The glutamine amidotransferase gene GAT1_2.1 is a marker of N status in Arabidopsis root, linked to a shoot branching phenotype. The protein has an N-terminal glutamine amidotransferase domain and a C-terminal extension with no recognizable protein domain. A purified, recombinant version of the glutamine amidotransferase domain was catalytically active as a glutaminase, with apparent K value of 0.66 mM and V value of 2.6 μkatal per mg. This form complemented an E. coli glutaminase mutant, ΔYneH. Spiking of root metabolite extracts with either the N-terminal or full length form purified from transformed tobacco leaves led to reciprocal changes in glutamine and ammonia concentration. No product derived from amido-N-labeled glutamine was identified. Visualization of GAT1_2.1-YPF transiently expressed in tobacco leaves confirmed its mitochondrial localization. gat1_2.1 exhibited reduced growth as compared with wild-type seedlings on media with glutamine as sole nitrogen source. Results of targeted metabolite profiling pointed to a possible activation of the GABA shunt in the mutant following glutamine treatments, with reduced levels of glutamic acid, 2-oxoglutarate and γ-aminobutyric acid and increased levels of succinic acid. GAT1_2.1 may act as a glutaminase, in concert with Glutamate Dehydrogenase 2, to hydrolyze glutamine and channel 2-oxoglutarate to the TCA cycle under high nitrogen conditions.