Biotechnology of Horticultural Crops, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Liesel-Beckmann-Straße 1, 85354 Freising, Germany.
Molecules. 2019 Apr 18;24(8):1531. doi: 10.3390/molecules24081531.
Feeding experiments with stable isotopes are helpful tools for investigation of metabolic fluxes and biochemical pathways. For assessing nitrogen metabolism, the heavier nitrogen isotope, [N], has been frequently used. In plants, it is usually applied in form of [N]-nitrate, which is assimilated mainly in leaves. Thus, methods for quantification of the [N]-nitrate/[N]-nitrate ratio in leaves are useful for the planning and evaluation of feeding and pulse-chase experiments. Here we describe a simple and sensitive method for determining the [N]-nitrate to [N]-nitrate ratio in leaves. Leaf discs (8 mm diameter, approximately 10 mg fresh weight) were sufficient for analysis, allowing a single leaf to be sampled multiple times. Nitrate was extracted with hot water and derivatized with mesitylene in the presence of sulfuric acid to nitromesitylene. The derivatization product was analyzed by gas chromatography-mass spectrometry with electron ionization. Separation of the derivatized samples required only 6 min. The method shows excellent repeatability with intraday and interday standard deviations of less than 0.9 mol%. Using the method, we show that [N]-nitrate declines in leaves of hydroponically grown , an African orphan crop, with a biological half-life of 4.5 days after transfer to medium containing [N]-nitrate as the sole nitrogen source.
稳定同位素喂养实验是研究代谢通量和生化途径的有用工具。为了评估氮代谢，通常使用较重的氮同位素 [N]。在植物中，它通常以 [N]-硝酸盐的形式应用，主要在叶片中被同化。因此，用于量化叶片中 [N]-硝酸盐/[N]-硝酸盐比率的方法对于喂养和脉冲追踪实验的规划和评估很有用。在这里，我们描述了一种简单灵敏的方法来测定叶片中的 [N]-硝酸盐与 [N]-硝酸盐的比率。叶片圆盘（直径 8 毫米，约 10 毫克鲜重）足以进行分析，允许对单个叶片进行多次采样。用热水提取硝酸盐，在硫酸存在下用间二甲苯将其衍生为硝基金刚烷。用电子电离气相色谱-质谱法分析衍生化产物。衍生化样品的分离仅需 6 分钟。该方法具有极好的重现性，日内和日间标准偏差小于 0.9 mol%。使用该方法，我们表明在水培生长的非洲孤儿作物 中，[N]-硝酸盐在转移到仅含 [N]-硝酸盐作为唯一氮源的培养基 4.5 天后会在叶片中下降，其生物半衰期为 4.5 天。
