DOE GRANT DEFG02-89ER14034: "Asparagine synthetase gene regulation and plant nitrogen metabolism" P.I. Gloria Coruzzi

ABSTRACT We are studying the structural and regulatory genes controlling asparagine metabolism (synthesis and catabolism) in the model plant, Arabidopsis thaliana. As asparagine serves as a N-storage and N-transport compound in plants, our long-term goals are to elucidate how the regulation of genes involved in asparagine metabolism controls nitrogen use, growth, and development. We also aim to isolate regulatory components affecting the expression of genes in this Asn metabolic pathway, which may be used to modify the efficiency of N-use in transgenic plants. We are accomplishing these goals by combining molecular-genetic approaches with novel Systems Biology and genomics-based approaches.

The significance of our studies to the DOE mission is two-fold:

1. Energy. Our Systems Biology approach will enable us to model and predictively alter Asn metabolic genes to enhance N-use efficiency, an energy conservation issue in agriculture.

2. Systems Biology. The Systems-based and genomics tools that we develop and test in the course of this project will be applicable to all areas of biological regulatory systems.

Thus, our studies on modeling the regulation of Asn metabolism in Arabidopsis may serve as a working example for modeling and modifying any other pathways in plants or other species. Our Specific Aims are the following:

Aim 1. To model regulation of Asn metabolism genes using a Systems Biology approach. For this, we will use Boolean Logic to model how signaling interactions of light, carbon and nitrogen affect the regulation of genes in the Asn metabolism pathway and to correlate these with changes in levels of Asn. These studies focus on modeling the regulation of genes involved in asparagine synthesis (Asparagine Synthtase, ASN1-3) and asparagine catabolism (Asparaginase, ANS1, ANS2).

Aim 2. To identify cis and trans regulatory components affecting expression of Asn metabolism genes by combining molecular-genetics and genomic analyses. We have used a positive genetic selection to identify lir mutants impaired in L/C repression of ASN1. We propose to map-base clone the affected gene to identify the nature of the signaling components involved in regulating the Asn metabolic pathway in plants. We will also use genomic chip analysis to identify circuits of genes mis-regulated in the lir mutants, including other genes involved in the metabolism of Asn substrates and products. We will identify cis-acting regulatory elements shared by these co-regulated genes in the Asn metabolism pathway using a microarray-enabled genomics approach combined with bioinformatics.

Aim 3. To determine how altering expression or regulation of Asn metabolism genes affects N-use, growth and development using mutants and transgenics. We will determine whether alterations in the regulation of genes in the Asn metabolic pathway affect the N-use and N-content of seed by subjecting mutants in regulatory and structural genes to a series of phenotypic effects to monitor N-use efficiency. These studies should enable us to define how the genes in the regulation of Asn metabolism control N-use efficiency in plants.