Computational protocol: Natural variation in herbivore-induced volatiles in Arabidopsis thaliana

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Protocol publication

[…] Dynamic headspace sampling was carried out in a climate room (20±2 °C, 70% RH; L8:D16 photoperiod, and 90–110 μmol photons m−2 s−1 PPFD at canopy height). Twenty-four hours before sampling, the pots were removed, roots and soil were carefully wrapped in aluminium foil, and four plants were placed together in a 2.5 l glass jar. The glass jars were then covered with insect-proof gauze. Just before headspace collection, the gauze was removed and jars were closed with a Viton-lined glass lid having an inlet and outlet. Inlet air was filtered by passing through a stainless steel cartridge (Markes, Llantrisant, UK) filled with 200 mg Tenax TA (20/35 mesh; Grace-Alltech, Deerfield, USA). Volatiles were trapped by sucking air out of the jar at a rate of 100 ml min−1 through a similar cartridge filled with 200 mg Tenax TA. Headspace volatiles for all treatments were collected for 3.5 h. Fresh weights of all rosettes were determined immediately after the experiments. On each experimental day, the headspaces of three or four accessions of each treatment were collected simultaneously. For each accession, 5 (An-1, C-24, Cvi, Eri-1, Kond, Kyo-1, Ler) or 6 (Col-0, WS) replicates for each experimental treatment (control, caterpillar-infested, or JA-treated) were analysed.Headspace samples were analysed with a Thermo Trace GC Ultra (Thermo Fisher Scientific, Waltham, USA) connected to a Thermo Trace DSQ (Thermo Fisher Scientific, Waltham, USA) quadrupole mass spectrometer. Before desorption of the volatiles, the Tenax cartridges were dry-purged with nitrogen at 30 ml min−1 for 20 min at ambient temperature to remove moisture. Volatiles were desorbed from the cartridges using a thermal desorption system at 250 °C for 3 min (Model Ultra Markes Llantrisant, UK) with a helium flow of 30 ml min−1. Analytes were focused at 0 °C on an electronically-cooled sorbent trap (Unity, Markes, Llantrisant, UK) and were then transferred in splitless mode to the analytical column (Rtx-5ms, 30 m, 0.25 mm i.d., 1.0 μm film thickness, Restek, Bellefonte, USA) by rapid heating of the cold trap to 250 °C. The GC was held at an initial temperature of 40 °C for 3.5 min followed by a linear thermal gradient of 10 °C min−1 to 280 °C and held for 2.5 min with a column flow of 1 ml min−1. The column effluent was ionized by electron impact ionization at 70 eV. Mass spectra were acquired by scanning from 45–400 m/z with a scan rate of 3 scans s−1. Compounds were identified using the deconvolution software AMDIS (version 2.64, NIST, USA) in combination with NIST 98 and Wiley 7th edition spectral libraries, and by comparing their retention indices with those from the literature (). For quantification, characteristic quantifier ions were selected for 84 compounds (see Supplementary Table S1 at JXB online). MetAlign software (PRI-Rikilt, Wageningen, the Netherlands) was used to remove baseline noise, to align the peaks of all chromatograms of the samples, and to integrate peak areas of quantifier ions. The peak areas of all compounds were corrected for the fresh weight of the leaf rosettes. To visualize differences between accessions and the different treatments, the total volatile profiles were analysed using principal component analysis (PCA, GeneMath XT 2.0). Data were preprocessed by a log10 transformation and subtracting the average value of all treatments.For a more detailed analysis, emitted quantities of individual volatiles were tested for significant changes between plant treatments using a t test. Individual volatiles were analysed for significant differences between accessions within each of the treatments, using one-way ANOVA followed by a Dunnett T3 post-hoc analysis (SPSS 15.0). Differences in emission of volatile compounds between treatments for each accession were analysed for significance using a one-way ANOVA. […]

Pipeline specifications

Software tools AMDIS, MetAlign
Application MS-based untargeted metabolomics
Organisms Arabidopsis thaliana, Pieris rapae, Diadegma semiclausum