Ćeranić Asja, Doppler Maria, Büschl Christoph, Parich Alexandra, Xu Kangkang, Koutnik Andrea, Bürstmayr Hermann, Lemmens Marc, Schuhmacher Rainer
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.
2Institute of Biotechnology in Plant Production, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), 3430 Tulln, Austria.
Plant Methods. 2020 Apr 6;16:46. doi: 10.1186/s13007-020-00590-9. eCollection 2020.
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 C and 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, C-labelling can be achieved by growth in CO atmosphere while global N-labelling needs N- containing salts in the watering/nutrient solution. Here, we present a method for the preparation of C and N-labelled plants by the use of closed growth chambers and hydroponic nutrient supply. The method is exemplified with durum wheat.
In total, 330 g of globally C- and 295 g of N-labelled wheat was produced during 87 cultivation days. For this, a total of 3.88 mol of CO and 58 mmol of N were consumed. The degree of enrichment was determined by LC-HRMS and ranged between 96 and 98 atom % for C and 95-99 atom % for N, respectively. Additionally, the isotopically labelled plant extracts were successfully used for metabolome-wide internal standardisation of native plants. Application of an isotope-assisted LC-HRMS workflow enabled the detection of 652 truly wheat-derived metabolites out of which 143 contain N.
A reproducible cultivation which makes use of climate chambers and hydroponics was successfully adapted to produce highly enriched, uniformly C- and 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 C-enriched biological samples when the necessary specific adaptations e.g. temperature and light regime, as well as nutrient supply are considered. Additionally, the N-labelling method can also be carried out under regular glasshouse conditions without the need for customised atmosphere.
稳定同位素标记的生物体在生命科学研究中有着广泛应用，包括植物生理学、植物胁迫与防御以及代谢相关科学领域。因此，可重复生产富含碳（C）、氮（N）等稳定同位素的植物材料备受关注。在植物生长/培育过程中通过持续供应底物可实现高度富集（>96原子%）且同位素均匀分布（全标记）。对于植物而言，在CO₂气氛中生长可实现碳标记，而全氮标记则需要在浇水/营养液中添加含氮盐。在此，我们介绍一种利用封闭生长室和水培营养液供应制备碳、氮标记植物的方法。该方法以硬粒小麦为例进行说明。
在87天的培育过程中，共生产出330克全碳标记和295克全氮标记的小麦。为此，总共消耗了3.88摩尔的CO₂和58毫摩尔的N。通过液相色谱-高分辨质谱（LC-HRMS）测定富集程度，碳的富集度在96至98原子%之间，氮的富集度在95至99原子%之间。此外，同位素标记的植物提取物成功用于天然植物的全代谢组内标标准化。采用同位素辅助的LC-HRMS工作流程能够检测出652种真正源自小麦的代谢物，其中143种含有氮。
利用气候室和水培法实现了可重复培育，成功生产出高度富集、全碳和全氮标记的小麦。所获得的植物材料适用于各类同位素辅助研究。当考虑到必要的特定适应性条件，如温度和光照条件以及营养供应时，所描述的技术设备和方案可轻松应用于其他植物以生产富含碳的生物样品。此外，氮标记方法也可在常规温室条件下进行，无需定制气氛。
