Determination of urea and ammonia in leaf extracts: application to ureide metabolism

Abstract

Methods are described for the routine determination of urea and ammonia in plant tissues. Ureido compounds such as allantoin and allantoate interfere with the urea assay but can be conveniently removed by ion-exchange chromatography prior to the color reaction with α-isonitrosopropiophenone. This assay was used to examine the effect of the urease inhibitor acetohydroxamate on leaf urea levels. A survey of methods for ammonia determination showed that for plant extracts and assay mixtures containing amides, microdiffusion followed by nesslerization provided a convenient and reliable technique. This procedure was used to determine asparaginase and glutaminase activities as well as ammonia levels in leaf tissue. In a study of detached soybean leaves, 5 mM acetohydroxamate, supplied via the transpiration stream, inhibited the production of ammonia normally observed in the presence of methionine sulfoximine (a glutamine synthetase inhibitor) and caused an accumulation of urea. The data were interpreted as evidence that leaf ammonia can arise from urea metabolism and supported a role for urea, probably derived from ureide breakdown, in the provision of nitrogen for amino acid and protein synthesis.