Plant metabolism of T-2 and HT-2 toxin in wheat, barley and oats

T2 toxin (T2) and HT2 toxin (HT2) are type-A trichothecenes produced by F. sporotrichioides, F. langsethiae and F. poae contaminating kernels of oats, barley and wheat. These toxins are of high concern to human health. T2 is a potent protein synthesis inhibitor and is rapidly metabolized in vivo to HT2 that induces similar adverse effects. Maximum levels for the sum of T2 and HT2 in cereals for human consumption are under discussion in the EU. A combined provisional TDI was already set at 0.06 microgr/kg. Plant metabolism of T2/HT2 may complicate risk evaluation with respect to human nutrition. T2- and HT2-3-O-glucosides and HT2-4-O-glucoside were detected in wheat and oats. Preliminary results show that wheat lines with the Fhb1 quantitative trait locus (QTL) are able to protect wheat against the phytotoxic effect of T2 and HT2. Fhb1 governs detoxification of deoxynivalenol (DON) to DON-3-O-glucoside. The detoxification mechanism of T2/HT2 is unknown and will be investigated. T2- and HT2-conjugates such as their glucosides could be "masked mycotoxins": they are not detectable by routine analysis but in the intestinal tract the native toxin could be released. Part of the mycotoxins may be bound to the plant matrix and can either be accessible by chemical extraction or be completely insoluble. These bound mycotoxins might be released in the digestive tract upon consumption of contaminated plant material. Knowledge on the metabolisation of T2 and HT2 by plants is very limited to date. In this project we want to 1) investigate the metabolic fate of T2/HT2 in planta, 2) develop suitable analytical methods for detection and quantification of possible new metabolites and 3) produce standards of such metabolites. To detect metabolites a 1:1 mixture of natural and U-[ 13C]-toxins will be applied on wheat/barley ears and oat panicles. At different time points after application both known (i.e. predicted) and unknown metabolites will be traced and identified by liquid chromatography high resolution mass spectrometry (LC-HRMS). Similar tests will be done with 14C-labeled toxins to assess the insoluble amount, measured by scintillation counting. A solvolysis procedure will be established to access the extractable biopolymer-bound and the non-soluble toxin fractions from the plant matrix. A molar sum of all metabolites will be estimated and compared to the total amount of toxins applied. To demonstrate the significance of the detected T2/HT2 biotransformation products, we will confirm their production after artificial inoculation with T2/HT2 producing Fusarium spp. and we will study their presence in naturally infected samples in the scope of a small survey. The resistance mechanism to T2/HT2 in wheat carrying Fhb1 is investigated using near-isogenic wheat lines with and without this QTL. To this end differential formation of T2 and HT2 metabolisation products will be studied by LC-HRMS.

The fungal toxins T-2 toxin (T2) and HT-2 toxin (HT2) are frequently detected contaminants of small grain cereals, such as barley, wheat and oats. In a defence reaction, plants metabolise these substances and form several modified derivatives of T2 and HT2. While the parent toxins are known to cause economic and human/animal health effects, the impact of T2/HT2 biotransformation products formed in planta is unclear, in particular, because they are largely unknown. Within this project, comprehensive T2/HT2 plant metabolism studies were performed using optimised metabolomics workflows. This revealed numerous novel T2/HT2 biotransformation products in barley, wheat and oats, such as hydroxylated, hydrolysed and conjugated (with glucose, acetic acid, putative malonic acid, malic acid and ferulic acid) derivatives. Independent quantitative time course experiments, performed for each plant species, enabled a deep insight into the kinetics and the extent of T2/HT2 biotransformation in planta. The experiments showed that the parent toxin T2 was transformed very fast and to a great extent into HT2 leading to a large overlap of T2 and HT2 biotransformation products. The main metabolic route of both type A trichothecenes was the rapid formation of HT2-3-O-?-D-glucoside in all investigated plant species, which was then further transformed and partly transported to non-treated plant tissues. Apart from that, another metabolic pathway was elucidated in T2-treated cereals, namely the direct conversion of intact T2 into tentatively identified T2-3-O-?-glucoside, 3-acetyl-T2 and putative feruloyl-T2.In the course of this project, metabolomics workflows were refined. Novel analytical methods were developed, which were tailored to detect, elucidate and quantify T2, HT2 and their derivatives and allow for the collection of occurrence data of those compounds in the future. Moreover, software programmes (MetExtract II and MetMatch) were refined/developed for the reliable recognition/annotation of tracer transformation products and the correction of analytical drifts, respectively. Our new findings and methods form an enhanced and sound basis to assess the risk of contaminated cereals. Further investigation of the detected derivatives is required to determine their toxicity, bioavailability for mammals, their behaviour during mammalian digestion and their occurrence in naturally contaminated plants. Another current topic is the resistance/susceptibility of plants to fungi, their toxins and related diseases, which might be directly associated with the ability to metabolise certain fungal toxins. Consequently, the knowledge, developed methods and findings from this project may also support resistance breeding of host plants.