OsBADH1–OsBADH2 Double Mutants Increase 2-Acetyl-1-Pyrroline Accumulation and Alter GABA-Associated Abiotic Stress Responses in Rice

Background/Objectives: Rice fragrance is mainly determined by 2-acetyl-1-pyrroline (2-AP), which is negatively regulated by OsBADH2. However, the contribution of its paralog OsBADH1 to aroma-associated metabolism and GABA-linked abiotic stress responses remains unclear. This study investigated whether simultaneous disruption of OsBADH1 and OsBADH2 further enhances 2-AP accumulation while affecting stress tolerance in rice. Methods: Independent osbadh1 and osbadh2 knockout lines were generated using CRISPR/Cas9 and crossed to obtain homozygous osbadh1 osbadh2 double mutants. Wild type, single mutants, and double mutants were compared for 2-AP accumulation, GABA content, agronomic traits, abiotic stress responses, and expression of genes associated with GABA metabolism and stress responses. Results: The osbadh2 mutant showed a marked increase in 2-AP, and the osbadh1 osbadh2 double mutant exhibited the highest level, corresponding to a 7.1-fold increase over the wild type. In contrast, the GABA content progressively decreased and reached 0.46-fold of the wild-type level in the double mutant. Under normal growth conditions, the double mutant showed no major agronomic defects. However, under salinity and drought stress, its survival declined to 0.41-fold and 0.40-fold of the wild-type levels, respectively. KEGG and expression analyses further indicated coordinated disruption of GABA-associated metabolic and stress-responsive pathways in the double mutant. Conclusions: Combined disruption of OsBADH1 and OsBADH2 enhanced aroma-associated metabolism but weakened GABA-linked abiotic stress tolerance, revealing a trade-off between increased fragrance and reduced stress resilience in rice.