Molecular and functional analyses support a role of Ornithine-{delta}-aminotransferase in the provision of glutamate for glutamine biosynthesis during pine germination.
We report the molecular characterization and functional analysis of a gene (PsdeltaOAT) from Scots pine (Pinus sylvestris) encoding Orn-delta-aminotransferase (delta-OAT; EC 2.6.1.13), an enzyme of arginine metabolism. The deduced amino acid sequence contains a putative N-terminal signal peptide for mitochondrial targeting. The polypeptide is similar to other delta-OATs from plants, yeast, and mammals and encoded by a single-copy gene in pine. PsdeltaOAT encodes a functional delta-OAT as determined by expression of the recombinant protein in Escherichia coli and analysis of the active enzyme. The expression of PsdeltaOAT was undetectable in the embryo, but highly induced at early stages of germination and seedling development in all different organs. Transcript levels decreased in later developmental stages, although an increase was observed in lignified stems of 90-d-old plants. An increase of delta-OAT activity was observed in germinating embryos and seedlings and appears to mirror the observed alterations in PsdeltaOAT transcript levels. Similar expression patterns were also observed for genes encoding arginase and isocitrate dehydrogenase. Transcripts of PsdeltaOAT and the arginase gene were found widely distributed in different cell types of pine organs. Consistent with these results a metabolic pathway is proposed for the nitrogen flow from the megagametophyte to the developing seedling, which is also supported by the relative abundance of free amino acids in embryos and seedlings. Taken together, our data support that delta-OAT plays an important role in this process providing glutamate for glutamine biosynthesis during early pine growth.
Cañas
Rafael A
RA
Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Campus Universitario de Teatinos, 29071-Málaga, Spain.
Villalobos
David P
DP
Díaz-Moreno
Sara M
SM
Cánovas
Francisco M
FM
Cantón
Francisco R
FR
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D018525:Germination; D018698:Glutamic Acid / Q000378:metabolism*; D005973:Glutamine / Q000096:biosynthesis*; D009953:Ornithine-Oxo-Acid Transaminase / Q000235:genetics / Q000378:metabolism*; D041605:Pinus sylvestris / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D036226:Seedlings / Q000378:metabolism; D012639:Seeds / Q000378:metabolism
D018076:DNA, Complementary; D000644:Quaternary Ammonium Compounds; D005973:Glutamine; D018698:Glutamic Acid; D009953:Ornithine-Oxo-Acid Transaminase