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. 2021 May 20;12:645030. doi: 10.3389/fpls.2021.645030

FIGURE 2.

FIGURE 2

Plant myrosinase activity influences glucosinolate sequestration in P. armoraciae. (A) Accumulation of 4-methylsulfinylbutyl (4MSOB) and 4-methylthiobutyl (4MTB) glucosinolates in P. armoraciae adults after feeding on Arabidopsis wild type and myrosinase-deficient tgg leaves (n = 28 per plant genotype). Glucosinolates in beetles and fed leaves were extracted, converted to desulfo-glucosinolates and analyzed by high performance liquid chromatography coupled to diode array detection (HPLC-DAD). The glucosinolate concentration per mg beetle fresh weight is expressed relative to the concentration per mg plant fresh weight in the corresponding fed leaf, which was set to 1. Boxplots show the median and the 25th and 75th percentile. Dots represent data points that lie outside the 10th and 90th percentiles. Data were compared by Mann-Whitney rank sum test (∗∗∗p < 0.001) (B) Relative composition of 4MSOB glucosinolate and hydrolysis products detected in bodies and feces of wild type- or tgg-fed adults (n = 5 per plant genotype). Beetles that had fed on the Arabidopsis myb mutant served as a control (n = 5). Glucosinolates and hydrolysis products were extracted with 50% methanol and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Metabolites were quantified using external standard curves. The average amounts of each metabolite detected in control beetles and their feces was subtracted from the corresponding samples of wild type and tgg-fed beetles. The proportions of hydrolysis products relative to the total amount of detected glucosinolate and hydrolysis products in bodies and feces of wild type and tgg-fed beetles (set to 100%, respectively) were compared by Student’s t-tests (beetles: ∗p = 0.025, feces ∗∗∗p < 0.001). Results of statistical analyses are shown in Supplementary Table 1. 4MSOB cyanide corresponds to the nitrile hydrolysis product of 4MSOB glucosinolate. Other isothiocyanate-derived metabolites comprise 4MSOB isothiocyanate-glutathione conjugate, 4MSOB isothiocyanate-cysteinylglycine conjugate, 4MSOB isothiocyanate-cysteine conjugate, 2-(4-(methylsulfinyl)butylamino)-4,5-dihydrothiazole-carboxylic acid, 4MSOB amine, and 4MSOB acetamide.