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United States Department of Agriculture

Agricultural Research Service

Title: Inhibition of Alfalfa Root Nodule Phosphoenolpyruvate Carboxylase Through An Antisense Strategy Impacts Nitrogen Fixation and Plant Growth

Authors
item Schulze, Joachim - UNIVERSITY OF MINNESOTA
item Shi, Lifang - UNIVERSITY OF MINNESOTA
item Blumenthal, Jurg - UNIVERSITY OF MINNESOTA
item SAMAC, DEBORAH
item Gantt, J - UNIVERSITY OF MINNESOTA
item VANCE, CARROLL

Submitted to: Phytochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 28, 1998
Publication Date: 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 compounds derived from sugars produced in the leaves, while the bacterium gives the plant nitrogen fertilizer. Before the nodule bacteria can use the energy from plant sugars, those sugars must be converted to a useful form. The plant enzyme phosphoenolpyruvate carboxylase (PEPC, an enzyme catalyst) is thought to play a major role in the conversion of sugars to energy useful for nitrogen fixing bacteria in root nodules. To test whether PEPC is required for nitrogen fixation we used a biotechnology approach to either reduce or prevent PEPC from forming. In this approach an extra copy of the PEPC gene in the opposite orientation (antisense) to the normal PEPC gene was transferred specifically to alfalfa nodules. Whe the extra copy of the PEPC gene is in the antisense orientation to the normal PEPC gene, the activity of PEPC 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 PEPC were reduced in PEPC activiy, nitrogen fixation, and growth as compared to the control plants. These data provide the first direct genetic evidence that PEPC is critical for nitrogen fixation. Our information is important because: (1) it shows that we can genetically engineer individual steps in root nodule nitrogen and energy metabolism and (2) it offers the possibility that we can use specific genes to improve nitrogen fixation.

Technical Abstract: Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) plays a paramount role in nodule metabolism and several reports have shown that PEPC provides substantial carbon for N2-fixation and N assimilation. To study the short- and long-term implications of reduced nodule CO2 fixation for N2 fixation in alfalfa, PEPC enzyme expression was reduced through an antisense strategy. The full-length root nodule-enhanced PEPC cDNA in antisense orientation driven by the nodule-enhanced AAT-2 promoter was transformed into alfalfa. Out of 105 transformed plants, 14 showed reduced in vitro nodule PEPC activity. Three plants were selected for further evaluation. RNA and protein blots showed reduced PEPC transcript and protein. Nodules of these plants also displayed reduced in vivo CO2 fixation. Total nitrogenase activity as measured by H2 evolution was reduced, although there was no change in apparent nitrogenase. The nodule electron allocation coefficient of antisense plants was reduced. All antisense plants accumulated less dry matter and nitrogen in a 6-week growing period under controlled conditions. The data confirm a strong interdependence of nodule PEPC and nitrogenase activity.

Last Modified: 9/10/2014
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