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

Abstract

Legumes obtain a substantial portion of their nitrogen (N) from symbiotic N₂fixation 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 sativaL.) 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 N₂ 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.