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

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+.