Overexpression of Glutamate Decarboxylase Gene SiGAD1 generates high levels of γ-aminobutyric acid (GABA) in foxtail millet grains

γ-Aminobutyric acid (GABA), an important non-protein amino acid, is involved in various physiological processes including plant growth and development, response to environmental stresses, and promoting human nutritional well-being. The synthesis of GABA from glutamate is primarily mediated by the enzyme glutamate decarboxylase (GAD), which serves as the rate-limiting catalyst in this biochemical pathway. In this study, based on transcriptomic data obtained from different developmental stages of foxtail millet grains, we identified SiGAD1 ( Seita.6G177000 ), which exhibits significantly higher expression at the early grain developmental stage. To investigate its function, we constructed two overexpression vectors carrying either the full-length SiGAD1 gene or a C-terminally truncated variant ( SiGAD1∆C ), both driven by the ubiquitin (Ubi) promoter, and successfully generated the corresponding transgenic lines, designated SiGAD1 ^OX and SiGAD1∆C ^OX , respectively. Molecular characterization and phenotypic analyses revealed that GAD enzyme activity was significantly enhanced in the SiGAD1 ^OX grains, leading to substantial accumulation of GABA and a concomitant reduction in glutamate levels compared to the wild type. This study provides valuable genetic resources and a solid theoretical foundation for molecular breeding strategies aimed at developing novel foxtail millet varieties with enhanced GABA content.