Physiological Roles of the ^-Substituted Alanine Synthase Gene Family in Arabidopsis1[wl[OA1

The ^-substituted alanine (Ala) synthase (Bsas) family in the large superfamily of pyridoxal 5 '-phosphate-dependent enzymes comprises cysteine (Cys) synthase (CSase) [O-acetyl-serine (thiol) lyase] and /3-cyano-Ala synthase (CASase) in plants. Nine genomic sequences encode putative Bsas proteins in Arabidopsis thaliana. The physiological roles of these Bsas isoforms in vivo were investigated by the characterization of T-DNA insertion mutants. Analyses of gene expression, activities of CSase and CASase, and levels of Cys and glutathione in the bsas mutants indicated that cytosolic Bsasl;l, plastidic Bsas2;l, and mitochondrial Bsas2;2 play major roles in Cys biosynthesis. Cytosolic Bsasl;l has the most dominant contribution both in leaf and root, and mitochondrial Bsas2;2 plays a significant role in root. Mitochondrial Bsas3;l is a genuine CASase. Nontargeted metabolome analyses of knockout mutants were carried out by a combination of gas chromatography time-offlight mass spectrometry and capillary electrophoresis time-of-flight mass spectrometry The level of y-glutamyl-/3-cyano-Ala decreased in the mutant bsas3;l, indicating the crucial role of Bsas3;l in /3-cyano-Ala metabolism in vivo.