Visualization of Glutamate Decarboxylase Activity in Barley Seeds under Salinity Stress Using Mass Microscope

γ-Aminobutyric acid (GABA) accumulates in plants in response to environmental stresses. The activity levels of glutamate decarboxylase (GAD), an enzyme involved in GABA biosynthesis, are reported to increase during germination under salinity stress. However, it is not clear which tissues of the plant seeds are affected by GAD activity in response to salinity stress. In this study, the effects of salinity stress on the distribution of barley seeds GAD activity during germination were investigated. The mass spectrometry imaging (MSI) method was optimized, and the distribution of GAD activity in germinated seeds exposed to salinity stress at different germination stages from 12 to 48 h after imbibition was investigated. In this study, MSI was successfully applied to enzyme histochemistry to visualize the relative GAD activity in germinating barley seeds for the first time. The salinity stress increased the GAD activity, mostly due to the increase in relative GAD activity in the embryo. Higher GAD activity was detected in seeds exposed to salinity stress in the scutellum or aleurone layer, which are difficult to separate for extraction. This method can be used to clarify the role of GABA shunts, including GAD enzyme responses, in barley seeds under stress.