PR001145 (Project)

Description:Carbon and Nitrogen balance in plant leaves, required for sustained growth, is achieved by inter-relationships between the processes of photosynthesis, respiration and amino acid metabolism in a photoperiod dependent manner. The GS/GOGAT cycle is one such mechanism and is highly elucidated in plants to serve as a crossroad between C and N metabolism. Non-photosynthetic tissues (e.g., roots, germinating seeds), however, lack a sufficient supply of carbon skeletons under high N conditions and hence may resort to other mechanisms, along with GS/GOGAT cycle, to achieve the aforementioned C/N balance. Here, we propose a potential role of an enzyme, GAT1_2.1, in hydrolyzing excess glutamine to Glu, which channels carbon skeletons to the TCA cycle, under high N conditions, using Arabidopsis as a model. GAT1_2.1, a class I glutamine amidotrasferase of unknown substrate specificity, was shown to be highly responsive to N status, localized in mitochondria and is highly co-expressed with Glutamate Dehydrogenase 2 (GDH2). Arabidopsis mutants lacking GAT1_2.1 have elevated GABA shunt pathway activity to replenish the depleted levels of Glu. This Glu may then be deaminated to 2-oxoglutarate by GDH2 and channeled into the TCA cycle thus providing a crossroad between C and N metabolism in root mitochondria. We use a metabolomics approach to demonstrate the difference in quantities of pathway intermediates between wild type Arabidopsis roots and gat1_2.1 mutants using glutamine as organic nitrogen treatment and KNO3 and Glu treatments as negative and positive controls, respectively. In addition, we used Arabidopsis root extracts, spiked with amide nitrogen labeled (15N1) Glutamine and a purified recombinant protein, both full length and glutaminase domain only versions, to determine the amido group acceptor, if any, in the glutamine amidotransferase reaction.