Physiological Roles of the β-Substituted Alanine Synthase Gene Family in Arabidopsis

The beta-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 beta-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 Bsas1;1, plastidic Bsas2;1, and mitochondrial Bsas2;2 play major roles in Cys biosynthesis. Cytosolic Bsas1;1 has the most dominant contribution both in leaf and root, and mitochondrial Bsas2;2 plays a significant role in root. Mitochondrial Bsas3;1 is a genuine CASase. Nontargeted metabolome analyses of knockout mutants were carried out by a combination of gas chromatography time-of-flight mass spectrometry and capillary electrophoresis time-of-flight mass spectrometry. The level of gamma-glutamyl-beta-cyano-Ala decreased in the mutant bsas3;1, indicating the crucial role of Bsas3;1 in beta-cyano-Ala metabolism in vivo.