A combined molecular-genetic approach is being used to study the genes encoding the multiple isoenzymes of aspartate aminotransferase (AspAT), a key enzyme in plant nitrogen metabolism. AspAT genes and AspAT deficient mutants of Arabidopsis thaliana are being characterized. The objectives are to determine the function that each AspAT isoenzyme serves in metabolism, and to determine whether the multiple AspAT isoenzymes perform distinct or overlapping roles using molecular biology and genetics. The molecular techniques used will define the expression pattern of each individual AspAT gene with regard to cell-type, development, and light. These molecular analyses combined with the ability to analyze the effects of a null mutation in a particular isoenzyme. Because AspAT is involved in mobilizing nitrogen within a plant, these findings have significance for improving plant nitrogen use. Four distinct AspAT cDNAs have been isolated. Characterization of full length cDNA clones will enable the determination of the primary structure of the encoded AspAT polypeptide and in vitro organellar uptake studies will establish which cDNAs encode chloroplast, mitochondrial, and cytosolic forms of AspAT. The expression pattern of each AspAT gene will be monitored and the results used to develop a model concerning the function of the encoded isoenzyme. Promoter elements for each AspAT gene will be fused to a reporter gene (GUS) and introduced into transgenic plants. Histochemical analysis for GUS activity will establish the cell-type in which each AspAT gene is expressed. This information will provide insight into the function of each gene. The ultimate test of the physiological function of each AspAT isoenzyme in vivo will be determined by monitoring the growth phenotype of plants which are specifically defective in one or more of the forms of AspAT. To date, three putative AspAT mutants have been identified out of 750 M2 plants screened. Genetic crosses between mutants will establish the number of different loci affected. Structural mutants will be identified by RFLP using the four cloned AspAT genes. Functional complementation of the AspAT mutants with the cloned AspAT genes will delimit the lesion to a particular AspAT gene. %%% Aspartate amino transferase is a key enzyme in higher plants which functions to shuttle carbon and nitrogen skeletons between organelles and between cells. The biochemistry of AspAT has been confused by the presence of multiple isoenzymes. We have taken a molecular-genetic approach to clone the four distinct genes encoding AspAT in a plant (arabidopsis thaliana). In addition, we have developed a technique to identify mutant plants which are specifically defective in one of the four major AspAT isoenzymes. The cloned AspAT genes will be used to study the structure, function, and regulation of each unique form of AspAT in plants. The mutants specifically defective in a particular AspAT isoenzyme will be evaluated with regard to the affected gene, the growth phenotype, and metabolite profile to determine the role that each AspAT enzyme plays in plant nitrogen metabolism. These experiments will define the function of each AspAT enzyme, the role of aspartate biosynthesis in plant growth, and also serve as a model for how molecular-genetics can be used to understand fundamental questions concerning plant metabolism.
