METABOLISM OF NITRATE AND AMMONIUM IN SEEDLINGS OF NORWAY SPRUCE (PICEA-ABIES) MEASURED BY IN-VIVO N-14 AND N-15 NMR-SPECTROSCOPY

In vivo N-15 and N-14 nuclear magnetic resonance spectroscopy was used to investigate the assimilation of nitrate and ammonium in seedlings of Norway spruce (Picea abies [L.] Karst.). The main objective was to study accumulation of free NH4+ and examine to what extent the nitroge n source affects the composition of the free amino acid pools in roots , stems and needles. NH4+ concentrations in plants growing in the pres ence of 0.5-50 mM ammonium were quantified using N-14 NMR. The NH4+ va lues in tissues ranged from 6 to 46 mu mol (g fresh weight)(-1), with highest concentrations in roots and needles. The tissue NHS peaked at 5.0 mM NH4+ in the medium, and failed to increase when NH4+ in the med ium was increased to 50 mM, indicating metabolic control of the concen tration of this cation in tissues. The N-14 NMR spectra were used to e stimate pH of the NH4+ storage pools. Based on the pH sensitivity of t he quintet of (NH4+)-N-14 resonance, we suggest that the pH of the amm onium storage compartments in the roots and stems should be 3.7-3.8, a nd in needles 3.4-3.5, representing extremely low pH values of the tis sue. N-15 from nitrate or ammonium was first incorporated into the ami de group of glutamine and then into alpha-amino groups, confirming tha t the glutamine synthetase/glutamate synthase cycle is the major route of nitrogen assimilation into amino acids and thus plays a role in lo wering the levels of NH4+ in the cytoplasm. NH4+ can also be assimilat ed in roots in plants growing in darkness. The main N-15-labelled amin o acids were glutamine, arginine and alanine. Almost no N-15 signals f rom needles were observed. Double labelling (delta N + omega,omega N) of arginine is consistent with the operation of the ornithine cycle, a nd enrichment indicates that this cycle is a major sink of newly assim ilated nitrogen. Nitrogen assimilation in roots in the presence of add ed methionine sulphoximine and glutamate indicated the catabolic actio n of glutamate dehydrogenase. The N-15 NMR spectra of plants grown on N-15-urea showed a marked increase in the labelling of ammonium and gl utamine, indicating high urease activity. Amino acids were also quanti fied using high pressure liquid chromatography. Arginine was found to be an important transport form of nitrogen in the stem.