Inhibition of alfalfa root nodule phosphoenolpyruvate carboxylase through an antisense strategy impacts nitrogen fixation and plant growth

Phosphoenolpyruvate carboxylase (PEPC, EC 411.1.31) 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.