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

Abstract

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. ResultsIn 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 wheatderived 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. BackgroundThe use of plants and plant-derived metabolites labelled with heavy stable isotopes offers an interesting perspective in phytochemical research. Some research fields where stable isotopes found

Unravelling Plant Responses to Stress—The Importance of Targeted and Untargeted Metabolomics

“…Subsequently, “light” plant metabolites were analyzed by targeted LC-MS, highlighting an infection reprogramming of plant signaling and primary metabolic pathways, including carbohydrates, nucleotides, and amino and organic acids. Alternatively, uniformly 13 C- and 15 N-labelled plants can be prepared, which will offer an accurate tool for metabolomic analysis under biotic stress conditions [ 170 ]. The growing interest in isotopically labelled biological material coincides with the development of additional analytical tools, such as NMR and high-throughput GC- and LC-HRMS, which allow the discrimination between native and labelled metabolites ( Figure 3 ).…”