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

Asja Ćeranić; Maria Doppler; Christoph Büschl; Alexandra Parich; Kangkang Xu; Andrea Koutnik; Hermann Bürstmayr; Marc Lemmens; Rainer Schuhmacher

doi:10.1186/s13007-020-00590-9

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

Plant Methods. 2020 Apr 6:16:46. doi: 10.1186/s13007-020-00590-9. eCollection 2020.

Authors

Affiliations

¹ 1Institute of Bioanalytics and Agro-Metabolomics, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), Konrad-Lorenz-Straße 20, 3430 Tulln, Austria.
² 2Institute of Biotechnology in Plant Production, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), 3430 Tulln, Austria.

^# Contributed equally.

Abstract

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 ¹³C and ¹⁵N 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 in ¹³CO_2(g) atmosphere while global ¹⁵N-labelling needs ¹⁵N- containing salts in the watering/nutrient solution. Here, we present a method for the preparation of ¹³C and ¹⁵N-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 ¹³C- and 295 g of ¹⁵N-labelled Triticum durum wheat was produced during 87 cultivation days. For this, a total of 3.88 mol of ¹³CO_2(g) and 58 mmol of ¹⁵N were consumed. The degree of enrichment was determined by LC-HRMS and ranged between 96 and 98 atom % for ¹³C and 95-99 atom % for ¹⁵N, 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 ¹³C- and ¹⁵N-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 ¹³C-enriched biological samples when the necessary specific adaptations e.g. temperature and light regime, as well as nutrient supply are considered. Additionally, the ¹⁵N-labelling method can also be carried out under regular glasshouse conditions without the need for customised atmosphere.

Keywords: 13CO2 atmosphere; 15N-containing nutrient solution; Cultivation of wheat; Internal standard; LC-HRMS; Metabolomics; Stable isotopic labelling.