Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Plants submitted to O-2 deficiency present a series of biochemical modifications, affecting overall root metabolism. Here, the effect of hypoxia on the metabolic fate of N-15 derived from (NO3)-N-15 (-), (NO2)-N-15 (-) and (NH4)-N-15 (+) in isolated soybean root segments was followed by gas chromatography-mass spectrometry, to provide a detailed analysis of nitrogen assimilation and amino acid biosynthesis under hypoxia. O-2 deficiency decreased the uptake of the nitrogen sources from the solution, as ratified by the lower (NO3)-N-15 (-) and (NH4)-N-15 (+) enrichment in the root segments. Moreover, analysis of endogenous NO2 (-) and (NH4)-N-15 (+) levels suggested a slower metabolism of these ions under hypoxia. Accordingly, regardless of the nitrogen source, hypoxia reduced total N-15 incorporation into amino acids. Analysis of N-15 enrichment patterns and amino acid levels suggest a redirecting of amino acid metabolism to alanine and gamma-aminobutyric acid synthesis under hypoxia and a differential sensitivity of individual amino acid pathways to this stress. Moreover, the role of glutamine synthetase in nitrogen assimilation both under normoxia and hypoxia was ratified. In comparison with (NH4)-N-15 (+), (NO2)-N-15 (-) assimilation into amino acids was more strongly affected by hypoxia and NO2 (-) accumulated in root segments during this stress, indicating that nitrite reductase may be an additional limiting step. NO2 (-) accumulation was associated with a higher nitric oxide emission. (NO3)-N-15 (-) led to much lower N-15 incorporation in both O-2 conditions, probably due to the limited nitrate reductase activity of the root segments. Overall, the present work shows that profound alterations of root nitrogen metabolism occur during hypoxic stress.442743755Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq [303931/2009-4]FAPESP [2009/17583-3]