Background: Stable isotopically labelled organisms have found wide application in life science research including plant physiology, plant stress and defense as well as metabolism related sciences. Therefore, the reproducible production of plant material enriched with stable isotopes such as¹³C and¹⁵N is of considerable interest. A high degree of enrichment (> 96 atom %) with a uniformly distributed isotope (global labelling) is accomplished by a continuous substrate supply during plant growth/cultivation. In the case of plants,¹³C-labelling can be achieved by growth in¹³CO_2(g)atmosphere while global¹⁵N-labelling needs¹⁵N- containing salts in the watering/nutrient solution. Here, we present a method for the preparation of¹³C and¹⁵N-labelled plants by the use of closed growth chambers and hydroponic nutrient supply. The method is exemplified with durum wheat.Results: In total, 330 g of globally¹³C- and 295 g of¹⁵N-labelledTriticum durumwheat was produced during 87 cultivation days. For this, a total of 3.88 mol of¹³CO_2(g)and 58 mmol of¹⁵N were consumed. The degree of enrichment was determined by LC-HRMS and ranged between 96 and 98 atom % for¹³C and 95–99 atom % for¹⁵N, respectively. Additionally, the isotopically labelled plant extracts were successfully used for metabolome-wide internal standardisation of nativeT.durumplants. Application of an isotope-assisted LC-HRMS workflow enabled the detection of 652 truly wheat-derived metabolites out of which 143 contain N.Conclusion: A reproducible cultivation which makes use of climate chambers and hydroponics was successfully adapted to produce highly enriched, uniformly¹³C- and¹⁵N-labelled wheat. The obtained plant material is suitable to be used in all kinds of isotope-assisted research. The described technical equipment and protocol can easily be applied to other plants to produce¹³C-enriched biological samples when the necessary specific adaptations e.g. temperature and light regime, as well as nutrient supply are considered. Additionally, the¹⁵N-labelling method can also be carried out under regular glasshouse conditions without the need for customised atmosphere.

The emerging mycotoxin fusaproliferin is produced by Fusarium proliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusarium proliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 (FUP2, FPRN_05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN_05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase (FUP4, FPRN_05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase (FUP5, FPRN_05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.

Stable isotope-assisted approaches can improve untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomics studies. Here, we demonstrate at the example of chemically stressed wheat that metabolome-wide internal standardization by globally ¹³C-labeled metabolite extract (GLMe-IS) of experimental-condition-matched biological samples can help to improve the detection of treatment-relevant metabolites and can aid in the post-acquisition assessment of putative matrix effects in samples obtained upon different treatments. For this, native extracts of toxin- and mock-treated (control) wheat ears were standardized by the addition of uniformly ¹³C-labeled wheat ear extracts that were cultivated under similar experimental conditions (toxin-treatment and control) and measured with LC-HRMS. The results show that 996 wheat-derived metabolites were detected with the non-condition-matched ¹³C-labeled metabolite extract, while another 68 were only covered by the experimental-condition-matched GLMe-IS. Additional testing is performed with the assumption that GLMe-IS enables compensation for matrix effects. Although on average no severe matrix differences between both experimental conditions were found, individual metabolites may be affected as is demonstrated by wrong decisions with respect to the classification of significantly altered metabolites. When GLMe-IS was applied to compensate for matrix effects, 272 metabolites showed significantly altered levels between treated and control samples, 42 of which would not have been classified as such without GLMe-IS.

Background Stable isotopically labelled organisms found wide application in life science research including plant physiology, plant stress and defense as well as metabolism related sciences. Therefore, the reproducible production of plant material enriched with stable isotopes such as 13 C and 15 N is of considerable interest. A high degree of enrichment (>96 atom%) with a uniformly distributed isotope (global labelling) is accomplished by a continuous substrate supply during plant growth/cultivation. In the case of plants, 13 C-labelling can be achieved by growth in 13 CO 2(g) atmosphere while global 15 N labelling needs 15 N- containing salts in the watering/nutrient solution. Here, we present a method for the preparation of 13 C and 15 N labelled plants by the use of closed growth chambers and hydroponic nutrient supply. The method is exemplified with durum wheat.Results In total, 330 g of globally 13 C- and 295 g of 15 N labelled T. durum wheat was produced during 87 cultivation days. For this, a total of 3.88 mol of 13 CO 2(g) and 58 mmol of 15 N were consumed. The degree of enrichment was determined by LC-HRMS and ranged between 96-98 atom% for 13 C and 95-99 atom% for 15 N, respectively. Additionally, the isotopically labelled plant extracts were successfully used for metabolome-wide internal standardisation of native T.durum plants. Application of an isotope-assisted LC-HRMS workflow enabled the detection of 652 truly wheat-derived metabolites out of which 143 contain N.Conclusion A reproducible cultivation which makes use of climate chambers and hydroponics was successfully adapted to produce highly enriched, uniformly 13 C- and 15 N-labelled wheat. The obtained plant material is suitable to be used in all kinds of isotope-assisted research. The described technical equipment and protocol can easily be applied to other plants to produce 13 C-enriched biological samples when the necessary specific adaptations e.g. temperature and light regime, as well as nutrient supply are considered. Additionally, the 15 N-labelling method can also be carried out under regular glasshouse conditions without the need for customised atmosphere.

Background Stable isotopically labelled organisms found wide application in life science research including plant physiology, plant stress and defense as well as metabolism related sciences. Therefore, the reproducible production of plant material enriched with stable isotopes such as 13 C and 15 N is of considerable interest. A high degree of enrichment (>96 atom%) with a uniformly distributed isotope (global labelling) is accomplished by a continuous substrate supply during plant growth/cultivation. In the case of plants, 13 C-labelling can be achieved by growth in 13 CO 2(g) atmosphere while global 15 N labelling needs 15 N- containing salts in the watering/nutrient solution. Here, we present a method for the preparation of 13 C and 15 N labelled plants by the use of closed growth chambers and hydroponic nutrient supply. The method is exemplified with durum wheat. Results In total, 330 g of globally 13 C- and 295 g of 15 N labelled T. durum wheat was produced during 87 cultivation days. For this, a total of 3.88 mol of 13 CO 2(g) and 58 mmol of 15 N were consumed. The degree of enrichment was determined by LC-HRMS and ranged between 96-98 atom% for 13 C and 95-99 atom% for 15 N, respectively. Additionally, the isotopically labelled plant extracts were successfully used for metabolome-wide internal standardisation of native T.durum plants. Application of an isotope-assisted LC-HRMS workflow enabled the detection of 652 truly wheat-derived metabolites out of which 143 contain N. Conclusion A reproducible cultivation which makes use of climate chambers and hydroponics was successfully adapted to produce highly enriched, uniformly 13 C- and 15 N-labelled wheat. The obtained plant material is suitable to be used in all kinds of isotope-assisted research. The described technical equipment and protocol can easily be applied to other plants to produce 13 C-enriched biological samples when the necessary specific adaptations e.g. temperature and light regime, as well as nutrient supply are considered. Additionally, the 15 N-labelling method can also be carried out under regular glasshouse conditions without the need for customised atmosphere.