We are using Arabidopsis thaliana as a model to identify the structural and regulatory genes controlling nitrogen assimilation into glutamine and glutamate using molecular, biochemical and genetic approaches. Nitrogen assimilation into these amino acids affects plant growth and ultimately seeds quantity and quality. Thus, our basic studies on the genes that control this process in plants relate indirectly to human and animal nutrition. The structural genes under investigation are: glutamine synthetase (GS), glutamate synthase (Fd-GOGAT or NADH-GOGAT), and glutamate dehydrogenase (GDH). The gene families for each enzyme in Arabidopsis contain independently regulated members encoding distinct isoenzymes. Despite decades of in vitro studies conducted in many species, the in vivo roles of GS, GOGAT and GDH isoenzymes in plants can only be conjectured. We propose to conduct the first systematic isolation of plant mutants specifically defective in each isoenzyme of GS, Fd-GOGAT, NADH-GOGAT or GDH, in a single species. We have shown that it is possible to isolate Arabidopsis mutants defective in nitrogen assimilatory genes using isoenzyme screens unbiased for growth phenotype. We propose a detailed characterization of the mutant progeny, to quantify the effects of the loss of a single isoenzyme on processes such as primary nitrogen assimilation, photorespiration, and nitrogen mobilization during seed set. This analysis will define the key and rate-limiting enzymes that control the efficiency of nitrogen use in plants. In addition we may also uncover mutants in regulatory genes. We have begun to investigate the mechanisms controlling the regulation of nitrogen assimilatory genes. Based on gene regulation studies, we have developed a """"""""metabolic control"""""""" model that proposes these nitrogen assimilatory genes are regulated in response to the ratio of carbon to nitrogen metabolites in a plant. We propose genetic screens to uncover the components of this machinery and have in hand two putative regulatory genes.
Our specific aims are: 1) Isolate additional Arabidopsis gdh mutants and mutants in chloroplastic GS2 by isoenzyme screening, 2) Characterize existing Arabidopsis mutants defective in one of two genes for Fd-GOGAT (gls1) and isolate mutants in the second gene, 3) Create mutants in cytosolic GS1 or NADH-GOGAT by expressing dominant-negative subunits in transgenic plants, 4) Test the metabolic control model and define the metabolites senses, 5) Define components of the regulatory pathway using genetic screens and define the in vivo function of candidate genes in hand. Our basic studies on the mechanisms that regulate nitrogen assimilation in Arabidopsis may have implications for improving nitrogen use in crops not amenable to such molecular-genetic studies. Furthermore, as metabolic signaling also occurs in animals, insights into this process may be more readily obtained using a molecular-genetic strategy in Arabidopsis.
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