Double knockout of NtBADH2a/NtBADH2b enhances 2-acetyl-1-pyrroline accumulation but reduces PEG-simulated drought stress and salt stress tolerance in tobacco

CRISPR/Cas9-mediated editing of betaine aldehyde dehydrogenase2 ( BADH2 ) has been shown to effectively enhance 2-acetyl-1-pyrroline (2-AP) content in several crops, yet the pleiotropic effects resulting from loss of BADH2 function remain inadequately elucidated. In this study, three independent ntbadh2a ntbadh2b double mutant lines were generated in tobacco using CRISPR/Cas9, and their performance was comprehensively evaluated across aroma production, abiotic stress tolerance, agronomic traits, and leaf quality. In cured middle leaves, 2-AP content reached 31.59 mg/kg dry weight (DW) in the mutants, a 6.4-fold increase over wild type (WT), whereas γ-aminobutyric acid (GABA) content decreased by up to 12.4%. The mutants exhibited differential stress responses: under PEG-simulated drought stress and salt stress, they showed visible wilting or leaf damage, with significantly elevated malondialdehyde (MDA), H₂O₂, and antioxidant enzyme activities compared with WT, while no phenotypic differences were detected under cold or heat stress, indicating that BADH2 inactivation specifically compromises drought and ionic stress tolerance. Encouragingly, key agronomic traits and major quality parameters remained largely unaffected. These findings indicate that fragrance enhancement via BADH2 editing comes at the cost of reduced stress tolerance, a trade-off likely associated with reduced GABA-dependent osmoprotection. Thus, deploying BADH2 mutations for aromatic tobacco improvement should be accompanied by tailored stress management, particularly under water-limited or saline growing conditions.