Evidence that class I glutamine amidotransferase, GAT1_2.1, acts as a glutaminase in roots of Arabidopsis thaliana

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 Km value of 0.66 mM and Vmax 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-15N-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.