|Miller, Susan - UNIVERSITY OF MINNESOTA|
|Gregerson, Robert - UNIVERSITY OF MINNESOTA|
|Robinson, D - UNIVERSITY OF MINNESOTA|
|Gantt, J - UNIVERSITY OF MINNESOTA|
Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 30, 1996
Publication Date: N/A
Interpretive Summary: Nitrogen (N2) fixation by legume plants, next to photosynthesis, is one of the more important biochemical processes. This process, which is a symbiosis between the soil bacteria Rhizobium and the plant, supplies the legume plant with a renewable source of N fertilizer. Thus, plants like alfalfa, clover, soybean and pea need little if any additional N fertilizer. Symbiotically fixed N from legumes also provides a significan source of N fertilizer to other crops when they are grown in rotation with legumes. The efficiency of symbiotic N2 fixation is dependent upon the interaction of both the Rhizobium and legume plant genes. Significant progress has been made in isolating and characterizing the bacterial genes associated with N2 fixation, but much less is known about the plant genes involved in this process. This report is the first documentation of the isolation of a plant gene responsible for making glutamate synthase (GOGAT), a key enzyme (protein catalyst) involved in the use of symbiotically fixed N. This gene was isolated from alfalfa. The information from this research will improve our understanding of how plants control the uptake and use of N. The GOGAT gene will also be used for mapping its position on the alfalfa chromosomes. The GOGAT gene will be useful in biotechnological approaches to improve the efficiency of plant N use.
Technical Abstract: Glutamate synthase (GOGAT), a key enzyme in ammonia (NH4+) assimilation, occurs as two forms in plants: a ferredoxin dependent form (Fd GOGAT) and an NADH dependent form (NADH GOGAT). These enzymes are encoded by distinct genes as evidenced by their cDNA and deduced amino acid sequences. This paper reports the isolation and characterization of a NADH GOGAT gene from alfalfa (Medicago sativa L.), the first GOGAT gene to be isolated from a eukaryote. RNase protection and primer extension experiments map the transcription start site of NADH GOGAT to nearly identical positions. The transcribed region of this gene, 12,214 bp, is comprised of 22 exons separated by 21 introns. The 2.7 kbp region 5 from the translation initiation site confers nodule specific reporter gene activity when used in a chimeric B glucuronidase construct and transformed into Lotus corniculatus and Medicago sativa. The abundance of NADH GOGAT transcripts increases substantially in developing nodules of plants infected with effective rhizobia. However, this increase is not observed when nodules are induced by a variety of ineffective rhizobial strains. Thus, unlike many other plant genes involved in root nodule NH4+ assimilation, high levels of NADH GOGAT expression are strictly associated with effective nodules indicating that NADH GOGAT plays a central role in the functioning of effective root nodules. An alfalfa Fd GOGAT PCR product showing greater than 85 percent identity to maize Fd GOGAT was isolated and used to investigate the contribution of this enzyme to NH4+ assimilation in nodules. Fd GOGAT mRNA was abundant in leaves and cotyledons but was not detected in alfalfa root nodules. Fd GOGAT in alfalfa does not appear to be important in symbiotic N2 fixation.