Genome-Wide Identification of the Barley GAD Gene Family and Molecular Characterization of HvGAD1

Glutamate decarboxylase (GAD) catalyzes the formation of γ-aminobutyric acid (GABA) and is a central enzyme of the plant GABA shunt. Six HvGAD genes were identified in the Morex V3 barley reference genome and characterized with respect to phylogeny, gene structure, conserved motifs, chromosomal distribution, and promoter cis-acting elements. Public transcriptome profiles were combined with reverse transcription quantitative PCR (RT-qPCR) analyses under drought, salt, and cold stress to identify broadly responsive family members, and the relationship between HvGAD1 expression and leaf GABA content was examined. HvGAD1 was broadly expressed and responded to all three stresses. Pearson analysis showed positive associations between HvGAD1 expression and GABA content in the pooled data and under drought and cold stress, although the association was not significant after adjustment for treatment and sampling time. Recombinant HvGAD1 was used to raise a rabbit polyclonal antibody that detected and enriched HvGAD-immunoreactive proteins from barley tissues. Immunoprecipitation–mass spectrometry (IP-MS) analysis identified six co-enriched candidate proteins, including cytoplasmic aspartate aminotransferase, probable serine acetyltransferase 5, and an Annexin D7-like protein. These findings support HvGAD1 as a candidate for further functional analysis of GABA metabolism and abiotic stress responses in barley. The antibody and six co-enriched proteins provide resources for subsequent protein-level studies.