Development and Validation of a Liquid Chromatography−Electrospray Ionization−Time-of-Flight Mass Spectrometry Method for Induced Changes in <i>Nicotiana attenuata</i> Leaves during Simulated Herbivory

Gaquerel, Emmanuel; Heiling, Sven; Schoettner, Matthias; Zurek, Gabriela; Baldwin, Ian Thomas

doi:10.1021/jf1017737

Cited by 61 publications

(109 citation statements)

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“…Herbivory by larvae of the specialist lepidopteran M. sexta was simulated by applying freshly collected oral secretions to mechanically wounded leaves (35). This procedure, hereafter referred to as W+OS treatment, elicits, in a highly reproducible manner, major changes in the secondary metabolites of N. attenuata leaves that can be profiled by UHPLC-ESI/qTOFMS (28,36). After mass feature (m/z signals detected for a specific retention time) extraction, alignment, and retention correction from the overall sample population, the resulting concatenated data matrix (Dataset S1) consisting of 1,044 m/z features (not including isotope peak features) was analyzed using principal component analysis (PCA) statistical modeling to explore the variance structure of across-individual metabolic profiles without categorizations by accession location (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

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Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis

Baldwin

Gaquerel

2015

Proc. Natl. Acad. Sci. U.S.A.

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Natural variation can be extremely useful in unraveling the determinants of phenotypic trait evolution but has rarely been analyzed with unbiased metabolic profiling to understand how its effects are organized at the level of biochemical pathways. Native populations of Nicotiana attenuata, a wild tobacco species, have been shown to be highly genetically diverse for traits important for their interactions with insects. To resolve the chemodiversity existing in these populations, we developed a metabolomics and computational pipeline to annotate leaf metabolic responses to Manduca sexta herbivory. We selected seeds from 43 accessions of different populations from the southwestern United Statesincluding the well-characterized Utah 30th generation inbred accession-and grew 183 plants in the glasshouse for standardized herbivory elicitation. Metabolic profiles were generated from elicited leaves of each plant using a high-throughput ultra HPLC (UHPLC)-quadrupole TOFMS (qTOFMS) method, processed to systematically infer covariation patterns among biochemically related metabolites, as well as unknown ones, and finally assembled to map natural variation. Navigating this map revealed metabolic branch-specific variations that surprisingly only partly overlapped with jasmonate accumulation polymorphisms and deviated from canonical jasmonate signaling. Fragmentation analysis via indiscriminant tandem mass spectrometry (idMS/MS) was conducted with 10 accessions that spanned a large proportion of the variance found in the complete accession dataset, and compound spectra were computationally assembled into spectral similarity networks. The biological information captured by this networking approach facilitates the mining of the mass spectral data of unknowns with high natural variation, as demonstrated by the annotation of a strongly herbivory-inducible phenolic derivative, and can guide pathway analysis.plant-insect interactions | metabolomics | mass spectrometry | natural variation E lucidating the structure of metabolites underlying complex traits and the factors that maintain their variation in natural populations are important challenges in plant ecological studies (1). Many studies have notably shown that stress-responsive pathways that produce secondary metabolites are sporadically found across different plant taxa with extensive diversification (2). This important diversification suggests that particular metabolic systems have been recruited through natural selection when the set of compounds that they produce address specific ecological needs. Interactions with insects are important selection pressures that have sculpted plant metabolism, and many plant metabolites protect against herbivore attack and physical damage (3-5). The timely production of particular secondary metabolites in response to insect attack benefits plants by decreasing the costs of constitutive metabolite production. Trade-offs between defense metabolite productions and the intrinsic growth-related functions of central metabolic pathways likely prov...

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Section: Resultsmentioning

confidence: 99%

“…Two microliters of the 40% methanol leaf extracts were separated using a Dionex rapid separation liquid chromatography system (Dionex) as previously described in ref. 36 (SI Appendix, Materials and Methods).…”

Section: Methodsmentioning

confidence: 99%

Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis

Baldwin

Gaquerel

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Elemental formulas of [1, 2-13 C]MeJA and 12-COOH-JA-Ile were verified by ultra-high-pressure LC time-of-flight MS using the method and calculation settings described in Gaquerel et al (2010).…”

Section: Phytohormone Analysismentioning

confidence: 99%

Ectopic Expression ofAtJMTinNicotiana attenuata: Creating a Metabolic Sink Has Tissue-Specific Consequences for the Jasmonate Metabolic Network and Silences Downstream Gene Expression

et al. 2011

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To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis (Arabidopsis thaliana) jasmonic acid O-methyltransferase (35S-jmt) and additionally silenced in other lines the N. attenuata methyl jasmonate esterase (35S-jmt/ir-mje) to reduce the deesterification of methyl jasmonate (MeJA). Basal jasmonate levels did not differ between transgenic and wild-type plants; however, after wounding and elicitation with Manduca sexta oral secretions, the bursts of JA, jasmonoyl-isoleucine (JA-Ile), and their metabolites that are normally observed in the lamina, midvein, and petiole of elicited wild-type leaves were largely absent in both transformants but replaced by a burst of endogenous MeJA that accounted for almost half of the total elicited jasmonate pools. In these plants, MeJA became a metabolic sink that affected the jasmonate metabolic network and its spread to systemic leaves, with major effects on 12-oxo-phytodieonic acid, JA, and hydroxy-JA in petioles and on JA-Ile in laminas. Alterations in the size of jasmonate pools were most obvious in systemic tissues, especially petioles. Expression of threonine deaminase and trypsin proteinase inhibitor, two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes that were uncoupled from the wounding and elicitation with M. sexta oral secretions-elicited JA-Ile gradient in elicited leaves. Taken together, this study provides support for a central role of the vasculature in the propagation of jasmonates and new insights into the versatile spatiotemporal characteristics of the jasmonate metabolic network.

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“…Most plants have an incredible diversity of defensive traits (Duffey and Stout 1996, Agrawal and Fishbein 2006, Gaquerel et al 2010. Hypotheses for the expression of multiple defensive v www.esajournals.org traits in a single species range from synergism or redundancy in their action, specificity of traits towards multiple attackers, to various nonadaptive explanations (Jones and Firn 1991, Berenbaum and Zangerl 1996, Agrawal 2011, Moles et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Tests of the coupled expression of latex and cardenolide plant defense in common milkweed (Asclepias syriaca)

2014

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Abstract. The coexpression of plant resistance traits suggests the hypothesis that they may have complementary functions in defense against herbivory. To address the extent to which defensive traits are necessarily coupled in plants grown under various conditions, we focused on latex and cardenolides, two potent defenses of milkweeds. We measured defenses across ontogenetic stages, different biotic and abiotic environments, and across genetic families of the common milkweed Asclepias syriaca. We first addressed the extent to which foliar cardenolides are derived from latex because latex actively flows through canals in leaves. We rinsed latex out of shredded leaves, which had no impact on foliar cardenolides, suggesting cardenolides are allocated to leaves independently of latex. Accordingly, there is potential for independent expression of the two traits. We next followed a cohort of plants from germination over three years; expression of both latex exudation and cardenolides increased annually, with the exception of a second year dip in cardenolides. Damage by monarch caterpillars induced '50% increases of both latex and cardenolides, with the former occurring rapidly within a day and the latter taking five days of herbivory; these responses were preceded by an earlier peak of the signaling hormones jasmonic acid and abscisic acid. Endogenous jasmonic acid showed an instantaneous positive correlation with latex exudation and foliar cardenolides. Under drought stress, latex and cardenolide expression were reversed, with water stress suppressing latex exudation, but nearly doubling cardenolide concentrations. These drought effects were not driven by phytohormones in the expected manner, as jasmonic acid was unaffected, salicylic acid was strongly suppressed, and abscisic acid tripled in response to drought. Finally, a meta-analysis of four previously published field studies representing 85 genetic families of A. syriaca revealed no evidence for a genetic correlation between latex exudation and foliar cardenolide concentrations. The same lack of a correlation was observed across 22 populations of A. syriaca when grown in a common environment. Thus, the two most important defensive traits of milkweeds, although often coexpressed, can become uncoupled during some ontogenetic stages, under some biotic and abiotic conditions, and there is no evidence that they evolve together.

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Development and Validation of a Liquid Chromatography−Electrospray Ionization−Time-of-Flight Mass Spectrometry Method for Induced Changes in Nicotiana attenuata Leaves during Simulated Herbivory

Cited by 61 publications

References 30 publications

Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis

Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis

Ectopic Expression ofAtJMTinNicotiana attenuata: Creating a Metabolic Sink Has Tissue-Specific Consequences for the Jasmonate Metabolic Network and Silences Downstream Gene Expression

Tests of the coupled expression of latex and cardenolide plant defense in common milkweed (Asclepias syriaca)

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