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

Abstract

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 Γ‐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 μM in a 10‐μl 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 N supplies, as even plants grown under mildly N‐deficient conditions contained NH4+.

Seasonal variation in nitrogen pools and <sup>15</sup>N/<sup>13</sup>C natural abundances in different tissues of grassland plants

“…Details of the analytical procedures can be obtained from Husted et al (2000a), Mattsson et al (2009) and Wang et al (2011). In brief, chlorophyll was extracted from frozen plant materials by the use of methanol.…”

“…The driver for NH 3 exchange between plants and the atmosphere is the stomatal compensation point for NH 3 , reflecting external N availability and internal plant processes involved in N assimilation and turnover (Mattsson and Schjoerring, 1996; al. Husted et al, 2000b;Massad et al, 2008). The stomatal compensation point for NH 3 is a function of leaf temperature and the concentrations of NH + 4 and H + in the leaf apoplastic solution.…”

Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine

“…Free ammonium was determined fluorometrically according to Husted et al (2000) following derivatization at 638C with 3 mM O-phthalaldehyde and 10 mM 2-mercaptoethanol (in 0.2 M Tris-HCl, pH 6.8) and quantification in an HPLC system (HP 1100; Hewlett-Packard). Ammoniun concentration was also determined as the amount of NADH oxidized via the aminating reaction of GDH at 340 nm at room temperature in a total reaction volume of 0.5 mL consisting of 100 mM Tris, pH 8.0, 15 mM 2OG, 1 mM CaCl 2 , 1 mM NADH, and 2 units of GDH (1 mg protein equals 46 units; Sigma-Aldrich).…”

High-throughput colorimetric method for the parallel assay of glyoxylic acid and ammonium in a single extract

“…2B). Husted and coworkers [16] reported that amino acids interfere with the Berthelot reaction when present in high concentration as compared with ammonium. We found that activated charcoal efficiently absorbed most of the amino acids from an aqueous solution (data not shown) This partial purification by charcoal makes the Berthelot reaction suitable for plant extracts.…”

“…The protocol presented in Ref. [16] requires elaborate instrumentation, whereas the novel extraction and purification protocol presented here allows reliable and efficient quantification of ammonium with a spectrophotometer. However, if this new protocol is to be adapted for different tissues or plant species, we recommend optimizing the relative amounts of extract to activated charcoal to minimize interference as described above.…”