Regulatory helix deletion in glutamate decarboxylase reduces GABA and enhances Agrobacterium-mediated transient expression in lettuce

Gamma-aminobutyric acid (GABA) is a metabolite involved in plant growth and stress responses, with its synthesis regulated by glutamate decarboxylase (GAD). Plant GAD enzymes have an autoinhibitory α-helix at the C-terminus, which calmodulin (CaM) binding typically relieves. Eliminating this C-terminal motif usually increases GABA levels in crops. In this case study, we generated a CRISPR/Cas9-edited lettuce line with a 14-amino acid deletion in the C-terminal helix of LsGAD2, the isozyme primarily expressed in most tissues. This targeted truncation removes CaM-binding residues while retaining the key Lys cluster (Lys489, Lys490, Lys491) responsible for autoinhibition, resulting in a significant reduction in GABA content without affecting growth. The LsGAD1/2-ΔC line showed a transcriptomic profile resembling stress responses in the wildtype under unstressed conditions. Reduced GABA levels appeared to upregulate genes involved in stress perception, signalling, and defense-related metabolic and hormonal changes, potentially mediated by WRKY-family transcription factors. Likely due to lower GABA levels and altered defense responses, LsGAD1/2-ΔC plants showed increased Agrobacterium-mediated transient expression of β-glucuronidase. Overall, our study suggests that targeted genetic manipulation of the C-terminal helix of GAD enzymes can reduce GABA levels while enhancing transformation efficiency in lettuce, thus presenting a means for engineering for such purposes.