Decreased NADH glutamate synthase activity in nodules and flowers of alfalfa (Medicago sativa L.) transformed with an antisense glutamate synthase transgene

Legumes obtain a substantial portion of their nitrogen (N) from symbiotic N-2 fixation in root nodules, The glutamine synthetase (GS, EC 6.3.1.2)/glutamate synthase (GOGAT) cycle is responsible for the initial N assimilation. This report describes the analysis of a transgenic alfalfa (Medicago sativa L.) line containing an antisense NADH-GOGAT (EC 1.4.1.14) under the control of the nodule-enhanced aspartate aminotransferase (AAT-2) promoter. In one transgenic line, NADH-GOGAT enzyme activity was reduced to approximately 50%, with a corresponding reduction in protein and mRNA, The transcript abundance for cytosolic GS, ferredoxin-dependent GOGAT (EC 1,4,7,1), AAT-2 (EC 2.6.1.1), asparagine synthase (EC 6.3.5,4), and phosphoenolpyruvate carboxylase (PEPC, EC 4,1,1.31) were unaffected, as were enzyme activities for AAT, PEPC and GS. Antisense NADH-GOGAT plants grown under symbiotic conditions were moderately chlorotic and reduced in growth and N content, even though symbiotic N-2 fixation was not significantly reduced. The addition of nitrate relieved the chlorosis and restored growth and N content. Surprisingly, the antisense NADH-GOGAT plants were male sterile resulting from inviable pollen, A reduction in NADH-GOGAT enzyme activity and transcript abundance in the antisense plants was measured during the early stages of flower development. Inheritance of the transgene was stable and resulted in progeny with a range of NADH-GOGAT activity, These data indicate that NADH-GOGAT plays a critical role in the assimilation of symbiotically fixed N and during pollen development.