9630604 Coruzzi A combined molecular-genetic and biochemical approach is being used to study the function of distinct isoenzymes of aspartate aminotransferase (AspAT) in the model genetic plant Arabidopsis thaliana. A major goal is to identify the in vivo role played by individual AspAT isoenzymes localized in the cytosol, chloroplast, mitochondria or peroxisome. Arabidopsis mutants deficient in either of the two major AspAT isoenzymes (cytosolic AAT2 or chloroplastic AAT3) have been isolated and partially characterized. Additional Arabidopsis mutants defective in the other "minor" AspAT isoenzymes, (mitochondrial AATl and peroxisomal AAT) will be created using a dominant-negative approach. Arabidopsis mutants defective in each AAT isoenzyme will be subject to physiological and metabolic analysis to define the in vivo role of the "missing" isoenzyme. In addition to these genetic studies, the cloned genes encoding each AspAT isoenzyme are also under investigation. RFLP mapping has been used to determine which ASP cloned genes co-segregate with an isoenzyme mutation. Preliminary results demonstrate that the mutants are deficient in cytosolic AAT2 are affected in the ASP2 gene for cytosolic AAT2. The phenotypic analysis of the aat2 mutant suggests that ASP2 encodes a key isoenzyme controlling the assimilation of nitrogen into aspartate used for intercellular nitrogen transport. Gene expression studies have also shown that ASP2 plays a key regulatory role as its expression is regulated by light and/or metabolic control. The specific aims of this project are to: 1) Demonstrate by in vitro organelle uptake that ASPl and ASP3 encode mitochondrial and peroxisomal forms of AspAT. 2) Determine the cell-specific expression pattern of each ASP gene by in situ hybridization and/or by using promoter-GUS fusions. 3) Elucidate the mechanism of metabolic regulation for ASP2. 4) Map all the ASP genes. 5) Determine whether the aat3 mutation deficient in chloroplast AAT3 is in an ASP structural or re gulatory gene. 6) Define the molecular lesion in each aat2 and aat3 mutant. 7) Isolate deletion alleles of aat2, aat3 and/or regulatory mutants. 8) Create mutants in mitochondrial AATl or peroxisomal AAT using anti-sense or dominant-negative transgenic technology. 9) Perform a detailed characterization of the aat mutants including genetic, physiological and biochemical tests. 10) Create transgenic plants which ectopically express specific ASP genes defined to be key by mutant analysis. %%% This project is a molecular genetic analysis that focuses on an important family of related enzymes, aspartate aminotransferase, (AspAT) involved in nitrogen metabolism in higher plants. AspAT functions to 1) convert assimilated nitrogen into aspartate for intercellular nitrogen transport to developing organs (e.g. seed), 2) act as the catalyst in innumerable transamination reactions to donate nitrogen for the synthesis of nitrogen containing compounds, 3) maintain a balance between organic nitrogen and carbon, 4) shuttle reducing equivalents between subcellular compartments. These molecular-genetic studies in Arabidopsis will help define which particular AspAT isoenzyme is involved in each of the above processes. Moreover, the engineering of the ASP genes in transgenic plants has potential for improving nitrogen-use in transgenic crop plants. Finally, these studies will also serve as a model for ways in which to dissect steps in other complex multi-isoenzyme pathways in plants. ***
