|Samac, Deborah - Debby|
Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/14/1999
Publication Date: N/A
Citation: N/A Interpretive Summary: Alfalfa plants living in symbiosis with the soil bacterium Rhizobium through a process called nitrogen fixation can convert atmospheric nitrogen gas to nitrogen fertilizer. This process occurs in wart-like structures termed nodules, that form on roots. The plant gives the bacterium energy from sugar produced in the leaves, while the bacterium gives the plant nitrogen fertilizer. Before that nitrogen fertilizer can be used by the plant it must be converted into a useful form. The plant enzyme glutamate synthase (GOGAT, a protein catalyst) is thought to play a major role in the conversion of symbiotically fixed nitrogen into amino acids. To test whether GOGAT is involved in assimilation of fixed nitrogen we used a biotechnology approach to either reduce or prevent GOGAT from forming. In this approach an extra copy of the GOGAT gene in the opposite orientation (antisense) to the normal GOGAT gene was transferred specifically to alfalfa nodules. When the extra copy of the GOGAT gene is in the antisense orientation to the normal GOGAT gene, the activity of GOGAT should be reduced. This is because the antisense gene reacts with the normal to block the gene activity. The results showed that plants having the antisense GOGAT were reduced in GOGAT activity and assimilation of symbiotically fixed nitrogen as compared to the control plants. These data provide the first direct genetic evidence that GOGAT is essential for assimilation of fixed nitrogen. Our information is important because: (1) it shows that we can genetically engineer individual steps in nitrogen assimilation; and (2) it offers the possibility that we can use specific genes to improve nitrogen assimilation.
Technical Abstract: Legumes obtain a substantial portion of their nitrogen (N) from symbiotic N2 fixation in root nodules. The glutamine synthetase (GS, EC 184.108.40.206)/ 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 220.127.116.11) 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 18.104.22.168), AAT-2 (EC 22.214.171.124), asparagine synthase (EC 126.96.36.199), and phosphoenolpyruvate carboxylase (PEPC, EC 188.8.131.52) 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 during pollen development.