-dependent manner. In other words, diurnal rhythms of JA signaling components in leaves may play a central role in modulating defense responses of plants depending on the time of day when they are attacked. Future work The timing of gene regulation has been intensively studied in Arabidopsis. However, a lack of association between diurnal rhythms and one of the major biotic stresses for plants, that of herbivore attack in which secondary metabolites play central roles, led us to examine how oscillating metabolites are influenced by herbivory. Recently, Kerwin et al. reported the reverse mechanism, that glucosinolate metabolism affects the output of the circadian clock and clock gene expression [54,55]. Mutations in glucosino- late biosynthetic genes result in the alteration of the circadian clock genes including the PSUDORESPONSIVE REGULATORs (PRRs). M. sexta attack elicits dramatic changes in N. attenuata metabolism [17,21,56]. These changes may also feedback on clock gene expression during herbivory. To test this hypothesis, in future work, we will examine the microarray data of irLOX2 (producing less green leaf volatiles), irLOX3 (deficient in JA biosynthesis), and irMYB8 (containing less phenylpropanoid-polyamine conjugates) of N. attenuata using single time point analysis described by Kerwin et al. [15,54,57]. Materials and Methods Plant material and treatment Wild type (WT), Nicotiana attenuata plants (30th inbred generation) were grown from seeds originating from a natural population in Utah. The original collection of the seeds was done on private lands and since N. attenuata is not an endangered plant species no specific permissions for seed collections were necessary. Moreover, all seeds used in this study were bred in the glasshouse at our institute. Seeds were sterilized and germinated on Gamborg’s B5 medium as previously described [58]. Ten-day old seedlings were transferred to small pot (TEKU JP 3050 104 pots, Po¨ppelmann GmbH & Co. KG, Lohne, Germany) with Klasmann plug soil (Klasmann-Deilmann GmbH, Geesten, Germany) and after 10 days, seedlings were transferred to 1 L pots with sand to facilitate sampling of roots. Plants were watered by flood irrigation system with 200 g CaNO34H2O, 200 g Flory B1 in 400 L water and grown in the glasshouse at 26–28uC under 16 h supplemental light from Master Sun-T PIA Agro 400 or Master Sun-T PIA Plus 600 W Na lights (Philips, Turnhout, Belgium). We used early elongated stage of WT N. attenuata plants for metabolomics and transcriptomic analyses. Six biological repli- cates (plants) were harvested every 4 h for 2 d from each treatment group. Before freezing the samples in liquid nitrogen, roots were washed in a water tank for a few seconds to remove sand. M. sexta oral secretions (OS) collected from fourth- or fifth-instar larvae were diluted 1:5 with deionized water. Metabolite analysis We used a 40% methanol extraction procedure optimized for the extraction of a wide range of our interesting metabolites of in N. attenuata [17]. 4 mL of the resulting leaf extracts and 6 mL of the resulting root extracts were injected onto a C18 column (Acclaim, 2.2 mm particle size, 150 mm62.1 mm inner diameter, Dionex Corporation, Sunnyvale, USA) and separated using a RSLC system (Dionex). Solvent A consisted of deionized water containing PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e26214