Nitrogen assimilation is critical for plant life because it constitutes the mechanism by which inorganic nitrogen is converted into amino acids and subsequently other nitrogenous organic compounds. Ammonia is a product of photorespiration and its assimilation is essential for land plants, as demonstrated by the lethal phenotypes of mutants deficient in ammonia assimilation. Ammonia is assimilated by reductive amination of 2-oxoglutarate, giving rise to glutamate. This reaction is localized in plastids and catalyzed by the enzymes glutamine synthetase (GS2) and glutamate synthase (Fd-GOGAT). Plastids are unable to synthesize 2-oxoglutarate; hence 2-oxoglutarate needs to be imported from the cytosol. The end product of ammonia assimilation, glutamate, needs to be exported to the cytosol. Two distinct translocators with overlapping substrate specificity catalyze these transport steps. In Arabidopsis, the corresponding genes form a small family of three members (DiT1, DiT2.1, DiT2.2). In this study, plastidic dicarboxylate transporters will be functionally dissected by testing whether: 1) the gene functions of DiT2.1 and DiT2.2 are redundant, 2) an oxolacetate/aspartate shuttle or a displacement in sub-cellular metabolite pools can compensate for deficiency in DiT1, and 3) altered dicarboxylate transporter activity impacts sub-cellular metabolite levels that trigger coordinated changes in the transcriptome. This project will improve the understanding of transport activities involved in ammonia assimilation in plants, and of the control that metabolite transporters exert over metabolic fluxes between intracellular compartments.
Broad Impact: Undergraduate and graduate students will receive cross-disciplinary training so they can think beyond their individual field of expertise towards an integrative view of plant biology. Junior scientists will be trained in handling membrane transporters, and graduate students and post-doctoral researchers will be involved in undergraduate teaching in the field of plant membrane transport. Through cooperation with other U.S. academic institutions and international partners, networks will be established and tools in the field of membrane biology will be made available to a broad range of scientists. Plant material generated in this project will be used to introduce K-12 teachers into genetic engineering of plants and K-12 teachers will be trained in modern molecular techniques in summer courses. Through the collaboration with faculty in Statistics and Probability, this project will foster the cross-disciplinary training of graduate students in mathematical and biological sciences.
