Double-truncated version of OsGADs leads to higher GABA accumulation and stronger stress tolerance in Oryza sativa L. var. japonica

Calmodulin binding domain truncation from OsGAD1 and OsGAD3 resulted in enhanced GABA accumulation, upregulated stress related genes, and improved tolerance to multiple abiotic stresses. Rice (Oryza sativa L.), a critical crop for global food security, faces significant challenges from abiotic stresses. Gamma-aminobutyric acid (GABA), synthesized by glutamate decarboxylase (GAD), plays a vital role in stress tolerance. Truncating the calmodulin-binding domain (CaMBD) in GAD enzymes enhances GAD activity and GABA production. In this study, we developed a hybrid line, Hybrid #78, by crossing two genome-edited lines, OsGAD1ΔC #5 and OsGAD3ΔC #8, with truncated CaMBD in OsGAD1 and OsGAD3, respectively. Hybrid #78 demonstrated significantly improved survival rates in cold (25%), salinity (33%), flooding (83%), and drought (83%) stress conditions, compared with wild-type Nipponbare (0-33%), OsGAD1∆C #5 (0-66%), and OsGAD3∆C #8 (0-50%). Hybrid #78 showed the highest GABA levels during stress, with increases of 3.5-fold (cold), 3.9-fold (salinity), 5-fold (flooding), and 5-fold (drought) relative to wild-type Nipponbare and up to 2-fold higher than that of the parent lines. RNA-seq analysis from shoot tissues in control conditions identified 975 differentially expressed genes between Hybrid #78 and wild-type Nipponbare, with 450 genes uniquely expressed in the hybrid. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that upregulation in nitrogen metabolism pathways likely contributes to enhanced GABA synthesis via increased glutamate production. Hybrid #78 also showed broader gene expression variability, suggesting enhanced adaptability to stress, especially upregulation of stress-related genes, such as OsDREB, OsHSP70, and OsNAC3. These findings highlight the potential of CaMBD truncation in OsGAD1 and OsGAD3 to develop rice lines with increased GABA accumulation and resilience to multiple abiotic stresses.