PRIMARY ASSIMILATION OF NITROGEN IN ALFALFA NODULES - MOLECULAR-FEATURES OF THE ENZYMES INVOLVED

The primary assimilation of symbiotically fixed nitrogen (N) in alfalf a root nodules involves complex intermingling with carbon (C) metaboli sm. Integrated functioning of both cytosolic and organelle-associated enzymes is required to link N assimilation with C metabolism. Understa nding how N and C metabolism are controlled in root nodules requires f undamental knowledge of how the plant genes involved are regulated. Wh ile significant progress has been made in understanding the regulation of glutamine synthetase (GS), much less is known about the genes cont rolling other enzymatic steps in this process. To that end we have iso lated, purified and characterized the root nodules enzymes aspartate a minotransferase (AAT), phosphoenolpyruvate carboxylase (PEPC) and glut amate synthase (NADH-GOGAT). Moreover the cDNAs encoding these crucial enzymes were isolated and characterized. While the most prominent for ms of GS associated with N assimilation in nodules are located in the cytosol, AAT and NADH-GOGAT appears to be organelle-associated. The de duced amino acid sequence suggested and immunogold labeling showed tha t nodule-enhanced AAT-2 is located in amyloplasts. Comparison of the d educed amino acid sequence of nodule-enhanced NADH-GOGAT to the N-term inal sequence of the processed protein indicated that NADH-GOGAT has a 101 amino acid presequence. However, it is unclear as to which organe lle NADH-GOGAT is targeted. Cytosolic phosphoenolpyruvate carboxylase (PEPC), which can be expressed in legume root nodules at levels compar able to those detected in leaves of C4 plants, provides a substantial amount of carbon for malate, aspartate and asparagine biosyntheses. RN A blots showed that GS, AAT, PEPC, and NADH-GOGAT mRNAs were enhanced about 15-fold during the development of effective alfalfa nodules. By comparison, the expression of GS, AAT and PEPC mRNAs was reduced by 65 % in ineffective nodules. NADH-GOGAT was different from GS, AAT, and P EPC in that expression had an absolute requirement for a factor(s) rel ated to effective nodules. The data suggest that NADH-GOGAT plays a ke y role in regulating N assimilation. Moreover, plastids in nodules pla y a major role not only in C metabolism but also in N metabolism.