Primary assimilation of nitrogen in alfalfa nodules: molecular features of the enzymes involved

The primary assimilation of symbiotically fixed nitrogen (N) in alfalfa root nodules involves complex intermingling with carbon (C) metabolism. Integrated functioning of both cytosolic and organelle-associated enzymes is required to link N assimilation with C metabolism. Understanding how N and C metabolism are controlled in root nodules requires fundamental knowledge of how the plant genes involved are regulated. While significant progress has been made in understanding the regulation of glutamine synthetase (GS), much less is known about the genes controlling other enzymatic steps in this process. To that end we have isolated, purified and characterized the root nodules enzymes aspartate aminotransferase (AAT), phosphoenolpyruvate carboxylase (PEPC) and glutamate synthase (NADH-GOGAT). Moreover the cDNAs encoding these crucial enzymes were isolated and characterized. While the most prominent forms of GS associated with N assimilation in nodules are located in the cytosol, AAT and NADH-GOGAT appears to be organelle-associated. The deduced amino acid sequence suggested and immunogold labeling showed that nodule-enhanced AAT-2 is located in amyloplasts. Comparison of the deduced amino acid sequence of nodule-enhanced NADH-GOGAT to the N-terminal sequence of the processed protein indicated that NADH-GOGAT has a 101 amino acid presequence. However, it is unclear as to which organelle ADH-GOGAT is targeted. Cytosolic phosphoenolpyruvate carboxylase (PEPC), which can be expressed in legume root nodules at levels comparable to those detected in leaves of C4 plants, provides a substantial amount of carbon for malate, aspartate and asparagine biosyntheses. RNA 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 PEPC in that expression had an absolute requirement for a factor(s) related to effective nodules. The data suggest that NADH-GOGAT plays a key role in regulating N assimilation. Moreover, plastids in nodules play a major role not only in C metabolism but also in N metabolism.