A critical experimental evaluation of methods for determination of NH₄⁺ in plant tissue, xylem sap and apoplastic fluid

Abstract

Ammonium (NH 4 + ) is a central intermediate in the N metabolism of plants, but the quantitative importance of NH 4 + in transporting N from root to shoot and the capability of plants to store NH 4 + 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 NH 4 + in plants. The most frequently used methods for determination of NH 4 + , 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 NH 4 + in plants. Results obtained by ion chromatography may overestimate the NH 4 + concentration due to co‐elution of NH 4 + with amines like methylamine, ethylamine, ethanolamine and the non‐protein amino acid Γ ‐aminobutyric acid. Derivatization of NH 4 + with o ‐phthalaldehyde at alkaline pH and subsequent quantification of NH 4 + 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, NH 4 + could be determined with a high selectivity and sensitivity down to a detection limit of 3.3 μ M in a 10‐μl sample volume. Derivatization was performed on‐line using a column‐less HPLC system, enabling rapid quantification of NH 4 + 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 NH 4 + during extraction and subsequent instrumental analysis if the samples were not stabilised. A simple and efficient stabilisation could be obtained by addition of 10 m M ice‐cold HCOOH to the plant extraction medium or to the samples of apoplastic fluid or xylem sap. We conclude that significant concentrations of NH 4 + , exceeding 1 m M , may occur in xylem sap, leaf apoplastic fluid and leaf tissue water of nitrate‐grown tomato and oilseed rape plants. The measured NH 4 + concentrations were not a result of excessive N supplies, as even plants grown under mildly N‐deficient conditions contained NH 4 + .