INHIBITION OF ALFALFA ROOT-NODULE PHOSPHOENOLPYRUVATE CARBOXYLASE THROUGH AN ANTISENSE STRATEGY IMPACTS NITROGEN-FIXATION AND PLANT-GROWTH

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) plays a paramount role in nodule metabolism and several reports have shown that PEPC pro vides substantial carbon for N-2-fixation and N assimilation. To study the short- and long-term implications of reduced nodule CO2 fixation for N-2 fixation in alfalfa, PEPC enzyme expression was reduced throug h an antisense strategy. The full-length root nodule-enhanced PEPC cDN A in antisense orientation driven by the nodule-enhanced AAT-2 promote r was transformed into alfalfa. Out of 105 transformed plants, 14 show ed reduced in vitro nodule PEPC activity. Three plants were selected f or further evaluation. RNA and protein blots showed reduced PEPC trans cript and protein. Nodules of these plants also displayed reduced in v itro CO2 fixation. Total nitrogenase activity as measured by H-2 evolu tion was reduced, although there was no change in apparent nitrogenase . The nodule electron allocation coefficient of antisense plants was r educed. All antisense plants accumulated less dry matter and nitrogen in a 6-week growing period under controlled conditions. The data confi rm a strong interdependence of nodule PEPC and nitrogenase activity. ( C) 1998 Elsevier Science Ltd. All rights reserved.