Transcriptomic Insights into GABA Accumulation in Tomato via CRISPR/Cas9-Based Editing of SlGAD2 and SlGAD3

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid with key roles in plant metabolism, stress responses, and fruit nutritional quality. In tomato (Solanum lycopersicum), GABA levels are dynamically regulated during fruit development but decline in the late ripening stages.

To enhance GABA accumulation, we used CRISPR/Cas9 to edit the calmodulin-binding domain (CaMBD) of SlGAD2 and SlGAD3, which encode glutamate decarboxylases (GADs). The resulting truncated enzymes were expected to be constitutively active. We quantified GABA content in leaves and fruits and performed transcriptomic analysis on edited lines at the BR+7 fruit stage.

CaMBD truncation significantly increased GABA levels in both leaves and fruits. In gad2 sg1 lines, GABA levels increased by 3.5-fold in leaves and 3.2-fold in BR+10 fruits; in gad3 sg3 lines, increases of 2.8- and 2.5-fold were observed, respectively. RNA-seq analysis identified 1383 DEGs in gad2 #1-5 and 808 DEGs in gad3 #3-8, with 434 DEGs shared across both lines. These shared DEGs showed upregulation of GAD, GABA-T, and SSADH, and downregulation of stress-responsive transcription factors including WRKY46, ERF, and NAC. Notably, total free amino acid content and fruit morphology remained unchanged despite elevated GABA.

CRISPR/Cas9-mediated editing of the CaMBD in SlGAD genes selectively enhances GABA biosynthesis in tomato without adverse effects on development or fruit quality. These lines offer a useful platform for GABA-centered metabolic engineering and provide insights into GABA's role in transcriptional regulation during ripening.