Nitrate is the predominant form of soil nitrogen available to plants. Nitrate, the highly oxidized form of inorganic nitrogen, must be reduced to ammonia prior to incorporation into amino acids. Thus, the nitrate reduction process, together with carbon fixation, are the two most important biological pathways by which plants replenish the biological food chain, converting inorganic matter into biological building blocks. Nitrate reductase (NR), the first enzyme in the nitrate reduction pathway is tightly regulated by nitrate and light. The goal of this proposal is to elucidate the regulatory pathway governing NR gene transcription. The processes are most easily conceptualized as DNA-binding proteins that interact with cis-acting sequences. This interaction is mediated by a host of other trans-acting factors; such as those involved in signal (light and/or nitrate) transduction. In order to molecularly characterize the cis-acting DNA sequences and the DNA-binding factors responsible for the regulated expression of NR genes, putative regulatory regions from Arabidopsis NR genes have been fused to a reporter gene and transformed into Arabidopsis. After plant regeneration, the reporter gene exhibited nitrate- and light-induced expression. The specific sequences required for this regulation will be defined by mutational analysis. The genes encoding the DNA-binding factors that interact with the cis-acting sequences will be sought. These factors will be characterized biochemically and subsequently cloned. To identify trans- acting factors other than DNA-binding proteins, I am developing genetic approaches that will allow me to select mutants defective in such factors. In concert, these two approaches will allow me to elucidate the molecular mechanisms regulating NR gene expression. The transcriptional regulation of NR genes in Arabidopsis offers an excellent model system to study transcriptional regulation in eucaryotes. The NR genes are regulated by two well defined stimuli: nitrate and light. Arabidopsis is an excellent experimental organism for genetic analysis. Arabidopsis has an extensive genetic map with only 5 linkage groups, a short generation time, it self fertilizes to allow recessive mutants to be easily recovered, it is compact and produces large numbers of seeds, making large scale mutant screens feasible, it has a small genome size with very little repetitive DNA to facilitate molecular biological manipulation, and it can be transformed by Agrobacterium-mediated transformation systems and transgenic plants regenerated with high efficiency. A powerful genetic scheme indigenous to NR offers a rare opportunity in an eucaryotic system to select for trans-acting factor mutants and for selection of revertants of such mutants.
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