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Title: NITROGEN ASSIMILATION IN ALFALFA: ISOLATION AND CHARACTERIZATION OF AN ASPARAGINE SYNTHETASE GENE SHOWING ENHANCED EXPRESSION IN ROOT NODULES AND DARK-ADAPTED LEAVES

Author
item SHI, LIFANG - UNIVERSITY OF MINNESOTA
item TWARY, SCOTT - UNIVERSITY OF MINNESOTA
item YOSHIOKA, HIROFUMI - UNIVERSITY OF MINNESOTA
item GREGERSON, ROBERT - LYON COLLEGE
item MILLER, SUSAN - UNIVERSITY OF MINNESOTA
item Samac, Deborah - Debby
item GANTT, J - UNIVERSITY OF MINNESOTA
item UNKEFER, PAT - LOS ALAMOS NATIONAL LAB
item Vance, Carroll

Submitted to: The Plant Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Mineral nutrients are critical for plant growth and reproduction. One of the most important mineral nutrients is nitrogen, which is used to form amino acids, proteins, enzymes, and other plant components. Plants in the bean family, such as alfalfa, are able to form a symbiotic relationship with a soil bacterium that allows them to use nitrogen gas from the atmosphere, in a process called nitrogen fixation, to form amino acids. Plants lacking this ability rely on nitrogen in the form of nitrate or ammonia that is usually found in only limited amounts in soil and must be added as fertilizer for crop production. An alfalfa gene for production of the amino acid asparagine, a reaction central to nitrogen fixation, was isolated. This is the first report of the isolation of this gene from a plant. This gene functions primarily in the root nodule, a plant organ formed in response to the symbiotic bacteria, and the site of nitrogen fixation. Initial gene activity coincides with the onset of nitrogen fixation, however, the signal for activation is still to be determined. The asparagine formed in the nodule travels through the rest of the plant, providing the nitrogen required for growth and development. The isolation of this gene will allow further studies on regulation of nitrogen fixation and modifications of plants for enhanced nitrogen fixation. Success in the improvement of nitrogen assimilation could have significant benefits for agriculture by reducing the need for and use of nitrogen fertilizer.

Technical Abstract: Asparagine, the primary assimilation product from N2 fixation in temperate legumes and the predominant nitrogen transport product in many plant species, is synthesized via asparagine synthetase (AS; EC 6.3.5.4). Here we report the isolation and characterization of a cDNA and a gene encoding the nodule-enhanced form of AS from alfalfa. The AS gene is comprised of 13 exons separated by 12 introns. The 5' flanking region of the AS gene confers nodule-enhanced reporter gene activity in transformed alfalfa. This region also confers enhanced reporter gene activity in dark-treated leaves. These results indicate that the 5' upstream region of the AS gene contains elements that affect expression in root nodules and leaves. Both AS mRNA and enzyme activity increased some 10- to 20-fold during the development of effective nodules. Ineffective nodules have strikingly reduced amounts of AS transcript. Alfalfa leaves have quite low levels of AS mRNA and protein, however, exposure to darkness resulted in a considerable increase in both. In situ hybridization with effective nodules and GUS staining of nodules from transgenic plants showed that AS is expressed in both infected and uninfected cells of the nodule symbiotic zone and in the nodule parenchyma. RNA gel blot analysis and in situ hybridization are consistent with the hypothesis that initial AS expression in nodules is independent of nitrogenase activity.