Ammonia Assimilation in the Roots of Nitrate- and Ammonia-Grown Hordeum Vulgare (cv Golden Promise)

Abstract

to be reached on the pathway of ammonia assimilation. Thus, 15N-feeding experiments with both root (17, 27) and shoot (8) tissues, and studies using 13N (13,22) in which glutamine was initially more highly labeled than glutamate, have been interpreted as being consistent with ammonia assimilation via the GS/glutamate synthase route. Similarly, experiments in which glutamate was initially more highly labeled than glutamine (6, 8) have been interpreted as being at least qualitatively consistent with assimilation via GDH. Such conclusions are, however, speculative in the absence of information on the number and relative sizes of the glutamate and glutamine pools in the tissues concerned.The purpose of these studies was to obtain kinetic labehling data from experiments in which Hordeum seedlings, growing under steady state conditions, were fed '5N-labeled ammonia or nitrate.Attempts were then made to apply a quantitative analysis to the 15N incorporation patterns in ammonia-fed barley roots. MATERIALS AND METHODSNumerous studies have demonstrated that both roots and leaves of higher plants possess the enzymic potential for the assimilation of ammonia either via the direct reductive amination of 2-oxoglutarate, catalyzed by GDH2 or via the combined action of GS and glutamate synthase (23). The now widely held belief that the GS/ glutamate synthase pathway is solely responsible for ammonia assimilation in these tissues (15) Growth of Plants. The growth system used was a 'continuous flow' water culture system. Seeds of Hordeum vulgare L. var.Golden Promise were soaked overnight in running tap water, with aeration, before sowing on sections of Netlon mesh covered with muslin and stretched over the top of lengths of plastic guttering filled with culture solution. Culture solution was cycled through the gutters from a large reservoir by means of a pump. The flow of nutrient solution through the gutters was adjusted to a rate sufficient to prevent the concentrations of nutrient in the gutters differing from those in the reservoir due to uptake by the plants. Seedlings were subjected to a 14-h daylight regime, with a light intensity of 4.95 J cm -2 h-' at the level of the plants, and a constant temperature of 23°C. The nutrient solution used was a modified one-quarter strength Long Ashton solution (4). Nitrogen was supplied either as potassium nitrate or ammonium chloride. The volume of the reservoir was chosen to prevent the concentration of ammonia or nitrate in the culture solution from being reduced by more than 10% over a 24-h period. Labeling studies were performed on 9-d-old seedlings. At the start of a '5N-labeling experiient, the pump was stopped, and the gutters were rapidly drained and refilled with culture solution containing the nitrogen source labeled at 96% 15N atom abundance. The reservoir was exchanged for one containing the labeled nitrogen source, and the pump was restarted. The plants were sampled by removing individual sections of Netlon mesh.Extraction of Soluble Nitrogen Pools. Root and shoot tissues...

Ammonia Assimilation by Aspergillus nidulans: [15N]Ammonia Study

“…nidulans differs from the previous observations on Lernna minor and barley roots (Fentem et al, 1983;Lewis et al, 1983;Rhodes et al, 1980). Although substantial activity of NADP-GDH was detected in these plants, ammonia was assimilated solely by the GS/GOGAT pathway.…”

“…However, the pattern of 5N labelling in the major amino acids in this study differed from the pattern of 5N labelling in major amino acids of ammonia assimilation by the GDH pathway in Candida utilis (Sims & Folkes, 1964), in which glutamate was initially more highly labelled than any other amino acid. Our results also differed from the pattern of labelling in the major amino acids of ammonia assimilation by the GSlGOGAT pathway in Lemna minor (Rhodes et al, 1980) and barley roots (Fentem et al, 1983), which showed that glutamine-amide was the first highly labelled compound.…”

“…Therefore, A. nidulans was grown on low ammonia and a quantitative analysis of the kinetics of l 5N incorporation into tissue amino acid pools was used. A similar approach has been successfully applied to the culture of Candida utilis, Lemna minor and Hordeum vulgare (Fentem et al, 1983; Rhodes et al, 1980; Sims & Folkes, 1964).…”

Dynamics of Nitrogenous Assimilate Partitioning between Cytoplasmic and Vacuolar Fractions in Carrot Cell Suspension Cultures

“…Despite considerable advances in our understanding of the major metabolic pathways of nitrogen assimilation in higher plants (9,12,21,23) …”

“…Despite considerable advances in our understanding of the major metabolic pathways of nitrogen assimilation in higher plants (9,12,21,23), there is still uncertainty as to the role played by intracellular partitioning of nitrogenous assimilates, particularly between the vacuolar and cytoplasmic compartments. Because of difficulties in measuring amino acid pools in vacuoles, approaches to this problem have been mostly indirect.…”