Detection and Functional Analysis of Promoter Elements of Genes Involved in N Assimilation in Plant
Nitrogen is the inorganic nutrient that plants require in greatest quantity and that most frequently limits productivity in agricultural systems. Most plants acquire nitrogen from the soil, as nitrate through commercial fertilizer or mineralization of indigenous organic matter. Plants, in general, cannot utilize free nitrogen which is present in abundant amounts in the atmosphere. Leguminous plants like alfalfa, however, are capable of forming a symbiotic relationship with a soil bacteria, resulting in the formation of the root nodule, where the symbiont can convert free nitrogen into NH3 which can be utilized by the host plant. To ensure high yields, farmers in the U.S. apply over 11 million metric tons of nitrogen fertilizer annually; manufacture and distribution of this fertilizer account for over one-third of the total energy expended in agriculture. Because of heavy use of fertilizers, the worldwide nitrogen pollution along with water shortage is becoming one of the major threats to human survival and the environment. Increasing the efficiency of nitrogen use would thus have an impact both on the cost of producing fertilizers and in minimizing problems associated with pollution. An alternative approach to using fertilizers would be to make all plants capable of using free nitrogen. A straightforward strategy for improving nitrogen (N) assimilation was assumed to be the enhancement of enzyme activities for N assimilation. However, N assimilation requires not only inorganic nitrogen but also the carbon (C) skeleton that is produced through sequential reactions from photoassimilates. Moreover, based on the results obtained with manipulating the levels of key enzymes in just the N assimilatory pathway or the C assimilatory pathway alone, we have concluded that the two pathways are metabolically connected. Thus, to accomplish our goal of increasing biomass, we have to understand the molecular and biochemical mechanisms associated with N-assimilation and to understand how the C-metabolic pathway influences N-metabolism.
We propose that improvement of the nitrogen assimilatory capability of plants would require the simultaneous modulation of expression of several of the genes in the pathway. Our hypothesis is that the entire repertoire of the key genes in the N assimilatory pathway are regulated by a similar signaling mechanism and that all these genes have similar regulatory motifs or elements to which the same regulatory protein/s (transcription factors) bind to activate expression. This would further imply that enhancing the N-assimilatory pathway could be accomplished by introducing copies of the transcription factor genes driven by a constitutive promoter. Thus, our experimental approach of identifying transcription factors that regulate the N assimilatory pathway genes involved in the N assimilation pathway is the first step towards our long term goal to engineer plants with a gene that would switch on the whole pathway, rather than individual genes, involved in N assimilation. The objectives of this collaborative research project is to use bioinformatics tools to identify the common cis regulatory elements in the genes involved in this common biochemical pathway leading from ammonia to amino acids and to demonstrate their regulatory role by functional analysis. Confirmation of the regulatory nature of the .cis. elements will be performed using genetic engineering tools and microscopy. The gene members that will be used for the analysis of .cis. elements will be those that are expressed in the root nodules of alfalfa which is the major site of nitrogen assimilation in the plant. The long term objective is to isolate the genes for the transcription factors that bind to these .cis. elements and use them for turning on the N assimilatory pathway in transgenic plants.