Tissue Specific Diurnal Rhythms of Metabolites and Their Regulation during Herbivore Attack in a Native Tobacco, Nicotiana attenuata Sang-Gyu Kim, Felipe Yon . , Emmanuel Gaquerel., Jyotasana Gulati, Ian T. Baldwin* Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Jena, Germany Abstract Ecological performance is all about timing and the endogenous clock that allows the entrainment of rhythms and anticipation of fitness-determining events is being rapidly characterized. How plants anticipate daily abiotic stresses, such as cold in early mornings and drought at noon, as well as biotic stresses, such as the timing of pathogen infections, is being explored, but little is known about the clock’s role in regulating responses to insect herbivores and mutualists, whose behaviors are known to be strongly diurnally regulated and whose attack is known to reconfigure plant metabolomes. We developed a liquid chromatography-mass spectrometry procedure and analyzed its output with model-based peak picking algorithms to identify metabolites with diurnal accumulation patterns in sink/source leaves and roots in an unbiased manner. The response of metabolites with strong diurnal patterns to simulated attack from the specialist herbivore, Manduca sexta larvae was analyzed and annotated with in-house and public databases. Roots and leaves had largely different rhythms and only 10 ions of 182 oscillating ions in leaves and 179 oscillating ions in roots were rhythmic in both tissues: root metabolites mainly peaked at dusk or night, while leaf metabolites peaked during the day. Many oscillating metabolites showed tissue-specific regulation by simulated herbivory of which systemic responses in unattacked tissues were particularly pronounced. Diurnal and herbivory-elicited accumulation patterns of disaccharide, phenylalanine, tyrosine, lyciumoside I, coumaroyl tyramine, 12-oxophytodienoic acid and jasmonic acid and those of their related biosynthetic transcripts were examined in detail. We conclude that oscillating metabolites of N. attenuata accumulate in a highly tissue- specific manner and the patterns reveal pronounced diurnal rhythms in the generalized and specialized metabolism that mediates the plant’s responses to herbivores and mutualists. We propose that diurnal regulation will prove to an important element in orchestrating a plant’s responses to herbivore attack. Citation: Kim S-G, Yon F, Gaquerel E, Gulati J, Baldwin IT (2011) Tissue Specific Diurnal Rhythms of Metabolites and Their Regulation during Herbivore Attack in a Native Tobacco, Nicotiana attenuata. PLoS ONE 6(10): e26214. doi:10.1371/journal.pone.0026214 Editor: Daniel J. Kliebenstein, University of California, United States of America Received July 2, 2011; Accepted September 22, 2011; Published October 18, 2011 Copyright: 2011 Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Max Planck Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: baldwin@ice.mpg.de . These authors contributed equally to this work. Introduction Timing is everything for ecological performances. The earth’s 24 h rotation on its tilted axis, geographical differences, and interactions with other organisms shape the specific diurnal rhythms of each organism. As sessile organisms, plants can entrain their physiology to abiotic condition of their environment such as day/night cycles and their associated temperature fluctuations. The endogenous plant clock (circadian clock) ‘wakes up’ the photosynthetic machinery just before sun rise to maximize energy harvesting and regulates guard cells at noon to minimize water loss [1]. It also increases cold tolerance of plants at night and dawn [2]. Plants also synchronize their physiology with tightly associated organisms. For instance, snapdragon flowers emit methyl benzoate during the day to attract day-active pollinating bees [3]. Pathogens that attack at dawn are anticipated by the clock in Arabidopsis [4]. Since the pioneering discoveries that the rhythmic behaviors of animals are encoded in their genomes, the molecular components and functions of the plant’s endogenous clock have been identified in the model plant, Arabidopsis thaliana [5]. Forward and reverse genetic approaches in Arabidopsis have revealed that many diurnal ‘behaviors’ are controlled by a few clock genes [1,5]. These clock genes regulate 30,40% of total gene expression in Arabidopsis [6]. Arrhythmic plants harboring mutations in the clock genes have reduced photosynthetic capacity, growth and competitive ability under normal conditions [7,8]. However, natural mutations in clock genes have been discovered that help entrain a particular accessions’ physiology to its local environment which enhances the plant fitness in that area [8]. Plants fix carbon in the shoot using light energy and make numerous metabolites from the products of photosynthesis. Plant metabolites therefore originate from a day/night cycle. Studies of primary metabolites have shown that sugars, starch, amino acids, most of organic acids involved in photosynthesis are circadian- regulated in leaves [9]. Although transcriptomic analyses show that many genes involved in secondary metabolite biosynthesis have diurnal expression patterns [5], the diurnal rhythms of secondary metabolite levels have been less studied in comparison with those of primary metabolites. PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26214