The research in this proposal will focus on host plant biochemical and molecular mechanisms that regulate phosphoenolpyruvate carboxylase and NADH-glutamate synthase, key enzymes involved in assimilation of symbiotically fixed nitrogen in alfalfa (Medicago sativa L.) root nodules. This proposal is based on two hypotheses. The first is that expression of the nodule PEPC and NADH-GOGAT genes is regulated by cis-acting element(s) that interact with one or more trans-acting factors and the activity of these factors is a function of one or more of the following: nodule organogenesis, effective bacteroids, ammonium ion availability and/or oxygen concentration. The second is that direct genetic alteration of nodule PEPC and/or NADH-GOGAT enzyme activities impacts upon nodule nitrogen fixation and ammonia assimilation. The specific objectives include: 1) isolation and sequence analysis of PEPC and NADH- GOGAT genomic clones and initial characterization of the promoter regions of these gene by deletion analysis and by protein-DNA binding studies: 2) localization in planta of PEPC and NADH- GOGAT proteins and mRNAs by immunogold staining and by in situ hybridization; and 3) alteration in planta of PEPC and NADH- GOGAT activities through direct modification of enzyme synthesis by the expression of transformed genes. Knowledge gained from these studies will enhance our fundamental understanding of plant genes involved in ammonia assimilation and may facilitate crop improvement through the development of strategies to modify nitrogen metabolism.
