A critical experimental evaluation of methods for determination of NH4+ in plant tissue, xylem sap and apoplastic fluid
A critical experimental evaluation of methods for determination of NH4+ in plant tissue, xylem sap and apoplastic fluid
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DOI：
10.1034/j.1399-3054.2000.100209.x
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发表时间：
2000-06-01
影响因子：
6.4
通讯作者：
Schjoerring, JK
中科院分区：
文献类型：
--
作者：
Husted, S;Hebbern, CA;Schjoerring, JK
铵离子（NH 4+）是植物氮代谢的重要中间体，但NH 4+在氮素从根向地上部运输中的数量重要性以及植物在叶片中储存NH 4+的能力仍存在很大争议。本文表明，这些争议中的一些必须与使用不充分的分析程序用于提取和量化的NH 4+在植物中。最常用的测定NH 4+的方法，即基于经典Berthelot反应的比色法，受到氨基酸、胺、酰胺和蛋白质的严重干扰。对于其中一些代谢物，干扰为阳性，而对于其他代谢物，干扰为阴性，因此无法进行校正。因此，比色法不适用于测定植物中的NH 4+。通过离子色谱法获得的结果可能会高估NH 4+浓度，因为NH 4+与甲胺、乙胺、乙醇胺和非蛋白质氨基酸γ-氨基丁酸等胺共洗脱。NH 4+与邻苯二甲醛在碱性pH下的衍生化和随后通过荧光光谱法对NH 4+的定量也与干扰相关。然而，当pH值降低到6.8在衍生化和2-巯基乙醇被用作还原剂时，NH 4+可以测定具有高选择性和灵敏度下降到3.3 μ M的检测限在10 μ l的样品体积。使用无柱HPLC系统在线进行衍生化，从而能够在几分钟内快速定量NH 4+。流动注射分析与在线气体透析，同样，不受干扰，除了当应用于高度衰老的植物材料含有挥发性胺。叶组织提取物，木质部汁液和质外体流体中的不稳定的N代谢产物降解为NH 4+在提取和随后的仪器分析，如果样品不稳定。一个简单而有效的稳定，可以通过添加10 mM冰冷的HCOOH的植物提取介质或样品的质外体流体或木质部汁液。我们的结论是显着浓度的NH 4+，超过1毫米，可能会发生在木质部汁液，叶质外体液和叶组织水的硝酸盐生长的番茄和油菜植物。所测得的NH 4+浓度不是过量IU供应的结果，因为即使在轻度缺氮条件下生长的植物也含有NH 4+。
Ammonium (NH4+) is a central intermediate in the N metabolism of plants, but the quantitative importance of NH4+ in transporting N from root to shoot and the capability of plants to store NH4+ in leaves are still matters of substantial controversy. This paper shows that some of these controversies have to be related to the use of inadequate analytical procedures used for extraction and quantification of NH4+ in plants. The most frequently used methods for determination of NH4+, viz. colorimetric methods based on the classical Berthelot reaction, suffered severely from interference caused by amino acids, amines, amides and proteins. For some of these metabolites the interference was positive, while for others it was negative, making correction impossible. Consequently, colorimetric analysis is inapplicable for determination of NH4+ in plants. Results obtained by ion chromatography may overestimate the NH4+ concentration due to co-elution of NH4+ with amines like methylamine, ethylamine, ethanolamine and the non-protein amino acid gamma-aminobutyric acid. Derivatization of NH4+ with o-phthalaldehyde at alkaline pH and subsequent quantification of NH4+ by fluorescence spectroscopy was also associated with interference. However, when pH was lowered to 6.8 during derivatization and 2-mercaptoethanol was used as reductant, NH4+ could be determined with a high selectivity and sensitivity down to a detection limit of 3.3 mu M in a 10-mu l sample volume. Derivatization was performed on-line using a column-less HPLC system, enabling rapid quantification of NH4+ in a few minutes. Flow injection analysis with on-line gas dialysis was, likewise, free from interference, except when applied on highly senescent plant material containing volatile amines. Labile N metabolites in leaf tissue extract, xylem sap and apoplastic fluid were degraded to NH4+ during extraction and subsequent instrumental analysis if the samples were not stabilised. A simple and efficient stabilisation could be obtained by addition of 10 mM ice-cold HCOOH to the plant extraction medium or to the samples of apoplastic fluid or xylem sap. We conclude that significant concentrations of NH4+, exceeding 1 mM, may occur in xylem sap, leaf apoplastic fluid and leaf tissue water of nitrate-grown tomato and oilseed rape plants. The measured NH4+ concentrations were not a result of excessive IU supplies, as even plants grown under mildly N-deficient conditions contained NH4+.