Biotransformation of the mycotoxin deoxynivalenol in fusarium resistant and susceptible near isogenic wheat lines

Abstract

In this study, a total of nine different biotransformation products of the Fusarium mycotoxin deoxynivalenol (DON) formed in wheat during detoxification of the toxin are characterized by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The detected metabolites suggest that DON is conjugated to endogenous metabolites via two major metabolism routes, namely 1) glucosylation (DON-3-glucoside, DON-di-hexoside, 15-acetyl-DON-3-glucoside, DON-malonylglucoside) and 2) glutathione conjugation (DON-S-glutathione, "DON-2H"-S-glutathione, DON-S-cysteinyl-glycine and DON-S-cysteine). Furthermore, conjugation of DON to a putative sugar alcohol (hexitol) was found. A molar mass balance for the cultivar 'Remus' treated with 1 mg DON revealed that under the test conditions approximately 15% of the added DON were transformed into DON-3-glucoside and another 19% were transformed to the remaining eight biotransformation products or irreversibly bound to the plant matrix. Additionally, metabolite abundance was monitored as a function of time for each DON derivative and was established for six DON treated wheat lines (1 mg/ear) differing in resistance quantitative trait loci (QTL) Fhb1 and/or Qfhs.ifa-5A. All cultivars carrying QTL Fhb1 showed similar metabolism kinetics: Formation of DON-Glc was faster, while DON-GSH production was less efficient compared to cultivars which lacked the resistance QTL Fhb1. Moreover, all wheat lines harboring Fhb1 showed significantly elevated D3G/DON abundance ratios.

Fig 1. EICs of DON and its corresponding biotransformation products.

EICs of accurate mass traces (± 3 ppm) of DON and its corresponding biotransformation products in a wheat sample harvested 96 hours post treatment with 1 mg DON. Due to low abundance, EIC intensities of DON-di-hexoside were multiplied by a factor of 10.

Fig 2. LC-HRMS/MS spectra of the DON biotransformation products.

a) DON-malonylglucoside, b) 15-acetyl-DON-3-β-D-glucoside and c) “DON-2H”-S glutathione (GSH), all LC-HRMS/MS spectra are shown with a proposed structure formula.

Fig 3. Overview on DON degradation.

Time course for the degradation of DON (1 mg) of wheat lines ‘CM-82036’, C1, C2, C3, C4 and ‘Remus’. Wheat ears were sampled 0, 12, 24, 48, and 96 hours after treatment (n = 5 biological replicates per time point and wheat line). a) Degradation rate of DON. b) boxplot of relative concentrations 96 h after DON treatment. c) DON-glucoside/DON ratio 96 h after DON treatment. * Significantly differing DON levels between wheat lines with and without resistance QTL Fhb1 based on a non-paired t-test (5% global significance threshold).

Fig 4. Detoxification of DON via glucosylation/sugar alcohol.

Glucose/sugar alcohol related detoxification of DON. Relative formation rates for the biotransformation products DON-glucoside (a), DON-malonyl-glucoside (b), 15-acetyl-DON-3-glucoside (c) and DON-hexitol (d). Additionally, for each biotransformation product boxplots for relative metabolite abundance observed 96 h after DON treatment were generated. * Significantly differing biotransformation product levels between wheat lines with and without resistance QTL Fhb1 based on a non-paired t-test (5% global significance threshold).

Fig 5. Detoxification of DON via glutathione pathway.

Glutathione pathway related detoxification of DON. Relative formation rates for biotransformation products DON-S-glutathione (a) and its related degradation products “DON-2H”-S-glutathione (b), DON-S-cysteinylglycine (c) and DON-S-cysteine (d). Additionally, for each biotransformation product boxplots for relative metabolite abundance observed 96 h after DON treatment were generated. * Significantly differing biotransformation product levels between wheat lines with and without resistance QTL Fhb1 based on a non-paired t-test (5% global significance threshold).