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Metabolism and Enzymology

Aspartate Aminotransferase in Alfalfa Root Nodules ¹

III. Genotypic and Tissue Expression of Aspartate Aminotransferase in Alfalfa and Other Species

Plant Science Research Unit, U.S. Department of Agriculture, Agricultural Research Service, University of Minnesota, St. Paul, Minnesota 55108, Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, Minnesota 55108

Aspartate aminotransferase (AAT) plays an important role in nitrogen metabolism in all plants and is particularly important in the assimilation of fixed N derived from the legume-Rhizoblum symbiosis. Two isozymes of AAT (AAT-1 and AAT-2) occur in alfalfa (Medicago sativa L.). Antibodies against alfalfa nodule AAT-2 do not recognize AAT-1, and these antibodies were used to study AAT-2 expression in different tissues and genotypes of alfalfa and also in other legume and nonlegume species. Rocket immunoelectrophoresis indicated that nodules of 38-day-old alfalfa plants contained about eight times more AAT-2 than did nodules of 7-day-old plants, confirming the nodule-enhanced nature of this isozyme. AAT-2 was estimated to make up 16, 15, 5, and 8 milligrams per gram of total soluble protein in mature nodules, roots, stems, and leaves, respectively, of effective N₂-fixing alfalfa. The concentration of AAT-2 in nodules of ineffective non-N₂-fixing alafalfa genotypes was about 70% less than that of effective nodules. Western blots of soluble protein from nodules of nine legume species indicated that a 40-kilodalton polypeptide that reacts strongly with AAT-2 antibodies is conserved in legumes. Nodule AAT-2 immunoprecipitation data suggested that amide- and ureide-type legumes may differ in expression and regulation of the enzyme. In addition, Western blotting and immunoprecipitations of AAT activity demonstrated that antibodies against alfalfa AAT-2 are highly cross-reactive with AAT enzyme protein in leaves of soybean (Glycine max L.), wheat (Triticum aestivum L.), and maize (Zea mays L.) and in roots of maize, but not with AAT in soybean and wheat roots. Results from this study indicate that AAT-2 is structurally conserved and localized in similar tissues among diverse species.

¹ Joint contribution from the Plant Science Research Unit, U.S. Department of Agriculture, Agricultural Research Service, and the Minnesota Agricultural Experiment Station (Paper No. 18,261, Scientific Journal Series). The research was supported in part by U.S. Department of Agriculture-Competitive Research Grants Office grant 87-CRCR-1-2588 and National Science Foundation: DCB grant 8905006.