Received May 14, 2008
Accepted July 3, 2008
Molecular and functional analyses support a role of ornithine-
-aminotransferase in the provision of glutamate for glutamine biosynthesis during pine germination
Rafael A. Canas , David P. Villalobos , Sara M. Diaz-Moreno , Francisco M. Canovas , and Francisco R. Canton *
Departamento de Biologia Molecular y Bioquimica, Facultad de Ciencias, Universidad de Malaga. Campus Universitario de Teatinos, 29071-Malaga, SPAIN
* Corresponding author; email: frcanton{at}uma.es.
We report the molecular characterization and functional analysis of a gene (Ps
OAT) from Scots pine (Pinus sylvestris L.) encoding ornithine--aminotransferase (-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 -OATs from plants, yeast and mammals and encoded by a single copy gene in pine. PsOAT encodes a functional -OAT as determined by expression of the recombinant protein in Escherichia coli and analysis of the active enzyme. The expression of PsOAT 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-day-old plants. An increase of -OAT activity was observed in germinating embryos and seedlings and appears to mirror the observed alterations in PsOAT transcript levels. Similar expression patterns were also observed for genes encoding arginase and isocitrate dehydrogenase. Transcripts of PsOAT 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 -OAT plays an important role in this process providing glutamate for glutamine biosynthesis during early pine growth.
