Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress

The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. 15NH4 labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized 15N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while 15N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt

Stimulation of alanine amino transferase (AlaAT) gene expression and alanine accumulation in embryo axis of the model legume Medicago truncatula contribute to anoxia stress tolerance

http://dx.doi.org/10.1111/j.1399-3054.2005.00449.xInternational audienc

Characterization of alanine aminotransferase (AlaAT) multigene family and hypoxic response in young seedlings of the model legume Medicago truncatula

International audienceFour alanine aminotransferases (AlaATs) are expressed in Medicago truncatula. In adult plants, two genes encoding mitochondrial isoforms m-AlaAT and alanine-glyoxylate aminotransferase (AGT), catalysing, respectively, reversible reactions of alanine/oxoglutarate glutamate/pyruvate and alanine/glyoxylate glycine/pyruvate, were expressed in roots, stems, and leaves. A gene encoding a cytosolic (c-AlaAT) isoform, catalysing the same reaction as m-AlaAT, was expressed specifically in leaves, while a gene encoding an isoform involved in branched chain amino acid metabolism was expressed in stems and roots. In young seedlings, only m-AlaAT and AGT were expressed in embryo axes. In hypoxic embryo axes, the amounts of transcript and putative protein of m-AlaAT (EC 2.6.1.2) increased while those of AGT (EC 2.6.1.44) decreased and in vivo enzyme activities changed as revealed by [N-15]alanine and [N-15]glutamate labelling. Under hypoxia, m-AlaAT catalysed only alanine synthesis while glutamate synthesis using alanine as amino donor was inhibited. As a result, alanine accumulated as the major amino acid in hypoxic seedlings instead of asparagine, in agreement with the involvement of the fermentative AlaAT pathway in hypoxia tolerance. Regulation of m-AlaAT at both the transcriptional and post-translational levels allowed for an increase in gene expression and orientation of the activity of the product of its transcription towards alanine synthesis under hypoxia. Labelling experiments showed that glycine synthesis occurred at the expense of either alanine or glutamate as amino donor, indicating that a glutamate-glyoxylate aminotransferase was operating together with AGT in Medicago truncatula seedlings. Both enzymes seemed to be inhibited by hypoxia, resulting in a very low amount of glycine in hypoxic seedling

NH labelling experiment for the determination of the fate of ammonium in seedlings under normoxia and hypoxic stress

Young seedlings (24 h) were fed with 2 mM (NH)SO (99% N atom excess) for 2, 10, and 24 h. Results are the mean of three replicates, corresponding to N atom excess in glutamate, alanine, aspartate, glutamine (single and double labelled), and asparagine (single and double labelled) (% N-amino acids) and the amounts of newly synthesized amino acids during the labelling period, expressed as nmol N-amino acids per embryo axis. Values followed by different letters, are significantly different according to ANOVA analysis and Newman–Keuls test (α = 5%).<p><b>Copyright information:</b></p><p>Taken from "Concerted modulation of alanine and glutamate metabolism in young seedlings under hypoxic stress"</p><p></p><p>Journal of Experimental Botany 2008;59(9):2325-2335.</p><p>Published online 26 May 2008</p><p>PMCID:PMC2423662.</p><p></p

NH labelling experiment for the determination of the fate of ammonium in seedlings fed excess ammonium under normoxia or hypoxic stress

Nineteen-hour germinated seedlings were split into two batches and maintained for 5 h either on sterile deionized water or aqueous MSX (5 mM), a glutamine synthetase inhibitor. Young seedlings (24 h) were fed with 30 mM (NH)SO (99% N atom excess) for 2, 10, and 24 h under either normoxia or hypoxic stress conditions. The results are the mean of three replicates, corresponding to N atom excess in glutamate, alanine, aspartate, glutamine (single and double labelled), and asparagine (single and double labelled) (% N-amino acids) and the amounts of newly synthesized amino acids during the labelling period, expressed as nmol N-amino acids per embryo axis. Values followed by different letters, are significantly different according to ANOVA analysis and Newman–Keuls test (α = 5%).<p><b>Copyright information:</b></p><p>Taken from "Concerted modulation of alanine and glutamate metabolism in young seedlings under hypoxic stress"</p><p></p><p>Journal of Experimental Botany 2008;59(9):2325-2335.</p><p>Published online 26 May 2008</p><p>PMCID:PMC2423662.</p><p></p