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Aspartate Aminotransferase in Alfalfa Nodules: Localization of mRNA During Effective and Ineffective Nodule Development and Promoter Analysis

April 1999 , Volume 12 , Number  4
Pages  263 - 274

Hirofumi Yoshioka , 1 , 2 Robert G. Gregerson , 3 Deborah A. Samac , 4 , 5 Kim C. M. Hoevens , 6 Gian Trepp , 1 , 7 J. Stephen Gantt , 8 and Carroll P. Vance 1 , 4

1Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul 55108, U.S.A.; 2Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan; 3Department of Biology, Lyon College, Batesville, AR 72503, U.S.A.; 4U.S. Department of Agriculture, Agricultural Research Service, Plant Science Research Unit, St. Paul, MN 55108, U.S.A.; 5Department of Plant Pathology, University of Minnesota, St. Paul 55108, U.S.A.; 6Higher Institute for Professional Education Venlo, Division Laboratory Science, 3-Decembersingel 36, 5822 BD Venlo, The Netherlands; 7Institut fuer Pflauzenwissenschaften ETH-Zurich, 8092 Zurich, Switzerland; 8Department of Plant Biology, University of Minnesota, St. Paul 55108, U.S.A.

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Accepted 15 December 1998.

Aspartate aminotransferase (AAT) plays a critical role in the assimilation of symbiotically fixed nitrogen into aspartate and asparagine in legume root nodules. The enzyme occurs as a cytosolic form (AAT1) and a plastid form (AAT2) in alfalfa nodules. To elucidate the functional role of each isozyme in root nodule metabolism further, in situ hybridization was used to determine the pattern of transcript accumulation from the two genes. AAT2 transcripts were localized to infected cells throughout the symbiotic zone of effective alfalfa nodules; however, expression was reduced in ineffective nodules. The AAT1 gene was expressed in the uninfected cells of the invasion zone and symbiotic zone, the nodule parenchyma, and nodule vascular bundles of both effective and ineffective nodules. The AAT1 and AAT2 promoters were evaluated in transgenic alfalfa plants containing promoter β-glucuronidase (GUS) gene fusions. Histochemical staining patterns agreed with results from in situ localization. The distribution pattern of gene transcripts suggests that AAT1 has a role in maintenance of the O2 diffusion barrier in nodules and that AAT2 plays a major role in assimilation of recently fixed nitrogen. Promoter deletion analysis of the AAT2 promoter revealed that nodule-specific expression was retained in a promoter fragment of 300 bp.

Additional keywords: malate dehydrogenase, Medicago sativa, nitrogen fixation, phosphoenolpyruvate carboxylase, Sinorhizobium meliloti.

The American Phytopathological Society, 1999