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 N2 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 amino-transferase (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.
Department of Agromomy and Plant Genetics, University of Minnesota, St Paul 55108, USA.
M A
MA
Schoenbeck
S J
SJ
Temple
G B
GB
Trepp
J M
JM
Blumenthal
D A
DA
Samac
J S
JS
Gantt
G
G
Hernandez
C P
CP
Vance
D016375:Antisense Elements (Genetics); D005970:Glutamate Synthase / Q000235:genetics / Q000378:metabolism*; D000455:Medicago sativa / Q000201:enzymology* / Q000235:genetics; D009243:NAD / Q000378:metabolism*; D018517:Plant Roots / Q000201:enzymology; D014170:Transformation, Genetic; D019076:Transgenes
D016375:Antisense Elements (Genetics); D009243:NAD; D005970:Glutamate Synthase