|VAN DE MORTEL, MARTIJN|
|Samac, Deborah - Debby|
Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/8/1999
Publication Date: N/A
Interpretive Summary: Nitrogen (N) is one of the primary fertilizer elements required for plant growth. Symbiotic nitrogen (N2) fixation is a major means world wide by which legume crops, such as alfalfa, obtain N. In legume plants, N2 fixation occurs in small wart-like outgrowths on roots, called nodules, which contain Rhizobium bacteria. Although a great deal is known about bacterial genes involved in N2 fixation and N assimilation, considerably less is known about the plant genes involved in this process. In this study we have isolated and characterized the plant gene from alfalfa that encodes a critical step in the assimilation of symbiotically fixed N in root nodules. This plant gene encodes the enzyme (protein catalyst) glutamate synthase that makes the amino acid glutamate. We identified specific regions in the gene that control its amount and location within nodules. Moreover, the work shows that a specific piece of DNA that is repeated at least twice in a row is a likely candidate for regulating the glutamate synthase gene. These findings are important because this is the first time the glutamate synthase gene has been characterized and the unique genetic element defined that controls expression. In addition, the isolation of the glutamate synthase gene will allow direct genetic modification of plant N assimilation by genetic engineering approaches.
Technical Abstract: NADH-dependent glutamate synthase (NADH-GOGAT; EC 126.96.36.199) is a key enzyme in primary nitrogen assimilation in alfalfa root nodules. Here we report that in alfalfa (Medicago sativa L.) a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type Saranac nodules the NADH-GOGAT gene is predominately expressed i infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH- GOGAT shifted during root nodule development. Until day 9 after planting, all infected cells appeared to express NADH-GOGAT. By day 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone, was observed. In 33-day-old nodules, expression was seen i only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript, but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric B-glucuronidase (GUS) promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88bp direct repeat.