Computational protocol: The Impact of the Absence of Aliphatic Glucosinolates on Insect Herbivory in Arabidopsis

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Protocol publication

[…] Detached leaf experiment: Arabidopsis plants (Col-0 and mutants) were grown in climate rooms with an 8 h light / 16 h darkness regime (light intensity 120 µmol m−2 s−1) at 20°C in soil. From 30-day old plants, leaves were detached with a sharp razor, and gently inserted pair-wise into 0.5 ml semi-solid water with 0.5% agar in a 0.5-ml reaction tube. For each line, twenty neonates of M. brassicae (Cabbage moth; Laboratory of Entomology, Wageningen University) were individually combined with leaves in sealed Petri dishes with ventilation holes, kept at room temperature and under natural daylight conditions. Every second day, leaf material was refreshed. Individual insects were weighed to the nearest 0.1 mg at day 14. Larval masses were log-transformed to meet assumptions of normality and homogeneity of variance. The log-transformed data were analyzed by ANOVA, followed by Tukey unequal N HSD analyses to identify significant differences between treatment groups.Whole-plant experiment: Seeds were sown in Petri dishes on water-saturated filter paper followed by a 4-days cold treatment at 4°C. They were then transferred to agar filled tubes and grown on hydroponics solution in trays of 50 plants. Plants were grown in a growth chamber with a 12 h light period at 20°C, 70% relative humidity and a light intensity of 35 W m−2. After 24 days of plant growth, Mamestra neonates were transferred to each tray of 50 plants. Insects weight was determined individually after 12 days. The larval mass data were log-transformed and analyzed with a nested ANOVA (tray nested in genotype) and Tukey unequal N HSD analysis. For statistical analyses Statistica 7.1 (Statsoft Inc., Tusla, OK, USA) software was used. Plant damage was determined by photographing the insect-exposed trays after 10 days. Photos were visually inspected for damage by five experienced observers and double blind ranked from low (value 1) to high (value 7) damage (10 replicates: two trays per line, five observers). The differences in ranks per plant line were analyzed by non-parametric Kruskal-Wallis ANOVA. [...] Leaves from five plants per line were snap-frozen in liquid nitrogen, snap frozen and ground to a fine powder, under continuous cooling. For metabolite profiling using LC-MS, 500 mg material was extracted using 5.0 ml 0.1% formic acid (v/v) in 75% aqueous-methanol, as described before .Extracts (3 µl) were subjected to a non-targeted LC-MS based metabolomics approach , using an Alliance HPLC system, a PDA detector and a high resolution quadrupole time-of-flight (QTOF) MS (Waters). Electrospray ionization in negative mode was used to ionize compounds separated by the reversed phase C18 column. Data were processed by extracting mass signals and aligning them across all samples in an unbiased manner using the dedicated Metalign™ software (, and a data matrix of intensities of all mass signals × samples was created. Mass signals with an intensity <10 times the local noise in all samples were filtered out. All analyses were performed using 5 biological replicates. For multivariate analysis, the LC-MS data were read into GeneMaths software (Applied Maths, Belgium) after 2log-transformation of mass signal intensities. Mass signals (variables) were normalized by dividing by the mean of each variable. […]

Pipeline specifications

Software tools Statistica, MetAlign
Applications Miscellaneous, MS-based untargeted metabolomics
Organisms Arabidopsis thaliana
Chemicals Glucosinolates