Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/14/2003
Publication Date: N/A
Citation: N/A Interpretive Summary: Legume plants such as alfalfa, bean, and pea in symbiosis with soil bacteria, collectively called rhizobia, can convert nitrogen (N) gas into N fertilizer through a process known as symbiotic nitrogen fixation (SNF). The process of SNF occurs in small wart-like structures (nodules) on legume roots. Within the nodule the plant gives the rhizobia bacteria food for growth while the rhizobia give the plant N fertilizer. A great deal is known about bacterial genes involved in SNF, however, much less is known about the plant genes. Here we describe experiments showing that the alfalfa gene which encodes the enzyme (protein catalyst) NADH-GOGAT is required for efficient SNF and N assimilation. Using a genetic approach, we suppressed the expression of the NADH-GOGAT gene which in turn caused a big reduction in NADH-GOGAT enzyme activity. Plants with reduced enzyme activity fixed less N symbiotically, had smaller more deformed nodules, and reduced growth. This is one of the first studies to conclusively demonstrate the role of NADH-GOGAT in plant function. This discovery is important because it shows specific plant genes are required for effective SNF and N assimilation. The NADH-GOGAT may be manipulated in a fashion that could result in increased activity giving better SNF.
Technical Abstract: Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation in root nodules. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impacts of specific reduction on nodule NADH-GOGAT on symbiotic performance of alfalfa (Medicago sativa L.). Four independent transgenic alfalfa lines, designated GA89, GA87, GA88, and GA82 (for GOGAT antisense), containing an antisense NADH-GOGAT cDNA fragment under the control of the soybean leghemoglobin (lbc3) promoter were evaluated. The GA plants were fertile and showed normal growth in non-symbiotic conditions. Clonally propagated plants were inoculated with Sinorhizobium meliloti after rooting. Analysis of the symbiotic phenotype was carried out 21 days post-inoculation. Nodules of each GA line presented diminished remaining NADH-GOGAT activity, ranging from 44 to 88% of control plants, that was accompanied by a decrease in RNA and protein levels. Plants from the GA89 line, with the lowest NADH-GOGAT activity (c. 40%), presented a strikingly altered symbiotic phenotype: activities of key enzyme for carbon and nitrogen assimilation were concertedly down-regulated; nodules were altered morphologically and showed less dry weight; lower nitrogenase activity and lower foliage dry weight and nitrogen content was observed; chlorotic symptoms related with nitrogen deprivation were observed; and the photosynthetic rate was reduced. Plants from the GA87 line with c. 60% remaining NADH-GOGAT activity also presented an altered symbiotic phenotype, albeit less impaired than the phenotype of GA89 plants. The reduction in nodule NADH-GOGAT activity limits ammonium assimilation and impairs the metabolic flux of carbon and nitrogen compounds.