|Trepp, Gian - UNIVERSITY OF MINNESOTA|
|Temple, Stephen - UNIVERSITY OF MINNESOTA|
|Bucciarelli, Bruna - UNIVERSITY OF MINNESOTA|
|Shi, Lifang - FORMER ARS EMPLOYEE|
Submitted to: Molecular Plant Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 26, 1999
Publication Date: N/A
Interpretive Summary: Legumes, like alfalfa, obtain a substantial amount of nitrogen (N) fertilizer through a process known as symbiotic nitrogen fixation. To initiate this process, soil bacteria invade the root causing wart-like growths, termed nodules, to form at the root. Within the nodule the bacteria take nitrogen gas (N2) and convert it to N fertilizer, which becomes available to the plant for growth. The plant gives the bacteria energy in the form of carbon. Although a great deal is known about bacterial genes involved in nodulation and N2 fixation, much less is known about the plant genes involved in this process. To ascertain where plant genes were expressed in nodules we mapped the location of expression within the root nodule of 11 alfalfa genes involved in nodule N and carbon metabolism. The location of expression was also measured for two bacterial genes involved in N2 fixation. The data showed that expression of four plant genes most closely linked to nodule N assimilation was localized to the area of greatest N2 fixation. This zone of N2 fixation was limited to 20 cell layers in 33-day-old nodules. Plant genes involved in carbon metabolism were expressed over a much broader region. Three particular genes were expressed in the older senescent zone of the nodule. These findings are important because they show that not all genes in root nodules are expressed uniformly, indicating that there are several factors that affect plant gene expression in nodules. The fact that only a small portion of the 33-day-old nodule is fixing and assimilating N suggests that one strategy for increasing N2 fixation in legumes would be to select plants having either delayed nodule senescence or a larger N2-fixing zone.
Technical Abstract: During root nodule development several key genes involved in nitrogen fixation and assimilation exhibit enhanced levels of expression. However, little is known about the temporal and spatial distribution pattern of these transcripts. In a systematic study the transcripts for 13 of the essential enzymes involved in alfalfa (Medicago sativa L.) root nodule nitrogen and carbon metabolism were localized using in situ hybridization. A serial section approach allowed the construction of a map that reflects the relative distribution of these transcripts. In 33-day-old root nodules, the expression of nifH, NADH-dependent glutamate synthase (NADH-GOGAT; EC 220.127.116.11) and a cytosolic isoform of glutamine synthetase (GS13; GS; EC 18.104.22.168) were localized predominantly in a 5- to 15-cell-wide region in the distal part of the nitrogen-fixing zone. This zone was also the region of high expression for leghemoglobin, a second cytosolic glutamine synthetase isoform (GS100), aspartate aminotransferase-2 (AAT-2; EC 22.214.171.124), asparagine synthetase (AS; 126.96.36.199), phosphoenolpyruvate carboxylase (PEPC; EC 188.8.131.52), and sucrose synthase (SuSy; EC 184.108.40.206). This suggests that in 33-day-old alfalfa root nodules, nitrogen fixation is restricted to this 5- to 15-cell-wide area. The continued significant expression of the GS100 subclass of GS and AS in the proximal part of the nitrogen-fixing zone implicates these gene products in nitrogen remobilization. A low constitutive expression of NADH glutamate dehydrogenase (NADH-GDH; EC 220.127.116.11) was observed throughout the nodule. The transcript distribution map will be used as a navigational tool to assist in developing strategies for the genetic engineering of alfalfa root nodules for enhanced nitrogen assimilation.