Preparation of uniformly labelled (13)C- and (15)N-plants using customised growth chambers.

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 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 Triticum 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 and 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.