Abstract BACKGROUND Gamma‐aminobutyric acid (GABA) is a naturally occurring bioactive component in plants. Our previous study demonstrated that ultrasound could increase the activity of glutamate decarboxylase (GAD) and effectively enhance GABA accumulation in coffee leaves. However, the mechanism underlying this action has yet to be explored. In this study, we investigated how ultrasound promotes GABA accumulation in coffee leaves by analyzing the relative contribution of the two GABA synthesis pathways, as well as changes in the key enzymes, signal molecules, and transcriptomes in coffee leaves under ultrasound treatment. RESULTS The mechanical extraction had a negligible effect on GABA levels in coffee leaves, and the substrate sodium glutamate was essential for GABA accumulation under ultrasound stress. Aminoguanidine pretreatment reduced GABA content by 31.02% under ultrasound treatment. Fluorescent imaging revealed increased intracellular Ca 2+ and H + levels, potentially contributing to enhanced GAD activity in ultrasound‐treated leaves. Transcriptomic analysis identified 1053 differentially expressed genes associated with multiple metabolic pathways, including carbohydrates, amino acids, flavonoids, and other primary and secondary metabolite biosynthesis. Further analysis indicated that ultrasound may enhance GABA accumulation by modulating Ca 2+ , reactive oxygen species, mitogen‐activated protein kinase, and phosphatidylinositol signaling pathways, as well as the glyoxylate and dicarboxylate metabolic pathways. CONCLUSION This study revealed that ultrasound promoted GABA accumulation mainly through the GABA shunt pathway, with mechanical extraction playing a minimal role. Ultrasound may enhance GAD activity by increasing intracellular Ca 2+ levels and lowering pH, while also regulating the expression of genes related to GABA biosynthesis. © 2026 Society of Chemical Industry.
Sun et al. (2026) studied this question.