DAO2 Modulates Gamma-Aminobutyric Acid Accumulation in Germinating Brown Rice

Gamma-aminobutyric acid (GABA) is a functional non-protein amino acid that regulates plant stress responses and signaling, while offering antihypertensive and hepatorenal benefits in humans. However, the genetic and molecular networks governing GABA accumulation during brown rice germination remain poorly understood. Here, we engineered DAO2 knockout rice lines using CRISPR/Cas9. Mutant brown rice exhibited significantly lower GABA levels alongside a marked accumulation of glutamate (Glu), glutamine (Gln), arginine (Arg), and proline (Pro) compared to the wild type (WT). Spatiotemporal profiling across seven germination intervals revealed that GABA titers peaked at 24 h in both the DAO2-1 mutant (24.24 mg/100 g) and WT (24.95 mg/100 g) before stabilizing. Notably, Glu levels were suppressed in DAO2-1 relative to WT during germination, whereas Pro showed the inverse trend. Transcriptomic analysis indicated substantial temporal variations in GABA metabolic gene expression between the two genotypes. The differences in expression level of OsGAD and OsGABA-T in DAO2-1 likely accounts for its diminished GABA capacity during early germination. These insights clarify the role of DAO2 within the regulatory network of rice GABA metabolism, providing a biochemical blueprint for optimized processing and the targeted breeding of GABA-enriched functional rice varieties.