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

Ammonium (NH4+) is a central intermediate in the N metabolism of plants, bu t 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 s ubstantial controversy. This paper shows that some of these controversies h ave to be related to the use of inadequate analytical procedures used for e xtraction and quantification of NH4+ in plants. The most frequently used me thods for determination of NH4+, viz. colorimetric methods based on the cla ssical Berthelot reaction, suffered severely from interference caused by am ino acids, amines, amides and proteins. For some of these metabolites the i nterference was positive, while for others it was negative, making correcti on impossible. Consequently, colorimetric analysis is inapplicable for dete rmination of NH4+ in plants. Results obtained by ion chromatography may ove restimate 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 alkalin e pH and subsequent quantification of NH4+ by fluorescence spectroscopy was also associated with interference. However, when pH was lowered to 6.8 dur ing derivatization and 2-mercaptoethanol was used as reductant, NH4+ could be determined with a high selectivity and sensitivity down to a detection l imit of 3.3 mu M in a 10-mu l sample volume. Derivatization was performed o n-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 tis sue extract, xylem sap and apoplastic fluid were degraded to NH4+ during ex traction and subsequent instrumental analysis if the samples were not stabi lised. A simple and efficient stabilisation could be obtained by addition o f 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 meas ured NH4+ concentrations were not a result of excessive IU supplies, as eve n plants grown under mildly N-deficient conditions contained NH4+.