|Simonsen, Anna Carina|
Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/13/1999
Publication Date: N/A
Citation: Interpretive Summary: Legume plants acquire nitrogen fertilizer through a process called symbiotic nitrogen fixation. This process occurs in small wart-like structures on roots, termed nodules. Within the nodule Rhizobium bacteria acquire carbon and nitrogen nutrients from the plant, while the plant receives nitrogen fertilizer in the form of ammonia from the bacterium. How this nutritional exchange between the plant and bacterium occurs is a key question to be resolved. In this report we have shown that a key plant enzyme (protein catalyst) aspartate aminotransferase-2 (AAT-2) is involved in this process. As the symbiotic Rhizobium bacteria acquire the capacity to convert atmospheric nitrogen gas into ammonia they become associated with the plant AAT-2 enzyme. Thus, it appears that the bacterium exchanges the ammonia produced by symbiotic nitrogen fixation for the plant produced amino acid aspartate. These findings are important because they identify a critical plant enzyme (AAT-2) that may be a target for approaches to improve nitrogen fixation and assimilation through biotechnology. We have recently isolated and characterized the alfalfa and pea genes that encode the AAT-2 enzyme.
Technical Abstract: Symbiosomes purified from pea (Pisum sativum L.) root nodules were used to study the targeting of aspartate aminotransferase (AAT; EC 18.104.22.168) to the interface between the symbionts in legume root nodules. An immunoblot analysis with polyclonal antibodies to nodule-enhanced AAT (AAT-2) indicated presence of AAT-2 in the peribacteroid space (PBS) fraction of purified symbiosomes. This suggests that AAT-2 may be a specific candidat for a plant protein targeted to the symbiosomes. To examine the hypothesis that AAT-2 is targeted to pea-Rhizobium symbiosomes we established an in vitro protein import assay. The plasmid pBSSKAAT2-3, encoding alfalfa AAT-2, was expressed in cell-free systems by in vitro transcription followed by in vitro translation in the presence of L-35S methionine. Purified symbiosomes were incubated with the radio-labeled pBSSKAAT2-3 expression products. Autoradiography of polyacrylamide gels with symbiosome subfractions, revealed association of the precursor form of AAT-2 with the symbiosome membrane (SM). A distinct band with a molecular weight corresponding to the processed form of AAT-2 (40 kD) was observed in the fraction that constitutes the interface between the symbionts. These results support the hypothesis that AAT-2 is targeted to the symbiosome and that the precursor form of AAT-2 is subjected to proteolytic cleavage during translocation across the SM.