Nitrate assimilation by plants, green algae, and some fungi and bacteria, is a complex, energy-consuming process which provides most of the organic nitrogen in the biosphere. The genes required for nitrate uptake and its reduction are tightly regulated, and respond to a variety of environmental and physiological signals. The long-term goal of the proposed research is to understand how the expression of genes involved in nitrate assimilation is regulated in photosynthetic organism. This study uses the unicellular green alga, Chlamydomonas, as a model experimental organism to identify and characterize genes that mediate control of nitrate assimilation. Initially, attention will be focused on understanding how ammonium, the product of nitrate assimilation, represses the expression of nitrate assimilation genes. Six genes involved in nitrate assimilation are clustered within a single region of the chromosome. Expression of these genes is coordinately blocked when ammonium is present, derepressed upon removal of ammonium, and induced to a high level by nitrate. Both derepression and induction of these genes requires the NIT2 gene product. The NIT2 gene appears to play a key role in mediating both positive and negative control of the pathway, since the NIT2 gene itself is repressed by ammonium. The ability to isolate mutants in Chlamydomonas that disrupt this regulation provides a powerful tool for identifying the cellular components required for perceiving and transducing the ammonium signal into a change in the pattern of gene expression. Mutants that express nitrate assimilation genes constitutively in the presence of ammonium have so far identified two negative regulatory genes, FARI and FAR2. Both genes are required for repression of the N1T2 gene by ammonium. The first specific aim of this proposal is to saturate the genome with mutations that define trans-acting factors required for negative control of nitrate assimilation. Mutants that disrupt this regulation will be informative for resolving whether repression occurs at one or more stages of gene expression and by one or more pathways. The second specific aim is to characterize the cloned FARI gene, and to use the collection of mutants to isolate and characterize the FAR2 gene and any additional genes identified in this study by transposon tagging.
