title: Characterization of alanine aminotransferase (AlaAT) multigene family and hypoxic response in young seedlings of the model legume Medicago truncatula.
abstract: Four 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 [(15)N]alanine and [(15)N]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 seedlings.
authors.affiliation: UMR INRA 1191, Physiologie Moléculaire des Semences, University of Angers, 2 Boulevard Lavoisier, F-49045 Angers cedex 01, France.
authors.forename: Claudie
authors.initials: C
authors.lastname: Ricoult
authors.forename: Luis Orcaray
authors.initials: LO
authors.lastname: Echeverria
authors.forename: Jean-Bernard
authors.initials: JB
authors.lastname: Cliquet
authors.forename: Anis M
authors.initials: AM
authors.lastname: Limami
mesh_terms: D000409:Alanine / Q000032:analysis / Q000378:metabolism; D000410:Alanine Transaminase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D015687:Cell Hypoxia; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D018698:Glutamic Acid / Q000032:analysis / Q000378:metabolism; D015151:Immunoblotting; D046913:Medicago truncatula / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D009587:Nitrogen Isotopes; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D018547:Plant Stems / Q000201:enzymology / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D024101:Mitochondrial Proteins; D009587:Nitrogen Isotopes; D010940:Plant Proteins; D020033:Protein Isoforms; D018698:Glutamic Acid; D000410:Alanine Transaminase; D000409:Alanine